MedWorm.com provides a medical RSS filtering service. Over 6000 RSS medical sources are combined and output via different filters. This feed contains the latest items from the 'Springer protocols feed by Protein Science' source. Thu, 09 Feb 2012 13:38:43 +0100 http://www.medworm.com/index.php?rid=5473453&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-379-0_1 Ribosome display is an in vitro evolution technology for proteins. It is based on in vitro translation, but prevents the newly synthesized protein and the mRNA encoding it from leaving the ribosome. It thereby couples phenotype and genotype. Since no cells need to be transformed, very large libraries can be used directly in selections, and the in vitro amplification provides a very convenient integration of random mutagenesis that can be incorporated into the procedure. This review highlights concepts, mechanisms, and different variations of ribosome display and compares it to related methods. Applications of ribosome display are summarized, e.g., the directed evolution of proteins for higher binding affinity, for higher stability or other improved biophysical parameters and enzymatic prop... Springer protocols feed by Protein Science news http://www.medworm.com/rss/comments.php?id=5473453 Mon, 05 Dec 2011 07:28:54 +0100 5473453 http://www.medworm.com/index.php?rid=5473452&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-379-0_2 Crude cell-free extracts are useful tools for investigating biochemical phenomena and exploiting complex enzymatic processes such as protein synthesis. Extracts derived from E. coli have been used for over 50 years to study the mechanism of protein synthesis. In addition, these S30 extracts are commonly used as a laboratory tool for protein production. The preparation of S30 extract has been streamlined over the years and now it is a relatively simple process. The procedure described here includes some suggestions for extracts to be used for ribosome display. (Source: Springer protocols feed by Protein Science) Springer protocols feed by Protein Science news http://www.medworm.com/rss/comments.php?id=5473452 Mon, 05 Dec 2011 07:28:54 +0100 5473452 http://www.medworm.com/index.php?rid=5473451&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-379-0_3 Ribosome display is a powerful in vitro technology for the selection and directed evolution of proteins. Cell-free translation is central to the ribosome display process and is performed in such a way that the ribosome provides the link between genotype and phenotype that allows genes encoding proteins with desired properties to be identified by selection. Prokaryotic cell-free translation reagents, based initially on E. coli cell extracts and more recently containing purified and recombinant factors, have dominated the ribosome display literature. Eukaryotic cell extracts are also suitable for ribosome display; however, protocols for prokaryotic ribosome display are not directly transferable to the use of eukaryotic cell extracts. This chapter describes an optimised methodology for the us... Springer protocols feed by Protein Science news http://www.medworm.com/rss/comments.php?id=5473451 Mon, 05 Dec 2011 07:28:54 +0100 5473451 http://www.medworm.com/index.php?rid=5473450&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-379-0_4 Ribosome display is a very effective and powerful technology for screening functional peptides or polypeptides in vitro. In ribosome display, each peptide or polypeptide (phenotype) links with its corresponding mRNA (genotype) through a ribosome. This link can be achieved by the absence of a stop codon in the mRNA, therefore stalling the ribosome at the end of translation with the nascent random sequence peptide extended by a spacer outside of the ribosome tunnel. In this chapter, we describe a method for the use of a further stabilized peptide–ribosome–mRNA complex for ribosome display. (Source: Springer protocols feed by Protein Science) Springer protocols feed by Protein Science news http://www.medworm.com/rss/comments.php?id=5473450 Mon, 05 Dec 2011 07:28:54 +0100 5473450 http://www.medworm.com/index.php?rid=5473449&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-379-0_6 mRNA display is a powerful technique that allows for covalent coupling of a translated protein with its coding mRNA. The resulting conjugation between genotype and phenotype can be used for the efficient selection and identification of peptides or proteins with desired properties from an mRNA-displayed peptide or protein library with high diversity. This protocol outlines the principle of mRNA display and the detailed procedures for the synthesis of mRNA–protein fusions. Some special considerations for library construction, generation, and purification are discussed. (Source: Springer protocols feed by Protein Science) Springer protocols feed by Protein Science news http://www.medworm.com/rss/comments.php?id=5473449 Mon, 05 Dec 2011 07:28:54 +0100 5473449 http://www.medworm.com/index.php?rid=5473448&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-379-0_5 Ribosome display is a cell-free display technology for in vitro selection and optimisation of proteins from large diversified libraries. It operates through the formation of stable protein-ribosome-mRNA (PRM) complexes and selection of ligand-binding proteins, followed by DNA recovery from the selected genetic information. Both prokaryotic and eukaryotic ribosome display systems have been developed. In this chapter, we describe the eukaryotic rabbit reticulocyte method in which a distinct in situ single-primer RT-PCR procedure is used to recover DNA from the selected PRM complexes without the need for prior disruption of the ribosome. (Source: Springer protocols feed by Protein Science) Springer protocols feed by Protein Science news http://www.medworm.com/rss/comments.php?id=5473448 Mon, 05 Dec 2011 07:28:54 +0100 5473448 http://www.medworm.com/index.php?rid=5473447&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-379-0_8 The cDNA display method is a robust in vitro display technology that converts an unstable mRNA–protein fusion (mRNA display) to a stable mRNA/cDNA–protein fusion (cDNA display) whose cDNA is covalently linked to its encoded protein using a well-designed puromycin linker. We provide technical details for preparing cDNA display molecules and for the synthesis of the puromycin linker for the purpose of screening the functional proteins and peptides. (Source: Springer protocols feed by Protein Science) Springer protocols feed by Protein Science news http://www.medworm.com/rss/comments.php?id=5473447 Mon, 05 Dec 2011 07:28:54 +0100 5473447 http://www.medworm.com/index.php?rid=5473446&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-379-0_7 SNAP display is based on the covalent reaction of the DNA repair protein AGT (O6-alkylguanine DNA alkyltransferase, the “SNAP-tag”) with its substrate benzylguanine (BG). Linear, BG-labelled template DNA is encapsulated in water-in-oil emulsion droplets with a diameter of a few micrometres (i.e. 1 mL of emulsion contains ∼1010 compartments). Each droplet contains only a single DNA copy, which is transcribed and translated in vitro. The expressed AGT fusion proteins attach to their coding DNA via the BG label inside the droplet, which ensures that a specific genotype–phenotype linkage is established. Subsequently, the emulsion is broken and protein-DNA conjugates, which constitute a DNA-tagged protein library, selected via affinity panning. This method will prove a use... Springer protocols feed by Protein Science news http://www.medworm.com/rss/comments.php?id=5473446 Mon, 05 Dec 2011 07:28:54 +0100 5473446 http://www.medworm.com/index.php?rid=5473445&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-379-0_11 The opportunity to enhance protein stability has a number of potential benefits for biological therapeutics – for example extending in vivo half-life, enabling a longer shelf life, reducing the propensity to aggregate, or enabling soluble expression. Engineering protein stability has been attempted empirically, rationally, and using directed evolution based on phage display. Ribosome display is a powerful in vitro technology for the selection and directed evolution of proteins. Ribosome display is typically used for the generation of high-affinity proteins and peptides. This method extends the utility of ribosome display to selecting for stability, defined as the propensity of a molecule to exist in its folded and active state. (Source: Springer protocols feed by Protein Science) Springer protocols feed by Protein Science news http://www.medworm.com/rss/comments.php?id=5473445 Mon, 05 Dec 2011 07:28:54 +0100 5473445 http://www.medworm.com/index.php?rid=5473444&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-379-0_10 Ribsosome display is a PCR-based in vitro display technology that it well suited for the selection and evolution of high-affinity antibodies. In particular, ribosome display lends itself to the evolution of functional characteristics, such as potency, and thereby facilitates the production of therapeutic antibodies from lead candidates. In this chapter, we describe how to mature large phage display antibody populations (>107) by performing increasingly stringent selections with decreasing antigen concentration. This process takes advantage of ribosome display’s intrinsic ability to evolve sequence during selection. Ribosome display can also be used as a complementary tool to phage display for isolating high-affinity antibodies from naïve libraries. Ultimately, maturation of l... Springer protocols feed by Protein Science news http://www.medworm.com/rss/comments.php?id=5473444 Mon, 05 Dec 2011 07:28:54 +0100 5473444 http://www.medworm.com/index.php?rid=5473443&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-379-0_12 A large antibody fragment library (>1012) has been generated in ribosome display format. The library was constructed in a two-step process. First, variable (V) genes were isolated from human B cells from a panel of 14 donors and cloned into designated ribosome display vectors to create a gene bank. Second, RD-VH and RD-VL genes from individual immunoglobulin families were combined in vitro resulting in 112 scFv ribosome display sub-libraries. These were subsequently pooled to form a master library. (Source: Springer protocols feed by Protein Science) Springer protocols feed by Protein Science news http://www.medworm.com/rss/comments.php?id=5473443 Mon, 05 Dec 2011 07:28:54 +0100 5473443 http://www.medworm.com/index.php?rid=5473442&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-379-0_13 Protein scaffolds containing some disulfide bonds (e.g., Knottin, Kunitz domain, etc.) are promising candidates for molecular recognition. cDNA display has been developed to screen functional disulfide-rich peptide aptamers from a vast library by promoting disulfide bond shuffling after the synthesis of peptides in a cell-free translation system. Here we present a detailed protocol for the selection of disulfide-rich peptide aptamers against interleukin 6 receptor (IL-6R) from a 35-amino acid peptide library containing 32 amino acids in the random region, which is linked to its genotype by cDNA display. (Source: Springer protocols feed by Protein Science) Springer protocols feed by Protein Science news http://www.medworm.com/rss/comments.php?id=5473442 Mon, 05 Dec 2011 07:28:54 +0100 5473442 http://www.medworm.com/index.php?rid=5473441&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-379-0_14 To demonstrate directed protein evolution or selection of functional polypeptides, ribosome display is one of the most ideal technologies of evolutionary engineering. Intrinsic components, such as nucleases in the cell extract-based cell-free protein synthesis systems, reduce the stability of the messenger RNA–ribosome–polypeptide ternary complex, thereby preventing the attainment of reliable results. To overcome this problem, we have developed an effective and highly controllable ribosome display system using the protein synthesizing using recombinant elements (PURE) system. Since the activities of nucleases and other inhibitory factors are very low in the PURE system, the ternary complex is highly stable and the selected mRNA can be reliably recovered. Using this system, we w... Springer protocols feed by Protein Science news http://www.medworm.com/rss/comments.php?id=5473441 Mon, 05 Dec 2011 07:28:54 +0100 5473441 http://www.medworm.com/index.php?rid=5473440&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-379-0_16 mRNA display is a powerful in vitro selection technique that can be applied toward the identification of peptides or proteins with desired properties. The physical conjugation between a protein and its own RNA presents unique challenges in manipulating the displayed proteins in an RNase-free environment. This protocol outlines the generation of synthetic peptide and natural proteome libraries as well as the steps required for generation of mRNA–protein fusion libraries, in vitro selection, and regeneration of the selected sequences. The selection procedures for the identification of Ca2+-dependent, calmodulin-binding proteins from synthetic peptide and natural proteome libraries are presented. (Source: Springer protocols feed by Protein Science) Springer protocols feed by Protein Science news http://www.medworm.com/rss/comments.php?id=5473440 Mon, 05 Dec 2011 07:28:54 +0100 5473440 http://www.medworm.com/index.php?rid=5473439&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-379-0_15 Ribosome display has proven to be a powerful in vitro selection and evolution method for generating high-affinity binders from libraries of folded proteins. It has been successfully applied to single-chain Fv fragments of antibodies and alternative scaffolds, such as Designed Ankyrin Repeat Proteins (DARPins). High-affinity binders with new target specificity can be obtained from highly diverse DARPin libraries in only a few selection rounds. In this protocol, the selection from the library and the process of affinity maturation and off-rate selection are explained in detail. (Source: Springer protocols feed by Protein Science) Springer protocols feed by Protein Science news http://www.medworm.com/rss/comments.php?id=5473439 Mon, 05 Dec 2011 07:28:54 +0100 5473439 http://www.medworm.com/index.php?rid=5473438&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-379-0_17 In vitro protein selection methods that are not biased by the context of living organisms allow the screening of genomic expression libraries against a large number of different ligands. As such, ribosome display is a powerful technology for the in vitro selection of proteins or peptides from large PCR-derived libraries. Libraries can be generated from the genomic fragments of pathogens, thus allowing potential vaccine genes and the immunologically relevant proteins of pathogens to be identified and mapped using ribosome display. This chapter describes a methodology for the use of ribosome display for the identification of potential vaccine genes for the bacterial pathogen APP-5. (Source: Springer protocols feed by Protein Science) Springer protocols feed by Protein Science news http://www.medworm.com/rss/comments.php?id=5473438 Mon, 05 Dec 2011 07:28:54 +0100 5473438 http://www.medworm.com/index.php?rid=5473437&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-379-0_18 We describe here the methodology for the construction of a library of Sac7d and its use for selection by ribosome display. (Source: Springer protocols feed by Protein Science) Springer protocols feed by Protein Science news http://www.medworm.com/rss/comments.php?id=5473437 Mon, 05 Dec 2011 07:28:54 +0100 5473437 http://www.medworm.com/index.php?rid=5473436&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-379-0_19 In vitro selection methods represent a powerful approach toward identifying high-affinity peptide ligands from highly diverse peptide libraries against a desired target. We herein describe a method for the display and selection of cyclic thioether peptide libraries. Reprogramming the initiation event from fMet to an N-chloroacetyl-amino acid by utilizing flexizyme to rapidly and efficiently prepare the aa-tRNA can be effectively used to initiate translation, upon which the thiol group of an inserted cysteine at the C terminus of the designed library spontaneously reacts to yield a nonreducible cyclic thioether peptide readily compatible with any in vitro display methods. Thus, cyclic peptides already in a nonreducible stable form can be selected directly against the target of interest. (So... Springer protocols feed by Protein Science news http://www.medworm.com/rss/comments.php?id=5473436 Mon, 05 Dec 2011 07:28:54 +0100 5473436 http://www.medworm.com/index.php?rid=5473435&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-379-0_9 Affinity optimisation of antibodies can be achieved with great success by using directed evolution approaches, that is, the creation of and selection from diverse libraries. Here, we describe in detail methods to optimise antibody affinity for an antigen through directed evolution using ribosome display. Diversification of antibody single chain variable (scFv) domains is carried out by error-prone PCR and oligonucleotide-directed mutagenesis to generate random and targeted libraries respectively. Subsequent libraries are converted to ribosome display format and taken through cycles of transcription, translation, and selection. Since the starting point and the recovered product are linear DNA, this can easily be manipulated further to allow accumulation of beneficial mutations through itera... Springer protocols feed by Protein Science news http://www.medworm.com/rss/comments.php?id=5473435 Mon, 05 Dec 2011 07:28:54 +0100 5473435 http://www.medworm.com/index.php?rid=5473434&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-379-0_23 The pursuit of more potent, safe, and cost-effective drugs has placed a greater emphasis on antibody optimisation within the drug discovery process. Technologies to rapidly improve antibody drug performance, such as phage display, ribosome display, and yeast display, are playing a key role in this effort. Among these ribosome display is a particularly powerful technology and has recently been applied to the affinity optimisation of a humanised anti-receptor for advanced glycation end products (anti-RAGE) antibody (Finlay et al., J Mol Biol 388:541–558, 2009). By using a combination of error-prone PCR with ribosome display each amino acid position within this humanised antibody was scanned for both its functional importance and its capacity to increase affinity resulting in both affin... Springer protocols feed by Protein Science news http://www.medworm.com/rss/comments.php?id=5473434 Mon, 05 Dec 2011 07:28:54 +0100 5473434 http://www.medworm.com/index.php?rid=5473433&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-379-0_22 In this case study, we describe the use of in vitro protein evolution with ribosome display to improve the potency of a human interleukin-13-neutralising antibody by a factor of over 200-fold and derive a therapeutic candidate, CAT-354, for the treatment of asthma. A combination of directed and random mutagenesis enabled the identification of highly potent neutralising antibodies and highlighted the advantage of the ribosome display protein evolution approach in identifying beneficial mutations across the entire sequence space. This chapter describes in detail the process followed to achieve a successful in vitro affinity maturation outcome using ribosome display technology. (Source: Springer protocols feed by Protein Science) Springer protocols feed by Protein Science news http://www.medworm.com/rss/comments.php?id=5473433 Mon, 05 Dec 2011 07:28:54 +0100 5473433 http://www.medworm.com/index.php?rid=5473432&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-379-0_21 The ribosomal synthesis of drug-like peptides containing unnatural amino acids is possible due to the broad substrate specificity of the ribosome. In this protocol, a reconstituted Escherichia coli ribosomal translation system (PURE) is adapted to incorporate unnatural amino acids into mRNA-displayed peptide libraries, which are used in in vitro selection. (Source: Springer protocols feed by Protein Science) Springer protocols feed by Protein Science news http://www.medworm.com/rss/comments.php?id=5473432 Mon, 05 Dec 2011 07:28:54 +0100 5473432 http://www.medworm.com/index.php?rid=5473431&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-379-0_20 The identification of peptide and protein ligands by directed evolution in vitro has been of enormous utility in molecular biology and biotechnology. However, the translation step in almost all polypeptide selection methods is performed in vivo or in crude extracts, restricting applications. These restrictions include a limited library size due to transformation efficiency, unwanted competing reactions in translation, and an inability to incorporate multiple unnatural amino acids (AAs) with high fidelity and efficiency. These restrictions can be addressed by “pure translation display” where the translation step is performed in a purified system. To date, all pure translation display selections have coupled genotype to phenotype in a ribosome display format, though other formats... Springer protocols feed by Protein Science news http://www.medworm.com/rss/comments.php?id=5473431 Mon, 05 Dec 2011 07:28:54 +0100 5473431 http://www.medworm.com/index.php?rid=5464973&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-445-2_6 Here, we describe an amino acid analysis that is based on the use of hydrophilic interaction liquid chromatography coupled with isotope dilution mass spectrometry for the accurate quantification of underivatized amino acids from hydrolyzed peptide/protein. Twelve underivatized amino acids were separated and detected during an 88-min runtime. The absolute limits of detection and limits of quantification (on column) of the four amino acids (isoleucine, phenylalanine, proline, and valine) were in the range of 6–80 and 20–200 fmol, respectively. As little as 25 pmol of peptide or protein hydrolysates is sufficient for determining absolute content. (Source: Springer protocols feed by Protein Science) Springer protocols feed by Protein Science news http://www.medworm.com/rss/comments.php?id=5464973 Fri, 02 Dec 2011 07:36:23 +0100 5464973 http://www.medworm.com/index.php?rid=5464972&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-445-2_5 (5-N-Succinimidoxy-5-oxopentyl)triphenylphosphonium bromide (SPTPP) is a highly sensitive and positively charged precolumn derivatization reagent for the analysis of amino acids in liquid chromatography–electrospray ionization-tandem mass spectrometry. The synthesis of this reagent and handling of the derivatization reaction are quite simple. It reacts with amino acids rapidly and with high efficiency. MS/MS analysis revealed that the SPTPP-derivatized amino acids formed strong product ions; thus, highly sensitive and selective detection is possible in the selected reaction monitoring mode. The limits of detection for the SPTPP-derivatized amino acids are in the sub-fmol range. The sensitivities of the derivatized amino acids increased about 500-fold, as compared to those of underiva... Springer protocols feed by Protein Science news http://www.medworm.com/rss/comments.php?id=5464972 Fri, 02 Dec 2011 07:36:23 +0100 5464972 http://www.medworm.com/index.php?rid=5464971&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-445-2_4 Analysis of underivatized amino acids is challenging regarding both separation and detection of this small, polar, and largely UV-inactive compounds. Additives for reversed phase chromatography such as ion-pairing reagents can hamper mass spectrometric detection. Zwitterionic hydrophilic interaction chromatography using MS compatible eluents together with tandem mass spectrometry in multiple reaction monitoring mode for selective detection is an attractive approach to overcome the abovementioned issues. (Source: Springer protocols feed by Protein Science) Springer protocols feed by Protein Science news http://www.medworm.com/rss/comments.php?id=5464971 Fri, 02 Dec 2011 07:36:23 +0100 5464971 http://www.medworm.com/index.php?rid=5464970&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-445-2_3 We present here our most recent method for the quantification of amino acids using isotope dilution LC-MS/MS. (Source: Springer protocols feed by Protein Science) Springer protocols feed by Protein Science news http://www.medworm.com/rss/comments.php?id=5464970 Fri, 02 Dec 2011 07:36:23 +0100 5464970 http://www.medworm.com/index.php?rid=5464969&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-445-2_2 Amino acid analysis is a powerful tool in life sciences. Current analytical methods used for the detection and quantitation of low abundance amino acids in complex samples face intrinsic challenges such as insufficient sensitivity, selectivity, and throughput. This chapter describes a protocol that makes use of AccQ∙Tag chemical derivatization combined with the exceptional chromatographic resolution of ultra performance liquid chromatography (UPLC), and the sensitivity and selectivity of tandem mass spectrometry (MS/MS). The method has been fully implemented and validated using different tandem quadrupole detectors, and thoroughly tested for a variety of samples such as Plasmodium falciparum, human red blood cells, and Arabidopsis thaliana extracts. Compared to currently available me... Springer protocols feed by Protein Science news http://www.medworm.com/rss/comments.php?id=5464969 Fri, 02 Dec 2011 07:36:23 +0100 5464969 http://www.medworm.com/index.php?rid=5464968&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-445-2_28 Analyzing individual cells allows detecting a minor group of abnormal cells present in a large population of normal cells. This ability can be essential to understanding diseases, such as cancer and diabetes. Microchip electrophoresis (MCE) is the technique of choice for single-cell analysis. However, since the channels in microfluidic devices are very small, achieving the desired assay sensitivity on a microfluidic platform remains a challenge. Here, we describe an MCE method with highly sensitive chemiluminescence detection for simultaneous determination of multiple amino acids present in single cells. (Source: Springer protocols feed by Protein Science) Springer protocols feed by Protein Science news http://www.medworm.com/rss/comments.php?id=5464968 Fri, 02 Dec 2011 07:36:23 +0100 5464968 http://www.medworm.com/index.php?rid=5464967&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-445-2_27 Single-compound analysis of stable or radio-isotopes has found application in a number of fields ranging from archaeology to forensics. Often, the most difficult part of these analyses is the development of a method for isolating the compounds of interest. (Source: Springer protocols feed by Protein Science) Springer protocols feed by Protein Science news http://www.medworm.com/rss/comments.php?id=5464967 Fri, 02 Dec 2011 07:36:23 +0100 5464967 http://www.medworm.com/index.php?rid=5464966&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-445-2_26 This chapter describes an accurate and user-friendly method for determining amino acid composition of wheat gluten proteins and their gliadin and glutenin fractions. The method consists of hydrolysis of the peptide bonds in 6.0 M hydrochloric acid solution at 110°C for 24 h, followed by evaporation of the acid and separation of the free amino acids by high-performance anion-exchange chromatography with integrated pulsed amperometric detection. In contrast to conventional methods, the analysis requires neither pre- or postcolumn derivatization, nor a time-consuming oxidation or derivatization step prior to hydrolysis. Correction factors account for incomplete release of Val and Ile even after hydrolysis for 24 h, and for losses of Ser during evaporation. Gradient conditions including an... Springer protocols feed by Protein Science news http://www.medworm.com/rss/comments.php?id=5464966 Fri, 02 Dec 2011 07:36:23 +0100 5464966 http://www.medworm.com/index.php?rid=5464965&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-445-2_25 In this chapter, we describe a method for quantification of 20 proteinogenic amino acids as well as 13 15N-labeled amino acids by liquid chromatography–mass spectrometry without the need for derivatization and use of organic solvents. Analysis of the underivatized amino acids is performed by liquid chromatography–electrospray ionization-tandem mass spectrometry (LC-ESI-MS-MS) in the positive ESI mode. Separation is achieved on a strong cation exchange (SCX) column (Luna 5 μm SCX 100 Å) with 30 mM ammonium acetate in water (A) and 5% acetic acid in water (B). Quantification is accomplished by use of d5-phenylalanine as internal standard achieving limits of detection of 0.1–3.0 μM. The method was successfully applied for the determination of proteinogenic and 15... Springer protocols feed by Protein Science news http://www.medworm.com/rss/comments.php?id=5464965 Fri, 02 Dec 2011 07:36:23 +0100 5464965 http://www.medworm.com/index.php?rid=5464964&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-445-2_24 This chapter describes a sequential injection chromatography method to automate the fluorimetric determination of amino acids after precolumn derivatization with o-phthaldialdehyde in presence of 2-mercaptoethanol using reverse-phase liquid chromatography in C18 silica-based monolithic column. The method is low-priced and based on six steps of isocratic elutions. At a flow rate of 30 μl/s, a 25 mm long-column coupled to 5-mm guard column is capable to separate aspartic acid (Asp), glutamic acid (Glu), asparagine (Asn), serine (Ser), glycine (Gly), threonine (Thr), citrulline (Ctr), arginine (Arg), alanine (Ala), tyrosine (Tyr), phenylalanine (Phe), ornithine (Orn), and lysine (Lys). Under these conditions, histidine (His) and glutamine (Gln), methionine (Met) and valine (Val), and isole... Springer protocols feed by Protein Science news http://www.medworm.com/rss/comments.php?id=5464964 Fri, 02 Dec 2011 07:36:23 +0100 5464964 http://www.medworm.com/index.php?rid=5464963&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-445-2_23 Multiple reaction monitoring (MRM) mass spectrometry may be regarded as the gold standard methodology for quantitative mass spectrometry and has been adopted for the analysis of small molecules especially within the pharmaceutical industry. It can also be applied to the analysis of peptides and proteins and to the measurement of the basic building blocks of proteins, amino acids. Here, we describe the application of MRM mass spectrometry to the measurement of hydroxyproline after acid hydrolysis of various animal tissues. We show that the measurement of hydroxyproline provides an accurate and reliable estimate of the collagen content of such tissues and may be a useful indicator of meat tenderness. (Source: Springer protocols feed by Protein Science) Springer protocols feed by Protein Science news http://www.medworm.com/rss/comments.php?id=5464963 Fri, 02 Dec 2011 07:36:23 +0100 5464963 http://www.medworm.com/index.php?rid=5464962&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-445-2_22 This report describes a rapid and innovative microwave procedure for protein hydrolysis coupled with high performance anion exchange chromatography and pulsed amperometric detection (HPAEC-PAD) to quantify the amino acid 4-hydroxyproline in meat and meat-based products. This innovative approach was successfully applied to determine collagen content (4-hydroxyproline × 8) as the index quality of meat material used in the preparation of typical meat-based foods. (Source: Springer protocols feed by Protein Science) Springer protocols feed by Protein Science news http://www.medworm.com/rss/comments.php?id=5464962 Fri, 02 Dec 2011 07:36:23 +0100 5464962 http://www.medworm.com/index.php?rid=5464961&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-445-2_21 Hydroxyproline (Hyp) is an imino acid post-translationally formed by sequence-specific hydroxylases in the repeating collagen Gly–Xaa–Yaa triad present in all collagen types of all species. In both Xaa- and Yaa-positions, Pro is the most common residue, often oxidized to 4-Hyp in the Yaa- and rarely to 3-Hyp in the Xaa-positions. Here, we describe the qualitative and quantitative analysis of 3- and 4-Hyp-isomers by separating the free imino acids either with hydrophilic interaction chromatography (HILIC) or after derivatization with reversed-phase chromatography (RPC). In both cases, the compounds were detected by electrospray ionization mass spectrometry (ESI-MS). (Source: Springer protocols feed by Protein Science) Springer protocols feed by Protein Science news http://www.medworm.com/rss/comments.php?id=5464961 Fri, 02 Dec 2011 07:36:23 +0100 5464961 http://www.medworm.com/index.php?rid=5464960&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-445-2_20 Reaction of reactive nitrogen species (RNS), such as peroxynitrite and nitryl chloride with soluble tyrosine and tyrosine residues in proteins produces soluble 3-nitro-tyrosine and 3-nitro-tyrosino-proteins, respectively. Regular proteolysis of 3-nitro-tyrosino-proteins yields soluble 3-nitro-tyrosine. 3-Nitro-tyrosine circulates in plasma and is excreted in the urine. Both circulating and excretory 3-nitro-tyrosine are considered suitable biomarkers of nitrative stress. Tandem mass spectrometry coupled with gas chromatography (GC–MS/MS) or liquid chromatography (LC–MS/MS) is one of the most reliable analytical techniques to determine 3-nitro-tyrosine. Here, we describe protocols for the quantitative determination of soluble 3-nitro-tyrosine in human plasma and urine by GC&ndas... Springer protocols feed by Protein Science news http://www.medworm.com/rss/comments.php?id=5464960 Fri, 02 Dec 2011 07:36:23 +0100 5464960 http://www.medworm.com/index.php?rid=5464959&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-445-2_1 Amino acids extracted from a biological matrix can be resolved and measured using a 6-min per sample method through high-performance liquid chromatography with a short C18 column and rapid gradient using the ion-pairing reagent perfluoroheptanoic acid. LC-tandem mass spectrometry with multiple reaction monitoring (MRM) transitions selective for each compound allows simultaneous quantification of the 20 proteinogenic amino acids and 5 metabolically related compounds. Distinct MRM transitions were also established for selective detection of the isomers leucine/isoleucine and threonine/homoserine. (Source: Springer protocols feed by Protein Science) Springer protocols feed by Protein Science news http://www.medworm.com/rss/comments.php?id=5464959 Fri, 02 Dec 2011 07:36:23 +0100 5464959 http://www.medworm.com/index.php?rid=5464958&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-445-2_19 Whole blood and/or plasma amino acids are useful for monitoring whole body protein and amino acid metabolism in an organism under various physiological and pathophysiological conditions. Various methodological procedures are in use for their measurement in biological fluids. From the time when capillary electrophoresis was introduced as a technology offering rapid separation of various ionic and/or ionizable compounds with low sample and solvent consumption, there were many attempts to use it for the measurement of amino acids present in physiological fluids. As a rule, these methods require derivatization procedures for detection purposes. (Source: Springer protocols feed by Protein Science) Springer protocols feed by Protein Science news http://www.medworm.com/rss/comments.php?id=5464958 Fri, 02 Dec 2011 07:36:23 +0100 5464958 http://www.medworm.com/index.php?rid=5464957&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-445-2_18 Enzyme deficiencies in amino acid (AA) metabolism affecting the levels of amino acids and their derivatives in physiological fluids may serve as diagnostically significant biomarkers for one or a group of metabolic disorders. Therefore, it is important to monitor a wide range of free amino acids simultaneously and to quantify them. This is time consuming if we use the classical methods and more than ever now that many laboratories have introduced Newborn Screening Programs for the semiquantitative analysis, detection, and quantification of some amino acids needed to be performed in a short time to reduce the rate of false positives. (Source: Springer protocols feed by Protein Science) Springer protocols feed by Protein Science news http://www.medworm.com/rss/comments.php?id=5464957 Fri, 02 Dec 2011 07:36:23 +0100 5464957 http://www.medworm.com/index.php?rid=5464956&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-445-2_17 We present here a modified reversed-phase LC of phenylthiocarbamyl amino acids in plasma deproteinated by the organic solvent acetonitrile. Specificity was monitored by UV-photodiode array detection and accuracy was controlled by a plasma spiking procedure with three internal standards. A dual-wavelength spectrophotometry (254, 283 nm) was used to quantify coeluting ornithine and tryptophan adducts. The method is simple and economical and enables the measure of most plasma amino acids for clinical research, also during therapeutic drug monitoring. Dual UV-fluorimetric detection solutions can improve its sensitivity. (Source: Springer protocols feed by Protein Science) Springer protocols feed by Protein Science news http://www.medworm.com/rss/comments.php?id=5464956 Fri, 02 Dec 2011 07:36:23 +0100 5464956 http://www.medworm.com/index.php?rid=5464955&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-445-2_16 The concentration of primary amino acids (AAs) in plasma can accurately be determined using high-performance liquid chromatography (HPLC). Before the analysis can be performed, several steps have to be regarded. First, the time and method of blood withdrawal, type of blood tube, use of medication, and differences in dietary intake are important factors that should be standardized. Second, the handling of and the way the blood is transported to the laboratory, the time between blood withdrawal and centrifugation, the method of centrifugation, and the temperature and time of plasma storage have to be noticed. Third, the methods used for deproteinization and derivatization may account for varying results between laboratories. (Source: Springer protocols feed by Protein Science) Springer protocols feed by Protein Science news http://www.medworm.com/rss/comments.php?id=5464955 Fri, 02 Dec 2011 07:36:23 +0100 5464955 http://www.medworm.com/index.php?rid=5464954&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-445-2_15 Two mass spectrometry-based methods for the quantitative analysis of free amino acids are described. The first method uses propyl chloroformate/propanol derivatization and gas chromatography–quadrupole mass spectrometry (GC–qMS) analysis in single-ion monitoring mode. Derivatization is carried out directly in aqueous samples, thereby allowing automation of the entire procedure, including addition of reagents, extraction, and injection into the GC–MS. The method delivers the quantification of 26 amino acids. The isobaric tagging for relative and absolute quantification (iTRAQ) method employs the labeling of amino acids with isobaric iTRAQ tags. The tags contain two different cleavable reporter ions, one for the sample and one for the standard, which are detected by fragmen... Springer protocols feed by Protein Science news http://www.medworm.com/rss/comments.php?id=5464954 Fri, 02 Dec 2011 07:36:23 +0100 5464954 http://www.medworm.com/index.php?rid=5464953&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-445-2_14 Plasma tryptophan (Trp) and other amino acids (AA) can be determined rapidly by gas (GC) or liquid (LC) chromatography using the Phenomenex EZ:Faast™ family of kits. Three kits are available: (1) GC-FID or -NPD, (2) GC-MS, (3) LC-MS. The two GC kits can determine 32 AA, whereas the LC-MS can determine five additional AA. All three kits, however, share the same experimental procedure up to and including the preparation of derivatised AA. The method is based on solid-phase extraction (SPE), thus saving time on prior removal of plasma or other proteins and interfering substances, and can be applied to other body fluids and experimental media and to supernatants of extracts of solid material. Briefly, SPE is performed using a proprietary cation-exchange mechanism. The acid solution of th... Springer protocols feed by Protein Science news http://www.medworm.com/rss/comments.php?id=5464953 Fri, 02 Dec 2011 07:36:23 +0100 5464953 http://www.medworm.com/index.php?rid=5464952&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-445-2_13 Gas chromatography (GC) is a commonly used technique in amino acid analysis (AAA). However, one of the requirements of the application of GC for AAA is a need for the polar analytes to be converted into their volatile, thermally stable derivatives. In the last two decades, alkyl chloroformates have become attractive derivatization reagents. The reagents react immediately with most amino acid functional groups in aqueous matrices and the process can easily be coupled with liquid–liquid extraction of the resulting less-polar derivatives into immiscible organic phase. Here, we describe a simple protocol for in situ derivatization of amino acids with heptafluorobutyl chloroformate followed by subsequent chiral as well as nonchiral GC/mass spectrometric analysis on a respective nonpolar f... Springer protocols feed by Protein Science news http://www.medworm.com/rss/comments.php?id=5464952 Fri, 02 Dec 2011 07:36:23 +0100 5464952 http://www.medworm.com/index.php?rid=5464951&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-445-2_12 Here, we describe two different amino acid analysis protocols based on the application of matrix-assisted laser desorption ionization time-of-flight (MALDI TOF) mass spectrometry (MS). First protocol describes a MALDI TOF MS-based method for a routine simultaneous qualitative and quantitative analysis of free amino acids and protein hydrolysates (Alterman et al. Anal Biochem 335: 184–191, 2004). Linear responses between the amino acid concentration and the peak intensity ratio of corresponding amino acid to internal standard were observed for all amino acids analyzed in the range of concentrations from 20 to 300 μM. Limit of quantitation varied from 0.03 μM for arginine to 3.7 μM for histidine and homocysteine. This method has one inherent limitation: the analysis of isomeri... Springer protocols feed by Protein Science news http://www.medworm.com/rss/comments.php?id=5464951 Fri, 02 Dec 2011 07:36:23 +0100 5464951 http://www.medworm.com/index.php?rid=5464950&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-445-2_11 Amino acid analysis is among the most accurate methods for absolute quantification of proteins and peptides. Here, we combine acid hydrolysis with the addition of isotopically labeled standard amino acids and analysis by mass spectrometry for accurate and sensitive protein quantitation. Quantitation of less than 10 fmol of protein standards with errors below 10% has been demonstrated using this method (1). (Source: Springer protocols feed by Protein Science) Springer protocols feed by Protein Science news http://www.medworm.com/rss/comments.php?id=5464950 Fri, 02 Dec 2011 07:36:23 +0100 5464950 http://www.medworm.com/index.php?rid=5464949&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-445-2_10 The combined use of a dual-UV detector as well as a fluorimetric and a multielectrode electrochemical detector (equipped with a dual electrode, consisting of a conventional size 3 mm diameter glassy carbon electrode (GCE) and of a pair of 30 μm thick carbon microfibers) is proposed for the detection of the following 15 underivatized amino acids: l-histidine (his), l-cysteine (cys), creatine (crn), S-methyl-l-cysteine (me-cys), dl-homocysteine (hcy), l-methionine (met), beta-(3,4-dihydroxyphenyl)-l-alanine (dopa), l-tyrosine (tyr), dl-m-tyrosine (m-tyr), l-a-methyl-dopa (me-dopa), l-phenylanine (phe), dl-alpha-methyltyrosine (me-tyr), 5-hydroxy-tryptophan (5htp), 3-nitro-l-tyrosine (NO2Tyr) and l-tryptophan (trp), as well as of two dipeptides: l-cystathionine (cysta), l-carnosine (car), ... Springer protocols feed by Protein Science news http://www.medworm.com/rss/comments.php?id=5464949 Fri, 02 Dec 2011 07:36:23 +0100 5464949 http://www.medworm.com/index.php?rid=5396475&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-364-6_20 Based on analyses of existing small organic drug molecules, a set of “rules-of-thumb” have been devised to assess the likeness of a small molecule under study to those existing drugs in terms of physicochemical and topological properties. These rules can be used to estimate the likelihood of a small molecule to possess the desired efficacy, pharmacokinetic/pharmacodynamic properties, and toxicity profiles to eventually become a drug, and therefore, whether it justifies further experimental work and development. These rules are particularly useful when selecting a chemical starting point for a given project or choosing a chemical series to focus when multiple series are available. Caution should be paid, however, not to overly rely on these rules for decision-making, since these... Springer protocols feed by Protein Science news http://www.medworm.com/rss/comments.php?id=5396475 Fri, 11 Nov 2011 07:28:33 +0100 5396475 http://www.medworm.com/index.php?rid=5396474&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-364-6_19 Chemical proteomics is concerned with the identification of protein targets interacting with small molecules. Hence, the availability of a high quality and free resource storing small molecules is essential for the future development of the field. The Chemical Entities of Biological Interest (ChEBI) database is one such database. The scope of ChEBI includes any constitutionally or isotopically distinct atom, molecule, ion, ion pair, radical, radical ion, complex, conformer, etc., identifiable as a separately distinguishable entity. These entities in question are either products of nature or synthetic products used to intervene in the processes of living organisms. In addition, ChEBI contains a chemical ontology which relates the small molecules with each other thereby making it easier for ... Springer protocols feed by Protein Science news http://www.medworm.com/rss/comments.php?id=5396474 Fri, 11 Nov 2011 07:28:33 +0100 5396474 http://www.medworm.com/index.php?rid=5396473&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-364-6_18 The handling of organic compounds in the laboratory requires the use of organic (co-) solvents to mediate solubility. Advantages and disadvantages of the widely used solvent dimethylsulfoxide (DMSO) are discussed, and guidelines for dissolution and storage of compounds are given. Finally, nephelometry is introduced as a fast method to determine the kinetic solubility of a compound. (Source: Springer protocols feed by Protein Science) Springer protocols feed by Protein Science news http://www.medworm.com/rss/comments.php?id=5396473 Fri, 11 Nov 2011 07:28:33 +0100 5396473 http://www.medworm.com/index.php?rid=5396472&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-364-6_17 Genome-wide association studies and genetic linkage studies have created a growing list of proteins related to disease. Small molecules can serve as useful probes of function for these proteins in a cellular setting or may serve as leads for therapeutic development. High-throughput and general binding assays may provide a path for discovering small molecules that target proteins for which little is known about structure or function or for which conventional functional assays have failed. One such binding assay involves small-molecule microarrays (SMMs) containing compounds that have been arrayed and immobilized onto a solid support. The SMMs can be incubated with a protein target of interest and protein–small molecule interactions may be detected using a variety of fluorescent readou... Springer protocols feed by Protein Science news http://www.medworm.com/rss/comments.php?id=5396472 Fri, 11 Nov 2011 07:28:33 +0100 5396472 http://www.medworm.com/index.php?rid=5396471&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-364-6_9 We describe the use of SILAC in identifying proteins that bind small-molecule probes and drugs in a cellular context. (Source: Springer protocols feed by Protein Science) Springer protocols feed by Protein Science news http://www.medworm.com/rss/comments.php?id=5396471 Fri, 11 Nov 2011 07:28:33 +0100 5396471 http://www.medworm.com/index.php?rid=5396470&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-364-6_16 The time-controlled transcardiac perfusion crosslinking (tcTPC) method differs from conventional perfusion fixation in that the crosslinking reagent is administered throughout the circulatory system for only a relatively short period of time, thereby allowing limited crosslinking to occur. Bait protein complexes are isolated by affinity capture (AC) under stringent conditions and are recovered from the AC matrix by acidic elution. Affinity-purified proteins are reduced, alkylated, and digested with a specific endoproteinase, such as trypsin. Subsequently, peptides are isotopically labeled, separated by reversed-phase chromatography and analyzed by quantitative tandem mass spectrometry (MS/MS). The proteins crosslinked to the bait protein during tcTPC are identified by database searches wit... Springer protocols feed by Protein Science news http://www.medworm.com/rss/comments.php?id=5396470 Fri, 11 Nov 2011 07:28:33 +0100 5396470 http://www.medworm.com/index.php?rid=5396469&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-364-6_15 Analyzing the effect of ligands on protein–protein interactions is important to better understand the cellular processes. In vitro characterization of these modulations remains challenging because of the drawbacks associated with the analysis of noncovalent interactions. To facilitate the analysis, stabilization of the protein complex by chemical cross-linking followed by High-Mass MALDI mass spectrometry is a recently developed method offering several advantages: No need for immobilization or special tags, the analysis is possible directly on wild-type protein complexes, no need for buffer exchange, large applicability range for any type of protein complex from 0 to 1,500 kDa. Using this method, we analyzed the effect of the inhibitors Nutlin-3a and Nutlin-3b on the protein complex ... Springer protocols feed by Protein Science news http://www.medworm.com/rss/comments.php?id=5396469 Fri, 11 Nov 2011 07:28:33 +0100 5396469 http://www.medworm.com/index.php?rid=5396468&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-364-6_14 This method describes the combination of chemical cross-linking and high-resolution mass spectrometry for analyzing conformational changes in target proteins that are induced by drug binding. Our approach is exemplified for detecting conformational changes within the peroxisome proliferator-activated receptor alpha upon binding of low-molecular weight compounds, proving that our strategy provides a basis to efficiently characterize target protein–drug interactions. (Source: Springer protocols feed by Protein Science) Springer protocols feed by Protein Science news http://www.medworm.com/rss/comments.php?id=5396468 Fri, 11 Nov 2011 07:28:33 +0100 5396468 http://www.medworm.com/index.php?rid=5396467&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-364-6_13 With the proteasome emerging as a therapeutic target for cancer treatment, accurate tools for monitoring proteasome (inhibitor) activity are in demand. In this chapter, we describe the synthesis and use of a fluorescent proteasome activity probe that allows for accurate profiling of proteasomal activity in cell lysates, intact cells, and murine and human patient-derived material, with high sensitivity using SDS-PAGE. The probe allows for direct scanning of the gel for fluorescent emission of the distinct proteasomal subunits and circumvents the use of Western blot analysis. Due to its suitable biochemical and biophysical properties, the fluorescent probe can also be used for confocal laser scanning microscopy and flow cytometry-based experiments. (Source: Springer protocols feed by Protein... Springer protocols feed by Protein Science news http://www.medworm.com/rss/comments.php?id=5396467 Fri, 11 Nov 2011 07:28:33 +0100 5396467 http://www.medworm.com/index.php?rid=5396466&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-364-6_12 Chemical proteomics is a versatile tool to investigate protein–small molecule interactions, but can be extended to probe also secondary binding investigating small molecule–protein 1–protein 2 interactions, providing insight into protein scaffolds. This application of chemical proteomics has in particular been applied extensively to cyclic nucleotide (cAMP, cGMP) signaling. cAMP regulates cellular functions primarily by activating cAMP-dependent protein kinase (PKA). Compartmentalization of PKA plays an important role in the specificity of cAMP signaling events and is mediated by interaction of the regulatory subunit (PKA-R) with A-kinase anchoring proteins (AKAPs), which often form the core of even larger protein machineries. The selective binding of AKAPs to one of the ... Springer protocols feed by Protein Science news http://www.medworm.com/rss/comments.php?id=5396466 Fri, 11 Nov 2011 07:28:33 +0100 5396466 http://www.medworm.com/index.php?rid=5396465&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-364-6_11 The high cost of drug discovery and development requires more efficient approaches to the identification and inhibition of tractable protein targets. One strategy is to pursue families of proteins that already possess affinity for a drug lead scaffold, where that scaffold plays the dual role of serving (a) when tethered to a resin, as a ligand to purify a subproteome of interest, and (b) as a lead molecule that has the potential for optimization for a given member of the subproteome. Here, we describe an example of the purification of a subproteome using a scaffold tailored to the dehydrogenase family of enzymes. Combined with modern LC-MS/MS methods and subsequent searching of proteome databases, such affinity chromatography strategies can be used to purify and identify any proteins with ... Springer protocols feed by Protein Science news http://www.medworm.com/rss/comments.php?id=5396465 Fri, 11 Nov 2011 07:28:33 +0100 5396465 http://www.medworm.com/index.php?rid=5396464&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-364-6_10 Chemical proteomics offers a unique approach for target identification of small molecule inhibitors directly from cell extracts, thus enabling characterization of target proteins under close to physiological conditions. Here, we describe a competition binding procedure that is based on affinity enrichment of potential target proteins on a probe matrix in the presence of increasing amounts of free test compound in solution. Reduced binding of target proteins to the probe matrix as a function of test compound concentration can be measured and thus, enables calculation of IC50 values. The method employs quantitative mass spectrometry using isobaric mass tags which enables determination of potency for a large number of target proteins in a single analysis. (Source: Springer protocols feed by P... Springer protocols feed by Protein Science news http://www.medworm.com/rss/comments.php?id=5396464 Fri, 11 Nov 2011 07:28:33 +0100 5396464 http://www.medworm.com/index.php?rid=5396463&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-364-6_8 We describe a generic approach based on trifunctional Capture Compounds, in which the initial equilibrium-driven interaction between a small molecule probe and target proteins is irreversibly fixed upon photo-crosslinking between an independent photo-activable reactivity function of the Capture Compound and the surface of the target protein(s). Subsequently, Capture Compound - protein conjugates are isolated from complex biological mixtures via the sorting function of the Capture Compound. Here, we describe the application of a trifunctional Capture Compound that carries the methyltransferase product inhibitor S-Adenosyl-l-homocysteine as the selectivity function for the isolation of methyltransferases from a complex lysate of Escherichia coli DH5α cells. Photo-activated crosslinking... Springer protocols feed by Protein Science news http://www.medworm.com/rss/comments.php?id=5396463 Fri, 11 Nov 2011 07:28:33 +0100 5396463 http://www.medworm.com/index.php?rid=5396462&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-364-6_7 We describe selective adduct capture using biotin affinity probes to enrich protein and peptide adducts for analysis by liquid chromatography-tandem mass spectrometry (LC-MS/MS). One approach employs biotinamidohexanoic acid hydrazide to covalently label residual carbonyl groups on adducts. The other employs alkynyl analogs of lipid electrophiles, which form adducts that can be postlabeled with azidobiotin tags by Cu+-catalyzed cycloaddition (Click chemistry). To enhance the selectivity of adduct capture, we use an azidobiotin reagent with a photocleavable linker, which allows recovery of adducted proteins and peptides under mild conditions. This approach allows both the identification of protein targets of lipid electrophiles and sequence mapping of the adducts. (Source: Springer protocol... Springer protocols feed by Protein Science news http://www.medworm.com/rss/comments.php?id=5396462 Fri, 11 Nov 2011 07:28:33 +0100 5396462 http://www.medworm.com/index.php?rid=5396461&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-364-6_6 Benzophenone photoprobes represent powerful tools for chemical proteomics. Upon UV irradiation, a benzophenone photoprobe can selectively form a covalent bond with its target protein in complex protein mixtures. Thus, photoprobes can be used to profile a wide variety of proteins in complex proteomes. This chapter describes simple protocols to derivatize fluorenylmethyloxycarbonyl (Fmoc)-protected peptide-nucleic-acid adenine (PNA adenine) into a benzophenone photoprobe and its application in photolabeling its target proteins. The method as described does not require specialized equipment for probe synthesis and photolabeling. In addition, the strategy is applicable to recognition motifs other than PNA adenine, such as peptides, to profile their target proteins in complex proteomes. (Source... Springer protocols feed by Protein Science news http://www.medworm.com/rss/comments.php?id=5396461 Fri, 11 Nov 2011 07:28:33 +0100 5396461 http://www.medworm.com/index.php?rid=5396460&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-364-6_5 Inherent complexity of the proteome often demands that it be studied as manageable subsets, termed subproteomes. A subproteome can be defined in a number of ways, although a pragmatic approach is to define it based on common features in an active site that lead to binding of a common small molecule ligand (e.g., a cofactor or a cross-reactive drug lead). The subproteome, so defined, can be purified using that common ligand tethered to a resin, with affinity chromatography. Affinity purification of a subproteome is described in the next chapter. That subproteome can then be analyzed using a common ligand probe, such as a fluorescent common ligand that can be used to stain members of the subproteome in a native gel. Here, we describe such a fluorescent probe, based on a catechol rhodanine ac... Springer protocols feed by Protein Science news http://www.medworm.com/rss/comments.php?id=5396460 Fri, 11 Nov 2011 07:28:33 +0100 5396460 http://www.medworm.com/index.php?rid=5396459&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-364-6_4 In affinity-based chemoproteomics strategies, the direct immobilization of small bioactive probe molecules to a solid support may pose problems with respect to the preservation of the functional activity toward the target proteins. Typically, immobilized molecules on solid supports exhibit lower affinity for target proteins compared to the free parent molecule. This may lead to a failure to specifically capture the target proteins or to unacceptable losses during the washing steps. To circumvent these shortcomings, we have devised small molecule-peptide conjugates (SMPCs), which enable wide-ranging experimental strategies for the capturing of protein targets of small molecules from cells or tissues. With the possibilities of synthesizing peptides of tailored biochemical and biophysical pro... Springer protocols feed by Protein Science news http://www.medworm.com/rss/comments.php?id=5396459 Fri, 11 Nov 2011 07:28:33 +0100 5396459 http://www.medworm.com/index.php?rid=5396458&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-364-6_3 Bioactive small molecules act through modulating a yet unpredictable number of targets. It is therefore of critical importance to define the cellular target proteins of a compound as an entry point to understanding its mechanism of action. Often, this can be achieved in a direct fashion by chemical proteomics. As with any affinity chromatography, immobilization of the bait to a solid support is one of the earliest and most crucial steps in the process. Interfering with structural features that are important for identification of a target protein will be detrimental to binding affinity. Also, many molecules are sensitive to heat or to certain chemicals, such as acid or base, and might be destroyed during the process of immobilization, which therefore needs to be not only efficient, but also... Springer protocols feed by Protein Science news http://www.medworm.com/rss/comments.php?id=5396458 Fri, 11 Nov 2011 07:28:33 +0100 5396458 http://www.medworm.com/index.php?rid=5396457&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-364-6_2 Real-world drug discovery and development remains a notoriously unproductive and increasingly uneconomical process even in the Omics era. The dominating paradigm in the industry continues to be target-based drug design, with an increased perception of the role of signaling pathways in homeostasis and in disease. Since proteins represent the major type of drug targets, proteomics-based approaches, which study proteins under relatively physiological conditions, have great potential if they can be reduced to practice such that they successfully complement the arsenal of drug discovery techniques. This chapter discusses examples of drug discovery processes where chemical proteomics-based assays using native endogenous proteins should have substantial impact. (Source: Springer protocols feed by... Springer protocols feed by Protein Science news http://www.medworm.com/rss/comments.php?id=5396457 Fri, 11 Nov 2011 07:28:33 +0100 5396457 http://www.medworm.com/index.php?rid=5396456&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-364-6_1 The term “chemical proteomics” refers to a research area at the interface of chemistry, biochemistry, and cell biology that focuses on studying the mechanism of action of bioactive small molecule compounds, which comprises the mapping of their target proteins and their impact on protein expression and posttranslational modifications in target cells or tissues of interest on a proteome-wide level. For this purpose, a large arsenal of approaches has emerged in recent years, many of which employing quantitative mass spectrometry. This review briefly summarizes major experiment types employed in current chemical proteomics research. (Source: Springer protocols feed by Protein Science) Springer protocols feed by Protein Science news http://www.medworm.com/rss/comments.php?id=5396456 Fri, 11 Nov 2011 07:28:33 +0100 5396456 http://www.medworm.com/index.php?rid=5374747&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-334-9_2 The complete binding cascade of human hemoglobin consists of a series of partially ligated intermediates. The individual intermediate binding constants cannot be distinguished in O2 binding curves, however, each constant can be determined from the O2-induced change in assembly constant for the α2β2 tetramer from its constituent αβ dimers. The characterization of these O2 binding constants has shown the Hb cascade to be asymmetric in nature, with binding dependent upon the specific distribution of O2 among the four hemesites. A stopped-flow approach to measuring the dissociation constant of a key doubly ligated intermediate, that in which one dimer is oxygenated and the other is not, is described. The intermediate is transiently formed in the absence of O2 and then all... Springer protocols feed by Protein Science news http://www.medworm.com/rss/comments.php?id=5374747 Wed, 05 Oct 2011 04:00:00 +0100 5374747 http://www.medworm.com/index.php?rid=5374746&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-334-9_1 Thermodynamic principles of cooperativity and allostery have long been used as a starting point to begin understanding the interplay between ligand binding events. Understanding the nature of allosteric effects requires an experimental technique that can be used to quantify ligand binding energies and simultaneously give experimental insights into the conformational dynamics at play upon ligand binding. CD spectroscopy provides macroscopic information about the relative secondary and tertiary structures present in a protein. Here, we use this spectroscopic technique with thermal shift assays wherein ligand binding constants can be quantified based on their stabilizing effect against thermally induced protein denaturation. Binding constants for two ligands are used to determine a pairwise c... Springer protocols feed by Protein Science news http://www.medworm.com/rss/comments.php?id=5374746 Wed, 05 Oct 2011 04:00:00 +0100 5374746 http://www.medworm.com/index.php?rid=5257040&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-319-6_23 In shotgun proteomics, peptide fractionation is essential for in-depth characterization of proteomes and the mapping of protein posttranslational modifications. Recently, a mix-mode chromatography [i.e., electrostatic repulsion hydrophilic interaction chromatography (ERLIC)] has been developed and found to be a versatile method in proteome characterization. Here, we use ERLIC to characterize the glycoproteome and phosphoproteome simultaneously. We also demonstrate that the ERLIC can be an alternative to the commonly used strong cation exchange chromatography for higher recovery of proteins during whole proteome analysis. These protocols can be easily adopted in most proteomics laboratories. (Source: Springer protocols feed by Protein Science) Springer protocols feed by Protein Science news http://www.medworm.com/rss/comments.php?id=5257040 Mon, 26 Sep 2011 23:43:11 +0100 5257040 http://www.medworm.com/index.php?rid=5257039&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-319-6_22 Although the snapshots of different in vitro conformational states have been intensively studied, current techniques such as nuclear magnetic resonance, X-ray crystallography, and electron microscope method cannot probe the in vivo conformational movements of integral membrane proteins on cell surfaces. Here, we describe a hydroxyl radical protein footprinting coupled to a mass spectrometry detection technique to probe the structural dynamics of a membrane protein directly on the native cell surface. This method uses in situ generation of hydroxyl radicals to oxidize and covalently modify integral membrane proteins on the cell surface. To explain this technique in detail, we use the porin OmpF as an example, although the method may be applied to study any membrane protein. Footprinting res... Springer protocols feed by Protein Science news http://www.medworm.com/rss/comments.php?id=5257039 Mon, 26 Sep 2011 23:43:11 +0100 5257039 http://www.medworm.com/index.php?rid=5257038&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-319-6_21 Protein phosphorylation plays a critical role in the regulation of many cellular functions. Phosphoproteomic analyses facilitate an in-depth understanding of such phosphorylation-dependent signaling networks. The use of mass spectrometry in phosphoproteomics has been especially successful, but the approach largely depends on an efficient method to enrich phosphopeptides from complex mixtures. We have developed a novel, effective soluble nanopolymer-based phosphopeptide enrichment technique, termed PolyMAC (polymer-based metal ion affinity capture). The homogenous, hyperfunctional nature of PolyMAC reagent makes it a more competent choice for highly efficient phosphopeptide binding and isolation, which was demonstrated through several applications with simple protein mixture and complex cel... Springer protocols feed by Protein Science news http://www.medworm.com/rss/comments.php?id=5257038 Mon, 26 Sep 2011 23:43:11 +0100 5257038 http://www.medworm.com/index.php?rid=5257037&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-319-6_20 Calcifying biologic nanoparticles (NPs) have been implicated as nucleation points for a number of ­pathologic events that include vascular calcification and the formation of kidney stones. In order to study these potential relationships, reproducible isolation of well-characterized biologic NPs is a necessity. Our group has isolated and propagated calcifying NPs from several human tissues and renal stones. Specific proteins that could nucleate a calcium phosphate shell under physiologic conditions have been identified as part of their structure, including elongation factor Tu (EF-Tu) and fetuin-A. Visualization, using advanced transmission electron microscopy (TEM), immunofluorescence microscopy, and nuclear and antibody staining in conjunction with flow cytometry, can further elucidat... Springer protocols feed by Protein Science news http://www.medworm.com/rss/comments.php?id=5257037 Mon, 26 Sep 2011 23:43:11 +0100 5257037 http://www.medworm.com/index.php?rid=5257036&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-319-6_19 The immunization of an animal with a whole proteome or the infection of an animal and the screening of the resulting antibody repertoire on either the same or different proteome(s) or the infecting agent(s), omits the laborious steps of recombinant protein expression and purification to obtain multiple antigen binders. This procedure allows the identification of antibodies that are specific to unique or common signatures of different proteomes without prior knowledge of these signatures. (Source: Springer protocols feed by Protein Science) Springer protocols feed by Protein Science news http://www.medworm.com/rss/comments.php?id=5257036 Mon, 26 Sep 2011 23:43:11 +0100 5257036 http://www.medworm.com/index.php?rid=5257035&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-319-6_18 Sensitive and quantitative analysis of proteins is central to disease diagnosis, drug screening, and proteomic studies. Among recent research advances exploiting new nanomaterials for biomolecule analysis, silicon nanowires (SiNWs), which are configured as field-effect transistors (FETs), have emerged as one of the most promising and powerful platforms for label-free, real-time, and highly sensitive electrical detection of proteins as well as many other biological species. Here, we describe a detailed protocol for realizing SiNW biosensors for protein detection that includes SiNW synthesis, FET device fabrication, surface receptor functionalization, and electrical sensing measurements. Moreover, incorporating both p-type and n-type SiNWs in the same sensor array provides a unique means of ... Springer protocols feed by Protein Science news http://www.medworm.com/rss/comments.php?id=5257035 Mon, 26 Sep 2011 23:43:11 +0100 5257035 http://www.medworm.com/index.php?rid=5257034&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-319-6_17 Immobilized metal affinity chromatography is a widely used method for the enrichment of phosphopeptides from proteolytic digests prior to mass spectrometric analysis. Here, we describe the selective enrichment of phosphopeptides from tryptic digests of proteins (α- and β-caseins) by zirconium phosphonate-magnetic Fe3O4/SiO2 (core/shell) nanoparticles for phosphoproteome analysis with MALDI-TOF mass spectrometry. (Source: Springer protocols feed by Protein Science) Springer protocols feed by Protein Science news http://www.medworm.com/rss/comments.php?id=5257034 Mon, 26 Sep 2011 23:43:11 +0100 5257034 http://www.medworm.com/index.php?rid=5257033&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-319-6_16 Smart multifunctional magnetic nanoparticles are popular candidates for several biological applications owing to their intrinsic magnetic property and diverse applications that range from rare protein separation and biomedical utilization to cancer therapy and diagnostics. A universal protocol, for the development of such nanocarriers, is highly desirable for scientists with different backgrounds so that custom-made multifunctional nanoparticles can be developed to address their needs, among which are the superparamagnetic iron oxide and manganese oxide nanoparticles that are synthesized through high temperature decomposition reactions. However, an interface is needed to present these inorganic materials to biomolecules to enhance their application for different biological use. This compat... Springer protocols feed by Protein Science news http://www.medworm.com/rss/comments.php?id=5257033 Mon, 26 Sep 2011 23:43:11 +0100 5257033 http://www.medworm.com/index.php?rid=5257032&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-319-6_15 In recent years, the majority of research on surface patterning, as a means of precisely controlling cell ­positioning and adhesion on surfaces, has focused on eukaryotic cells. Such research has led to new insights into cell biology, advances in tissue engineering, and cell motility. In contrast, considerably less work has been reported on tightly controlled patterning of bacteria, despite its potential in a wide variety of applications, including fabrication of in vitro model systems for studies of bacterial processes, such as quorum sensing and horizontal gene transfer. This is partly due to their small size – often 1–3 μm or less. To study these processes, microscale and nanoscale engineered material surfaces must be developed to create in vitro bacteria arrays, whic... Springer protocols feed by Protein Science news http://www.medworm.com/rss/comments.php?id=5257032 Mon, 26 Sep 2011 23:43:11 +0100 5257032 http://www.medworm.com/index.php?rid=5257031&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-319-6_14 Mass spectrometry (MS) provides quantitative information toward accurate mass identification of unknown molecules and has become a powerful and widely used technique for bioanalysis. In this chapter, we describe the fabrication and MS analysis steps for a high sensitivity matrix-free laser desorption/ionization mass spectrometry (LDI-MS) target substrate termed nanofilament silicon (nSi). Unlike other nanostructured porous silicon surfaces, nSi possesses an open pore morphology with associated benefits including efficient transport of sample into the nanopores and effective removal of solvent from the surface. The utility of nSi targets for LDI-MS analysis lies primarily in the analysis of small molecules, which are typically less than 5,000 Da with a detection sensitivity in the attomole ... Springer protocols feed by Protein Science news http://www.medworm.com/rss/comments.php?id=5257031 Mon, 26 Sep 2011 23:43:11 +0100 5257031 http://www.medworm.com/index.php?rid=5257030&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-319-6_13 Alterations in protein phosphorylation, a posttranslational modification (PTM) that regulates many ­processes in living cells, is a fundamental mechanism of many diseases, including cancer. Phosphoproteomics, with the combined use of affinity chromatography and electrospray ionization (ESI) or matrix-assisted laser desorption/ionization (MALDI) mass spectrometry, is shedding light into phosphorylation signaling pathways at the proteome level and helps to solve difficulties related to sample complexity and phosphopeptide enrichment. One of the most frequent and efficient methods used to enrich samples for the phosphorylated components is titanium dioxide chromatography. Titanium dioxide has a high affinity for phosphopeptides and can also be selective in specific experimental conditions... Springer protocols feed by Protein Science news http://www.medworm.com/rss/comments.php?id=5257030 Mon, 26 Sep 2011 23:43:11 +0100 5257030 http://www.medworm.com/index.php?rid=5257029&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-319-6_12 A sample preparation method to detect small molecules in laser desorption/ionization time-of-flight mass spectrometry (LDI-TOF MS) was developed using bare gold nanoparticles (AuNPs) as matrices. In this sample preparation method, the analyte is deposited first and then followed by the bare AuNPs. Neutral steroids and carbohydrates, which are difficult to ionize, using organic matrices, are cationized efficiently by combining AuNP-assisted LDI-TOF MS with this sample preparation method. As compared to the dried-droplet method (i.e., analyte and bare AuNPs are mixed and dried together), this method offers distinct advantages for improving shot-to-shot reproducibility, increasing the ionization efficiency of the analyte, and reducing sample preparation time. (Source: Springer protocols feed ... Springer protocols feed by Protein Science news http://www.medworm.com/rss/comments.php?id=5257029 Mon, 26 Sep 2011 23:43:11 +0100 5257029 http://www.medworm.com/index.php?rid=5257028&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-319-6_9 Nanoscale liquid chromatography coupled to tandem mass spectrometry (nano LC–MS/MS) has become an essential tool in the field of proteomics. In fact, its sensitivity has advantages over conventional LC–MS/MS that allow the analysis of peptide mixtures in sample-limited situations (e.g., proteolytically digested proteins isolated by two-dimensional gel electrophoresis). Technical challenges, associated with low flow rates of the chromatographic separation, make this technology still difficult to run routinely. Here, we describe a nano LC–MS/MS setup that allows several weeks of continuous operation for the analysis of peptides derived by enzymatic digestion of either purified proteins or moderately complex protein mixtures. (Source: Springer protocols feed by Protein Scien... Springer protocols feed by Protein Science news http://www.medworm.com/rss/comments.php?id=5257028 Mon, 26 Sep 2011 23:43:11 +0100 5257028 http://www.medworm.com/index.php?rid=5257027&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-319-6_8 Mass spectrometry can provide a very sensitive and rapid analysis of protein expression and can be used as an alternative to immunochemical methods to study protein–protein interaction and protein posttranslational modifications. In many circumstances, a functional study, such as one that aims to elucidate a specific signaling pathway or disease state, will require the detection and quantification of a specific set of proteins and their modifications. Very often, there will be no available antibody for some of the proteins in the set, and mass spectrometry will be the only option. This chapter describes a robust and efficient protocol for a small-scale sample preparation and a suite of separation and mass spectrometry techniques that allow the quantitative analysis of low femtomolar ... Springer protocols feed by Protein Science news http://www.medworm.com/rss/comments.php?id=5257027 Mon, 26 Sep 2011 23:43:11 +0100 5257027 http://www.medworm.com/index.php?rid=5257026&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-319-6_7 A method is detailed for the global profiling of underivatized N-linked glycans that are derived from complex protein mixtures. The method consists of five main steps that include the following: (1) protein denaturation; (2) enzymatic digestion; (3) solid phase extraction; (4) nanoLC MS analysis; and (5) data interpretation. Materials, methods, and algorithms for the identification of both glycan composition and structure are summarized. In addition, potential problems and their resolutions are addressed. (Source: Springer protocols feed by Protein Science) Springer protocols feed by Protein Science news http://www.medworm.com/rss/comments.php?id=5257026 Mon, 26 Sep 2011 23:43:11 +0100 5257026 http://www.medworm.com/index.php?rid=5257025&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-319-6_6 The profound biological relevance of protein and lipid glycosylation has made glycomics (i.e., the comprehensive study of all glycans in a cell or organism), an indispensable field of research in the life sciences. Consequently, numerous strategies have been developed for a high-throughput analysis of complex glycan mixtures, with mass spectrometry (MS) playing a key role. In particular, nanoelectrospray ionization (ESI-) MS n , employing multiple cycles of isolation and fragmentation of native or derivatized precursor ions, is recognized as a highly valuable tool in this context, as it allows, at least in part, structural characterization of glycans without prior fractionation. This chapter describes suitable work flows for this purpose and illustrates both advantages and... Springer protocols feed by Protein Science news http://www.medworm.com/rss/comments.php?id=5257025 Mon, 26 Sep 2011 23:43:11 +0100 5257025 http://www.medworm.com/index.php?rid=5257024&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-319-6_5 The introduction of electrospray ionization (ESI) and in particular nano-electrospray (nESI) has enabled the routine mass spectrometric (MS) analysis of large protein complexes in native aqueous buffers. Time-of-flight (ToF) mass spectrometers, in particular the hybrid quadrupole time-of-flight (Q-ToF) instruments, are well suited to the analysis of large protein complexes. When ionized under native-MS conditions, protein complexes routinely exhibit multiple charge states in excess of m/z 6,000, well above the standard mass range of many quadrupole or ion cyclotron-based instruments. The research area of native MS has expanded considerably in the last decade and has shown particular relevance in the area of protein structure determination. Researchers are now able to routinely measure inta... Springer protocols feed by Protein Science news http://www.medworm.com/rss/comments.php?id=5257024 Mon, 26 Sep 2011 23:43:11 +0100 5257024 http://www.medworm.com/index.php?rid=5257023&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-319-6_4 To sensitively analyze complex protein mixtures by mass spectrometry-based shotgun proteomics, researchers have employed platforms that couple orthogonal peptide fractionation methods using nanoscale HPLC. Commonly used platforms have coupled either strong cation exchange (SCX) HPLC or preparative isoelectric focusing (IEF) with nanoscale reversed-phase (nanoRP) HPLC fractionation of peptides. Coupling two dimensions of peptide fractionation, prior to mass spectrometric analysis, increases the sensitivity for identifying low abundance proteins. However, the large dynamic range of protein abundance and high level of complexity of protein mixtures derived from many biological sources, such as bodily fluids, require additional steps of peptide fractionation. To address this shortcoming, we ha... Springer protocols feed by Protein Science news http://www.medworm.com/rss/comments.php?id=5257023 Mon, 26 Sep 2011 23:43:11 +0100 5257023 http://www.medworm.com/index.php?rid=5257022&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-319-6_3 We describe two protocols to characterize high molecular weight peptides (>3 kDa) and intact proteins involving on-line trypsin digestion, separation of the digests by nano-HPLC, and analysis by mass spectrometry using two different ionization sources (matrix-assisted laser desorption and electrospray ionization). These protocols have the advantage of promoting protein denaturation in an aqueous-organic solvent, which reduces the derivatization of the sample and facilitates an in-depth analysis for detection and identification of proteins. Additional advantages include the following: (1) integration of these protocols into standard proteomic workflows after the preprocessing of samples and separation; (2) use of high-resolution monolithic columns; and (3) the ability to acquire informat... Springer protocols feed by Protein Science news http://www.medworm.com/rss/comments.php?id=5257022 Mon, 26 Sep 2011 23:43:11 +0100 5257022 http://www.medworm.com/index.php?rid=5257021&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-319-6_11 Selected reaction monitoring (SRM) is one of the most powerful techniques for the relative and absolute quantification of proteins from complex protein mixtures. In contrast to traditional protein quantification methods such as ELISAs or RIAs, the SRM method uses mass spectrometry for detection. Further benefits of SRM are as follows: (1) high specificity and sensitivity; (2) large linear dynamic range of at least three orders of magnitude; and (3) the possibility to quantify multiple proteins simultaneously in a single MS run from an individual sample. To perform SRM-based protein quantification reliably, a careful design of the assay is essential, and several pitfalls must be avoided. The aim of this chapter is to help SRM newcomers to establish SRM-based protein quantification assays an... Springer protocols feed by Protein Science news http://www.medworm.com/rss/comments.php?id=5257021 Mon, 26 Sep 2011 23:43:11 +0100 5257021 http://www.medworm.com/index.php?rid=5257020&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-319-6_10 Compared with conventional gel-based techniques, such as gel electrophoresis, which are routinely used for bioseparation in biology and biomedical laboratories, nanofluidic devices with regular engineered sieving structures offer the potential for faster separation, better resolution, higher throughput, and more convenient sample recovery. Here, we detail the fabrication process of a two-dimensional nanofluidic filter array device and its implementation for rapid continuous-flow separation of biomolecules such as proteins. (Source: Springer protocols feed by Protein Science) Springer protocols feed by Protein Science news http://www.medworm.com/rss/comments.php?id=5257020 Mon, 26 Sep 2011 23:43:11 +0100 5257020 http://www.medworm.com/index.php?rid=5257019&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-319-6_2 Epithelial lining fluid (ELF) forms a thin fluid layer that covers the mucosa of the alveoli, the small airways, and the large airways. Since it constitutes the first barrier between the lung and the outer world, it is an interesting target for proteomics studies that focus on lung disease. Bronchoscopic microprobe (BMP) sampling of ELF uses small probes with an absorptive tip that are introduced bronchoscopically. In contrast to other methods used so far for the collection of biofluids from the lung (e.g., bronchoalveolar lavage fluid, induced sputum), this technique has the advantage that ELF is not diluted and contains high concentrations of biomolecules. In addition, the investigated location in the tracheobronchial tree is well defined, and there is no contamination with oropharyngeal... Springer protocols feed by Protein Science news http://www.medworm.com/rss/comments.php?id=5257019 Mon, 26 Sep 2011 23:43:11 +0100 5257019 http://www.medworm.com/index.php?rid=5257018&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-319-6_1 We describe the semiautomated capture of serum peptides/small proteins with magnetic beads that harbor C18 alkyl chains, the deposition of captured material on a target plate for MALDI-TOF mass spectrometry, as well as, general guidelines for data acquisition. We also include, in a separate note, a short manual version of the capture procedure. Overall, the serum sample processing protocol, reported here, is reproducible and robust. In conjunction with MALDI-TOF mass spectrometry, this protocol allows for the profiling of several hundreds of serum peptides. (Source: Springer protocols feed by Protein Science) Springer protocols feed by Protein Science news http://www.medworm.com/rss/comments.php?id=5257018 Mon, 26 Sep 2011 23:43:11 +0100 5257018 http://www.medworm.com/index.php?rid=5203884&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-286-1_25 A review is provided of contact-printing technologies for the fabrication of planar protein microarrays. The key printing performance parameters for creating protein arrays are reviewed. Solid pin and quill pin technologies are described and their strengths and weaknesses compared. (Source: Springer protocols feed by Protein Science) Springer protocols feed by Protein Science news http://www.medworm.com/rss/comments.php?id=5203884 Sun, 11 Sep 2011 03:16:56 +0100 5203884 http://www.medworm.com/index.php?rid=5203883&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-286-1_24 Protein chips are becoming a key technology in proteomic research and medical diagnostics. Surface chemistry for immobilization of proteins forms the basis for assay design and determines the properties of protein microarrays. Optimal substrates provide a homogeneous environment for probes, preventing loss of biological activity and unspecific adsorption. Numerous immobilization approaches, based on covalent binding, affinity, or adsorption, have been proposed thus far, and these represent the toolbox for choosing optimized strategies for each individual application. (Source: Springer protocols feed by Protein Science) Springer protocols feed by Protein Science news http://www.medworm.com/rss/comments.php?id=5203883 Sun, 11 Sep 2011 03:16:56 +0100 5203883 http://www.medworm.com/index.php?rid=5203882&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-286-1_23 A significant proportion of protein microarray researchers would like the arrays they develop to become widely used research, screening, validation or diagnostic devices. For this to be achievable the arrays must be compatible with high-throughput techniques that allow manufacturing scale production. In order to simplify the transition from laboratory bench to market, Arrayjet have developed a range of inkjet microarray printers, which, at one end of the scale, are suitable for R&D and, at the other end, are capable of true high-throughput array output. To maintain scalability, all Arrayjet microarray printers utilise identical core technology comprising a JetSpyder™ liquid handling adaptor, which enables automated loading of an industry standard inkjet printhead compatible with ... Springer protocols feed by Protein Science news http://www.medworm.com/rss/comments.php?id=5203882 Sun, 11 Sep 2011 03:16:56 +0100 5203882 http://www.medworm.com/index.php?rid=5203881&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-286-1_22 Profiling the autoantibody (AAb) repertoire in serum has been routinely used for many years for the diagnosis of autoimmune diseases, including rheumatoid arthritis, scleroderma, and lupus. In recent years, AAb profiling of cancers has become a prominent field in oncology research. Protein arrays enable high-throughput screening of clinical samples, characterising the serum profile using low volumes of samples. This chapter describes the use of a protein array comprising 37,200 redundant proteins (containing over 10,000 non-redundant human recombinant proteins) for identification of the proteins bound by the antibodies in human sera using a test set of serum samples. The proteins identified have the potential to be candidate biomarkers. These recombinant proteins are expressed, purified, a... Springer protocols feed by Protein Science news http://www.medworm.com/rss/comments.php?id=5203881 Sun, 11 Sep 2011 03:16:56 +0100 5203881 http://www.medworm.com/index.php?rid=5203880&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-286-1_21 Protein microarrays have many potential applications in the systematic, quantitative analysis of protein function. However, simple, reproducible, and robust methods for array fabrication that are compatible with the study of large, custom collections of potentially unrelated proteins are required. Here, we discuss different routes to array fabrication and describe in detail one approach in which the purification and immobilisation procedures are combined into a single step, significantly simplifying the array fabrication process. We illustrate this approach by reference to the creation of an array of human protein kinases and discuss methods for assay and data analysis on such arrays. (Source: Springer protocols feed by Protein Science) Springer protocols feed by Protein Science news http://www.medworm.com/rss/comments.php?id=5203880 Sun, 11 Sep 2011 03:16:56 +0100 5203880 http://www.medworm.com/index.php?rid=5203879&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-286-1_20 We describe the identification of a high affinity interaction between calmodulin and the single-pass transmembrane proteins STIM1 and STIM2 that localise to the ER. Translocation of STIM1 and STIM2 from the endoplasmic reticulum to the plasma membrane is a key step in store operated calcium entry in the cell. (Source: Springer protocols feed by Protein Science) Springer protocols feed by Protein Science news http://www.medworm.com/rss/comments.php?id=5203879 Sun, 11 Sep 2011 03:16:56 +0100 5203879 http://www.medworm.com/index.php?rid=5203878&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-286-1_19 Endocytosis is one of the most essential cellular processes, which enables cells to internalise diverse ­material. It is crucial for regulation of receptor activity and signalling, cell polarisation, attachment and motility, and a great number of other cellular functions. A number of diverse endocytosis pathways are described by now; however, their specificity for different cellular cargoes is poorly resolved. Only few of endocytosis regulators are well-characterised and even less are attributed to the specific cargo. That is very true for the integrin endocytosis pathway, which is a key process in cell migration, adhesion, and signalling. The recent advent of quantitative fluorescent microscopy and cell arrays opened an exciting possibility to systematically characterise molecules pla... Springer protocols feed by Protein Science news http://www.medworm.com/rss/comments.php?id=5203878 Sun, 11 Sep 2011 03:16:56 +0100 5203878 http://www.medworm.com/index.php?rid=5203877&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-286-1_18 The development of protein microarrays makes possible interaction-based protein assays in miniaturised, multiplexed formats. A major requirement determining their uptake and use is the availability and stability of purified, functional proteins for immobilisation. With conventional methods, involving individual expression and purification of recombinant proteins, the cost of commercial high-content protein arrays is often found to be prohibitively high. Moreover, due to the need for specialised microarray production equipment, custom-made protein arrays containing more focussed sets of proteins of interest are also in little use. In the DNA array to protein array technology described herein, repeated economical printing of protein microarrays from a reusable template DNA microarray is perf... Springer protocols feed by Protein Science news http://www.medworm.com/rss/comments.php?id=5203877 Sun, 11 Sep 2011 03:16:56 +0100 5203877 http://www.medworm.com/index.php?rid=5203876&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-286-1_17 Affinity proteomics, mainly represented by antibody microarrays, has in recent years been established as a powerful tool for high-throughput (disease) proteomics. The technology can be used to generate detailed protein expression profiles, or protein maps, of focused set of proteins in crude proteomes and potentially even high-resolution portraits of entire proteomes. The technology provides unique opportunities, for example biomarker discovery, disease diagnostics, patient stratification and monitoring of disease, and taking the next steps toward personalized medicine. However, the process of designing high-performing, high-density antibody micro- and nanoarrays has proven to be challenging, requiring truly cross-disciplinary efforts to be adopted. In this mini-review, we address one of t... Springer protocols feed by Protein Science news http://www.medworm.com/rss/comments.php?id=5203876 Sun, 11 Sep 2011 03:16:56 +0100 5203876 http://www.medworm.com/index.php?rid=5203875&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-286-1_16 To expedite the development of personalized medicine, new and reliable biomarkers are required to facilitate early diagnosis, to determine prognosis, predict response or resistance to different therapies, and to monitor disease progression or recurrence. Human body fluids, such as blood, present a promising resource for biomarker discovery, in every sense. Microspot immunoassays allow the simultaneous quantification of multiple analytes from a minute amount of samples in a single measurement. The experimental design of microspot immunoassays is based on antibody pairs recognizing different epitopes of the analyte. The first antibody is used to capture the analyte from the complex sample, and the second antibody is used for detection. As with traditional enzyme-linked immunosorbent assays, ... Springer protocols feed by Protein Science news http://www.medworm.com/rss/comments.php?id=5203875 Sun, 11 Sep 2011 03:16:56 +0100 5203875 http://www.medworm.com/index.php?rid=5203874&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-286-1_15 This article provides detailed descriptions of the use of ALSA, with a particular focus on biomarker research. The preparation and selection of antibodies and lectins, the preparation and use of the arrays and samples, and special considerations for using the platform for biomarker research are covered. (Source: Springer protocols feed by Protein Science) Springer protocols feed by Protein Science news http://www.medworm.com/rss/comments.php?id=5203874 Sun, 11 Sep 2011 03:16:55 +0100 5203874 http://www.medworm.com/index.php?rid=5203873&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-286-1_14 Antibody microarrays are a multiplexing technique for the analyses of hundreds of different analytes in parallel from small sample volumes of few microlitres only. With sensitivities in the picomolar to femtomolar range, they are gaining importance in proteomic analyses. These sensitivities can be obtained for complex protein samples without any pre-fractionation or signal amplification. Also, no expensive or elaborate protein depletion steps are needed. As with custom DNA-microarrays, the implementation of a dual-colour assay adds to assay robustness and reproducibility and was therefore a focus of our technical implementation. In order to perform antibody microarray experiments for large sets of samples and analytes in a robust manner, it was essential to optimise the experimental layout... Springer protocols feed by Protein Science news http://www.medworm.com/rss/comments.php?id=5203873 Sun, 11 Sep 2011 03:16:55 +0100 5203873 http://www.medworm.com/index.php?rid=5203872&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-286-1_13 Protein microarrays are an ideal technology platform which allow for a robust and standardized profiling of the cellular proteome. Many cellular functions are not simply controlled by the presence of certain proteins, especially the propagation of external stimuli, which depend on transient post-translational modifications that determine whether a protein is in its active or inactive state. Thus, complex biological processes require the availability of a sound set of quantitative and time-resolved measurements to be understood. For this reason, new assay platforms which allow for the investigation of several proteins in parallel are necessary. The current best understood mode of cellular regulation occurs via phosphorylation and dephosphorylation processes, which are mediated via a large p... Springer protocols feed by Protein Science news http://www.medworm.com/rss/comments.php?id=5203872 Sun, 11 Sep 2011 03:16:55 +0100 5203872 http://www.medworm.com/index.php?rid=5203871&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-286-1_12 With the increasing collection of affinity reagents, their validation in terms of functionality and binding specificity becomes a challenge. To match this growing need, miniaturized and parallelized platforms have become available to corroborate the applicability for a broad range of binder scaffolds. Among the ­commonly used systems, planar microarrays have been a platform of choice for a long time but alternative systems are emerging, of which one is based on color-coded beads for the creation of arrays in suspension. The latter systems offer to perform a two-dimensional multiplexing by now analyzing up to 384 samples against up to 500 analytes in a single experiment. While the analyte parameter is flexible in terms of its composition, an extended screening can be facilitated without... Springer protocols feed by Protein Science news http://www.medworm.com/rss/comments.php?id=5203871 Sun, 11 Sep 2011 03:16:55 +0100 5203871 http://www.medworm.com/index.php?rid=5203870&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-286-1_11 The cancer biomarkers field is being enriched by molecular profiling obtained by high-throughput approaches. Targeted antibody arrays are strongly contributing to the identification of protein cancer ­biomarker candidates and functional proteomic analyses. Due to their versatility, novel technological and experimental design implementations are broadening the applications of antibody arrays. However, the cancer biomarker candidates delivered to date using this technology are still in their early developmental phase, requiring validation with high number of specimens focusing on specific clinical endpoints. Innovative strategies multiplexing protein measurements of protein extracts of cultured cells, tissue and body fluids using antibody arrays combined with appropriate validation appro... Springer protocols feed by Protein Science news http://www.medworm.com/rss/comments.php?id=5203870 Sun, 11 Sep 2011 03:16:55 +0100 5203870 http://www.medworm.com/index.php?rid=5203869&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-286-1_9 In most hospitals around the world FFPE (formalin fixed, paraffin embedded) tissues have been used for diagnosis and have subsequently been archived since decades. This has lead to a sizeable pool of this kind of tissues. Till quite recently it was not possible to use this congeries of samples for protein analysis, but now several groups described successful protein extraction from FFPE tissues. In this chapter, we describe a protein extraction protocol established in our laboratory combined with the use of reverse phase protein microarray. (Source: Springer protocols feed by Protein Science) Springer protocols feed by Protein Science news http://www.medworm.com/rss/comments.php?id=5203869 Sun, 11 Sep 2011 03:16:55 +0100 5203869 http://www.medworm.com/index.php?rid=5203868&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-286-1_8 Formalin-fixed and paraffin-embedded (FFPE) tissues are used routinely everyday in hospitals world-wide for histopathological diagnosis of diseases like cancer. Due to formalin-induced cross-linking of proteins, FFPE tissues present a particular challenge for proteomic analysis. Nevertheless, there has been recent progress for extraction-based protein analysis in these tissues. Novel tools developed in the last few years are urgently needed because precise protein biomarker quantification in clinical FFPE tissues will be crucial for treatment decisions and to assess success or failure of current and future personalized molecular therapies. Furthermore, they will help to conceive why only a subset of patients responds to individualized treatments. Reverse phase protein array (RPPA) is a ver... Springer protocols feed by Protein Science news http://www.medworm.com/rss/comments.php?id=5203868 Sun, 11 Sep 2011 03:16:55 +0100 5203868 http://www.medworm.com/index.php?rid=5203867&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-286-1_7 In this study, we have used reverse protein arrays to profile kinase inhibitors in various cellular pathways in order to unravel their MoA. Multiplexing and simultaneous analysis of several phospho-proteins within the same lysate allows (1) the estimation of inhibitor concentrations needed to shut down an entire pathway, (2) the estimation of inhibitor selectivity, and (3) the comparison of inhibitors of different kinases within one assay. For example, parallel analysis of p-InsR, p-PKB, p-GSK-3, p-MEK, p-ERK, and p-S6rp in insulin treated A14 cells allows profiling for inhibitors of the InsR, PI3K, PKB, mTor, RAF, and MEK. Selective kinase inhibitors revealed different specific inhibitory pattern of the analyzed phospho-read outs. Altogether, multiplexed analysis of reverse (phase) protei... Springer protocols feed by Protein Science news http://www.medworm.com/rss/comments.php?id=5203867 Sun, 11 Sep 2011 03:16:55 +0100 5203867 http://www.medworm.com/index.php?rid=5203866&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-286-1_6 Theoretical models describing complex biological phenomena have been accumulating. However, most of these models have been created with hypothetical parameter determination without seeing actual cell reactions. The parameter determination requires high-dimensional data monitoring, particularly at the protein level. It has been a difficult task to develop the standard model system because of the lack of an appropriate validation technique. Reverse-phase protein lysate microarray (RPA) is one of the most potent technologies for high-dimensional proteomic monitoring. Therefore, proteomic monitoring by RPA may contribute substantially to develop theoretical protein network models based on experimental validation. (Source: Springer protocols feed by Protein Science) Springer protocols feed by Protein Science news http://www.medworm.com/rss/comments.php?id=5203866 Sun, 11 Sep 2011 03:16:55 +0100 5203866 http://www.medworm.com/index.php?rid=5203865&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-286-1_5 Reverse phase protein array (RPPA) techniques allow the quantitative analysis of signal transduction events in a high-throughput format. Sensitivity is important for RPPA-based detection approaches, since numerous signaling proteins or posttranslational modifications are present at low levels. Especially, the proteomic analysis of clinical samples exposes its own challenges with respect to sensitivity. Antibody-mediated signal amplification (AMSA) is a novel strategy relying on sequential incubation steps with fluorescently labeled secondary antibodies reactive against each other. AMSA is a simple extension of the standard quantification in the near-infrared range and is highly specific and robust. In this chapter, we present the amplification protocol and application examples for the time... Springer protocols feed by Protein Science news http://www.medworm.com/rss/comments.php?id=5203865 Sun, 11 Sep 2011 03:16:55 +0100 5203865 http://www.medworm.com/index.php?rid=5203864&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-286-1_4 Reverse phase protein arrays (RPPAs) emerged as a very useful tool for high-throughput screening of protein expression in large numbers of small specimen. Similar to other protein chemistry methods, antibody specificity is also a major concern for RPPA. Currently, testing antibodies on Western blot for specificity and applying serial dilution curves to determine signal/concentration linearity of RPPA signals are most commonly employed to validate antibodies for RPPA applications. However, even the detection antibodies fulfilling both requirements do not always give the expected result. Chemically synthesized small interfering RNAs (siRNAs) are one of the most promising and time-efficient tools for loss-of-function studies by specifically targeting the gene of interest resulting in a reduct... Springer protocols feed by Protein Science news http://www.medworm.com/rss/comments.php?id=5203864 Sun, 11 Sep 2011 03:16:55 +0100 5203864 http://www.medworm.com/index.php?rid=5203863&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-286-1_3 Phosphorylated proteins reflect the activity of specific cell signaling nodes in biological kinase protein networks. Cell signaling pathways can be either activated or deactivated depending on the phosphorylation state of the constituent proteins. The state of these kinase pathways reflects the in vivo activity of the cells and tissue at any given point in time. As such, cell signaling pathway information can be extrapolated to infer which phosphorylated proteins/pathways are driving an individual tumor’s growth. Reverse phase protein microarrays (RPMAs) are a sensitive and precise platform that can be applied to the quantitative measurement of hundreds of phosphorylated signal proteins from a small sample of tissue. Pre-analytical variability originating from tissue procurement and ... Springer protocols feed by Protein Science news http://www.medworm.com/rss/comments.php?id=5203863 Sun, 11 Sep 2011 03:16:55 +0100 5203863 http://www.medworm.com/index.php?rid=5203862&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-286-1_2 Careful selection of well-qualified antibodies is critical for accurate data collection from reverse phase protein arrays (RPPA). The most common way to qualify antibodies for RPPA analysis is by Western blotting because the detection mechanism is based on the same immunodetection principles. Western blots of tissue or cell lysates that result in single bands and low cross-reactivity indicate appropriate antibodies for RPPA detection. Western blot conditions used to validate antibodies for RPPA experiments, including blocking and detection reagents, have significant effects on aspects of antibody performance such as cross-reactivity against other proteins in the sample. We have found that there can be a dramatic impact on antibody behavior with changes in blocking reagent and detection met... Springer protocols feed by Protein Science news http://www.medworm.com/rss/comments.php?id=5203862 Sun, 11 Sep 2011 03:16:55 +0100 5203862 http://www.medworm.com/index.php?rid=5203861&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-286-1_1 Phosphorylated proteins represent one of the most important constituents of the proteome and are under intense analysis by the biotechnology and pharmaceutical industry because of their central role for cellular signal transduction. Indeed, alterations in cellular signaling and control mechanisms that modulate signal transduction, functionally underpin most human cancers today. Beyond their central role as the causative components of tumorigenesis, these proteins have become an important research focus for discovery of predictive and prognostic biomarkers. Consequently, these pathway constituents comprise a powerful biomarker subclass whereby the same analyte that provides prediction and/or prognosis is also the drug target itself: a theranostic marker. Reverse phase protein microarrays ha... Springer protocols feed by Protein Science news http://www.medworm.com/rss/comments.php?id=5203861 Sun, 11 Sep 2011 03:16:55 +0100 5203861 http://www.medworm.com/index.php?rid=5203860&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-286-1_10 Leukemias are well suited to proteomic profiling by RPPA due to the ready accessibility of blasts from the blood or marrow. In this review, we review methodological and procedural issues that affect the quality of RPPA data. We recommend contact printers that minimize sample quantities and evaporation and maximize sample per slide. The impact of sample selection and handling is reviewed as well. Protein is best prepared fresh on the date of acquisition as cryopreservation changes protein expression levels in some diseases. Rapid processing is also required to avoid changes in phosphorylation over time. Sample source, blood vs. marrow does not seem to affect results as long as leukemic blast enrichment procedures are utilized. The choice of the correct “normal” control is import... Springer protocols feed by Protein Science news http://www.medworm.com/rss/comments.php?id=5203860 Sun, 11 Sep 2011 03:16:55 +0100 5203860 http://www.medworm.com/index.php?rid=4870544&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-148-2_9 Membrane protein profiling and characterization is of immense importance for the understanding of vital processes taking place across cellular membranes. Traditional techniques used for soluble proteins, such as 2D gel electrophoresis, are sometimes not entirely applicable to membrane protein targets, due to their low abundance and hydrophobic character. New tools have been developed that will accelerate research on membrane protein targets. Lipid-based protein immobilization (LPI) is the core technology in a new approach that enables immobilization and digestion of native membrane proteins inside a flow cell format. The presented method is described in the context of comparing the method to traditional approaches where the sample amount that is digested and analyzed is the same. (Source: ... Springer protocols feed by Protein Science news http://www.medworm.com/rss/comments.php?id=4870544 Fri, 27 May 2011 22:08:32 +0100 4870544 http://www.medworm.com/index.php?rid=4870543&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-148-2_8 Proteomics has significantly contributed to improve our understanding of cell structures and functions in the last decade. The possibility to identify large sets of proteins from minute amount of material, linked with the isolation of cellular organelles using various cell fractionation methods, has provided unique insights into the molecular mechanisms governing cell functions in health and disease. The success of this approach relies on the isolation of highly enriched cell fractions enabling the separation of organelles with minimal contamination by other cellular structures. (Source: Springer protocols feed by Protein Science) Springer protocols feed by Protein Science news http://www.medworm.com/rss/comments.php?id=4870543 Fri, 27 May 2011 22:08:32 +0100 4870543 http://www.medworm.com/index.php?rid=4870542&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-148-2_7 In the field of analytical chemistry, stable isotope dilution assays are extensively used in combination with liquid chromatography-mass spectrometry (LC-MS) to provide confident quantification results. Over the last decade, the principle of isotope dilution has been adopted by the proteomic community in order to accurately quantify proteins in biological samples. In these experiments, a protein’s concentration is deduced from the ratio between the MS signal of a tryptic peptide and that of a stable isotope-labeled analog, which serves as an internal standard. The first isotope dilution standards introduced in proteomics were chemically synthesized peptides incorporating a stable isotope-tagged amino acid. These isotopically labeled peptide standards, which are currently widely used,... Springer protocols feed by Protein Science news http://www.medworm.com/rss/comments.php?id=4870542 Fri, 27 May 2011 22:08:32 +0100 4870542 http://www.medworm.com/index.php?rid=4870541&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-148-2_6 The chemical modification of protein thiols by reduction and alkylation is common in the preparation of proteomic samples for analysis by mass spectrometry (MS). Modification at other functional groups has received less attention in MS-based proteomics. Amine modification (Lys, N-termini) by reductive dimethylation or by acylation (e.g., iTRAQ labeling) has recently gained some popularity in peptide-based approaches (bottom-up MS). Modification at acidic groups (Asp, Glu, C-termini) has been explored very minimally. Here, we describe a sequential labeling strategy that enables complete modification of thiols, amines, and acids on peptides or small intact proteins. This method includes (1) the reduction and alkylation of thiols, (2) the reductive dimethylation of amines, and (3) the amidati... Springer protocols feed by Protein Science news http://www.medworm.com/rss/comments.php?id=4870541 Fri, 27 May 2011 22:08:32 +0100 4870541 http://www.medworm.com/index.php?rid=4870540&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-148-2_5 The quantitative comparison of proteome level changes across biological samples has become an essential feature in proteomics that remains challenging. We have recently introduced isobaric peptide termini labeling (IPTL), a novel strategy for isobaric quantification based on the derivatization of peptide termini with complementary isotopically labeled reagents. Unlike non-isobaric quantification methods, sample complexity at the MS level is not increased, providing improved sensitivity and protein coverage. The distinguishing feature of IPTL when comparing it to more established isobaric labeling methods (iTRAQ and TMT) is the presence of quantification signatures in all sequence-determining ions in MS/MS spectra, not only in the low mass reporter ion region. This makes IPTL a quantificati... Springer protocols feed by Protein Science news http://www.medworm.com/rss/comments.php?id=4870540 Fri, 27 May 2011 22:08:32 +0100 4870540 http://www.medworm.com/index.php?rid=4870539&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-148-2_4 Stable isotope labeling in combination with mass spectrometry has emerged as a powerful tool to identify and quantify thousands of proteins within complex protein mixtures. Isotope-coded protein label (ICPL) is capable of high-throughput quantitative proteome profiling on a global scale. Since ICPL is based on stable isotope tagging at the free amino groups of intact proteins, it is applicable to any protein sample, including extracts from tissues or body fluids. After labeling of up to four different proteome states, the samples can be combined and the complexity reduced by any separation method currently employed in protein chemistry. After enzymatic cleavage of the protein fractions the ratios of peptides in the different proteome states can be calculated by simple MS-based mass spectro... Springer protocols feed by Protein Science news http://www.medworm.com/rss/comments.php?id=4870539 Fri, 27 May 2011 22:08:31 +0100 4870539 http://www.medworm.com/index.php?rid=4870538&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-148-2_3 Stable isotope labeling based on relative peptide/protein abundance measurements is commonly applied for quantitative proteomics. Recently, trypsin-catalyzed oxygen-18 labeling has grown in popularity due to its simplicity, cost-effectiveness, and its ability to universally label peptides with high sample recovery. In 18O labeling, both C-terminal carboxyl group atoms of tryptic peptides can be enzymatically exchanged with 18O, thus providing the labeled peptide with a 4 Da mass shift from the 16O-labeled sample. Peptide 18O labeling is ideally suited for generating a labeled “universal” reference sample used for obtaining accurate and reproducible quantitative measurements across large number of samples in quantitative discovery proteomics. (Source: Springer protocols fee... Springer protocols feed by Protein Science news http://www.medworm.com/rss/comments.php?id=4870538 Fri, 27 May 2011 22:08:31 +0100 4870538 http://www.medworm.com/index.php?rid=4870537&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-148-2_2 Quantitative proteomics aims to identify and quantify proteins in cells or organisms that have been obtained from different biological origin (e.g., “healthy vs. diseased”), that have received different treatments, or that have different genetic backgrounds. Protein expression levels can be quantified by labeling proteins with stable isotopes, followed by mass spectrometric analysis. Stable isotopes can be introduced in vitro by reacting proteins or peptides with isotope-coded reagents (e.g., iTRAQ, reductive methylation). A preferred way, however, is the metabolic incorporation of heavy isotopes into cells or organisms by providing the label, in the form of amino acids (such as in SILAC) or salts, in the growth media. The advantage of in vivo labeling is that it does not suffe... Springer protocols feed by Protein Science news http://www.medworm.com/rss/comments.php?id=4870537 Fri, 27 May 2011 22:08:31 +0100 4870537 http://www.medworm.com/index.php?rid=4870536&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-148-2_25 MS-driven proteomics has evolved over the past two decades to a high tech and high impact research field. Two distinct factors clearly influenced its expansion: the rapid growth of an arsenal of instrument and proteomic techniques that led to an explosion of high quality data and the development of software tools to analyze and interpret these data which boosted the number of scientific discoveries. In analogy with the benchmarking of new instruments and proteomic techniques, such software tools must be thoroughly tested and analyzed. Recently, new tools were developed for automatic peptide quantification in quantitative proteomic experiments. Here we present a case study where the most recent and frequently used tools are analyzed and compared. (Source: Springer protocols feed by Protein ... Springer protocols feed by Protein Science news http://www.medworm.com/rss/comments.php?id=4870536 Fri, 27 May 2011 22:08:31 +0100 4870536 http://www.medworm.com/index.php?rid=4870535&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-148-2_24 Mass spectrometry-based proteomics has become an essential part of the analytical toolbox of the life sciences. With the ability to identify and quantify hundreds to thousands of proteins in high throughput, the field has contributed its fair share to the data avalanche coming from the so-called omics fields. As a result, the challenges involved in processing and managing this flood of data have grown as well. This chapter will point out and discuss these challenges, starting from the processing of raw mass spectrometry data into peaks, over the identification of peptides and proteins, to the quantification of the identified molecules. Finally, the informatics aspects of the nascent field of targeted proteomics are outlined as well. (Source: Springer protocols feed by Protein Science) Springer protocols feed by Protein Science news http://www.medworm.com/rss/comments.php?id=4870535 Fri, 27 May 2011 22:08:31 +0100 4870535 http://www.medworm.com/index.php?rid=4870534&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-148-2_23 Modification by ubiquitin (Ub) and ubiquitin-like proteins (UbLs) is involved in the regulation of numerous cellular processes and has therefore become an important subject of research in various areas of biomedicine. The large number of components of the system (∼1,500), most of them being ligases (∼800) that recognize their target substrates specifically, along with the complexity of the ubiquitination process, mostly the synthesis of the hallmark polyubiquitin chains, has rendered studies of many of the processes related to the activity of the system resistant to detailed mechanistic analysis. Thus, our knowledge of the modes of recognition of target substrates by ligases and of consensus ubiquitination sites is sparse. We also lack basic tools such as antibodies directed agains... Springer protocols feed by Protein Science news http://www.medworm.com/rss/comments.php?id=4870534 Fri, 27 May 2011 22:08:31 +0100 4870534 http://www.medworm.com/index.php?rid=4870533&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-148-2_22 This paper describes a cyclic on-column procedure for the sequential degradation of complex O-glycans on proteins by periodate oxidation of sugars and cleavage of oxidation products by elimination. Glycoproteins are immobilized to alkali-stable, reversed-phase Poros 20 beads, desialylated by treatment with dilute trifluoroacetic acid, and de-O-glycosylated by two degradation cycles before the eluted apoproteins are digested with trypsin for analysis by liquid chromatography electrospray ionization-mass spectrometry. Even complex glycan moieties are removed under mild conditions with only minimal effects on structural integrity of the peptide core by fragmentation, dehydration, or racemization of lysine and arginine residues. The protocol is also applicable on gel-immobilized glycoproteins ... Springer protocols feed by Protein Science news http://www.medworm.com/rss/comments.php?id=4870533 Fri, 27 May 2011 22:08:31 +0100 4870533 http://www.medworm.com/index.php?rid=4870532&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-148-2_21 Glycosylation is one of the many post-translational protein modifications that regulate several biological processes of proteins and lipids. In particular aberrant sialylation, at the terminal position of the glycan structures of cell surface proteins, occurs in numerous diseases such as cancer metastasis and viral infections. Methodological improvements in the sample preparation and analysis currently enable the detailed identification of the glycosylation sites and glycan structure characterization. In this context, the aim of this chapter is to describe a methodology to identify the glycosylation site of N-linked sialylated glycoproteins. The method relies on the specificity of titanium dioxide affinity chromatography to isolate sialic acid-containing glycopeptides. After enzymatic rele... Springer protocols feed by Protein Science news http://www.medworm.com/rss/comments.php?id=4870532 Fri, 27 May 2011 22:08:31 +0100 4870532 http://www.medworm.com/index.php?rid=4870531&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-148-2_20 Glycosylations represent major and essential co- and post-translational modification forms of proteins and facilitate a multitude of functions such as cell–cell interactions as well as protein folding and stability. The analysis of protein glycosylation is still an enormous task due to the vast heterogeneity and multitude of different possible carbohydrate structures. The elucidation of glycosylation sites – the attachment points of carbohydrate structures to the polypeptide backbone – is often among the first necessary steps of analysis. Therefore, we here present a simple protocol for charge-based enrichment of sialylated glycopeptides by strong cation exchange chromatography and subsequent analysis of glycosylation sites by mass spectrometry. (Source: Springer protocol... Springer protocols feed by Protein Science news http://www.medworm.com/rss/comments.php?id=4870531 Fri, 27 May 2011 22:08:31 +0100 4870531 http://www.medworm.com/index.php?rid=4870530&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-148-2_1 Proteins are reckoned to be the key actors in a living organism. By studying proteins, one engages into deciphering a complex series of events occurring during a protein’s life span. This starts at the creation of a protein, which is tightly controlled on both a transcriptional (Williams and Tyler, 2007, Curr Opin Genet Dev 17, 88–93) and a translational level (Van Der Kelen et al., 2009, Crit Rev Biochem Mol Biol 44, 143–168). During translation, a primary strand of amino acids undergoes a complex folding process in order to obtain a native three-dimensional protein structure (Gross et al., 2003, Cell 115, 739–750). Proteins take on a plethora of functions, such as complex formation, receptor activity, and signal transduction, which ultimately ... Springer protocols feed by Protein Science news http://www.medworm.com/rss/comments.php?id=4870530 Fri, 27 May 2011 22:08:30 +0100 4870530 http://www.medworm.com/index.php?rid=4870529&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-148-2_19 Glycosylation has been recognized as one of the most important modifications on proteins. The interactions between proteins and glycans are known to play an important role in many biological processes. Lectins are carbohydrate-binding proteins that can specifically interact with and select for carbohydrate structures. The technique of lectin affinity chromatography takes advantage of this specific interaction and enables the selection and purification of glycoproteins with carbohydrate structures complementary to the lectin-binding site. Depending on the carbohydrate specificity of the lectin glycoprotein fractions enriched for example, high mannose or complex N-glycans or O-glycans can be obtained. Afterward both the protein part and the glycan part can be analyzed in more detail allowing... Springer protocols feed by Protein Science news http://www.medworm.com/rss/comments.php?id=4870529 Fri, 27 May 2011 22:08:30 +0100 4870529 http://www.medworm.com/index.php?rid=4870528&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-148-2_18 Determining the sequence of protein N-termini and their modifications functionally annotates proteins since translation isoforms, posttranslational modifications, and proteolytic truncations direct localization, activity, and the half-life of most proteins. Here we present in detail the steps required to perform our recently described approach we call Terminal Amine Isotopic Labeling of Substrates (TAILS), a combined N-terminomics and protease substrate discovery degradomics platform for the simultaneous quantitative and global analysis of the N-terminome and proteolysis in one MS/MS experiment. By a 3-day procedure with flexible α- and ɛ-amine labeling and blocking options, TAILS removes internal tryptic and C-terminal peptides by binding to a dendritic polyglycerol aldehyde po... Springer protocols feed by Protein Science news http://www.medworm.com/rss/comments.php?id=4870528 Fri, 27 May 2011 22:08:30 +0100 4870528 http://www.medworm.com/index.php?rid=4870527&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-148-2_17 Protease specificity profiling using proteome-derived, database-searchable peptide libraries is a novel approach to define the active site specificity of proteolytic enzymes we call PICS (Proteomic Identification of protease Cleavage Sites). Proteome-derived peptide libraries are generated by trypsin, GluC, or chymotrypsin digestion of biologically relevant proteomes, such as cytosolic lysates, to generate three separate libraries that each differ from the others in their C-terminal amino acid residues according to the protease specificity. Primary amines of all peptides are then chemically protected so that after incubation with a test protease, the neo-N-termini of the prime-side cleavage products with exposed α-amines can be specifically biotinylated, enriched, and identified by l... Springer protocols feed by Protein Science news http://www.medworm.com/rss/comments.php?id=4870527 Fri, 27 May 2011 22:08:30 +0100 4870527 http://www.medworm.com/index.php?rid=4870526&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-148-2_16 Proteases play vital roles in many cellular processes and signaling cascades through specific limited cleavage of their targets. It is important to identify what proteins are substrates of proteases and where their cleavage sites are so as to reveal the molecular mechanisms and specificity of signaling. We have developed a method to achieve this goal using a strategy that chemically tags the substrate’s alpha amine generated by proteolysis, enriches for tagged peptides, and identifies them using liquid chromatography-coupled tandem mass spectrometry (LC-MS/MS). Peptide MS/MS data are searched against a database to reveal what proteins are cleaved, whereby peptide N-termini demarcate sites of protease cleavage. (Source: Springer protocols feed by Protein Science) Springer protocols feed by Protein Science news http://www.medworm.com/rss/comments.php?id=4870526 Fri, 27 May 2011 22:08:30 +0100 4870526 http://www.medworm.com/index.php?rid=4870525&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-148-2_15 One strategy to reduce complexity in proteome analysis is through rational reduction of the proteolytic peptides that constitute the analyte for mass spectrometric analysis. Methods for selective isolation of C- and N-terminal peptides have been developed. In this chapter, we outline the context and variety of methods for selective isolation of N-terminal peptides and detail one method based on negative selection through differential removal of internal peptides. (Source: Springer protocols feed by Protein Science) Springer protocols feed by Protein Science news http://www.medworm.com/rss/comments.php?id=4870525 Fri, 27 May 2011 22:08:30 +0100 4870525 http://www.medworm.com/index.php?rid=4870524&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-148-2_14 Over the past decade phosphoproteomics has become an emerging discipline within proteomics research, focusing on detection of the reversible modification of proteins by phosphorylation of serine, threonine, and tyrosine residues. For successful analysis, phosphopeptide enrichment is often a prerequisite due to their low stoichiometry, heterogeneity, and low abundance. The enrichment of phosphopeptides is often performed manually, which is inherently labor intensive and a major hindrance in large-scale analyses. Automation of the enrichment method would vastly improve reproducibility and thereby facilitate “high-throughput” phosphoproteomics research. Here, we describe the setup of a simple, robust, and automated online TiO2-based nanoscale chromatographic approach to selectivel... Springer protocols feed by Protein Science news http://www.medworm.com/rss/comments.php?id=4870524 Fri, 27 May 2011 22:08:30 +0100 4870524 http://www.medworm.com/index.php?rid=4870523&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-148-2_13 We present an overview of sensitive and robust analytical methods for phosphopeptide analysis, including calcium phosphate precipitation and affinity enrichment methods such as IMAC and TiO2. We then discuss various tandem mass spectrometry approaches for phosphopeptide sequencing and quantification, and we consider aspects of phosphoproteome data analysis and interpretation. Efficient integration of these stages of phosphoproteome analysis is highly important to ensure a successful outcome of large-scale experiments for studies of phosphorylation-mediated protein regulation. (Source: Springer protocols feed by Protein Science) Springer protocols feed by Protein Science news http://www.medworm.com/rss/comments.php?id=4870523 Fri, 27 May 2011 22:08:30 +0100 4870523 http://www.medworm.com/index.php?rid=4870522&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-148-2_12 Measuring protein synthesis and degradation rates on a proteomic scale is an important step toward modeling the kinetics in complicated cellular response networks. A gel-free method, able to quantify changes in the formation of new proteins on a 15 min timescale, compatible with mass spectrometry is described. The methionine analogue, azidohomoalanine (azhal), is used to label newly formed proteins during a short pulse-labeling period following an environmental switch in Escherichia coli. Following digestion a selective reaction against azhal-containing peptides is applied to enrich these peptides by diagonal chromatography. This technique enables quantitation of hundreds of newly synthesized proteins and provides insight into immediate changes in newly synthesized proteins on a prote... Springer protocols feed by Protein Science news http://www.medworm.com/rss/comments.php?id=4870522 Fri, 27 May 2011 22:08:30 +0100 4870522 http://www.medworm.com/index.php?rid=4870521&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-148-2_11 The current advances in mass spectrometry technology have led to the possibility of analyzing more complex biological samples such as entire proteomes. Here, we describe a new and powerful methodology that combines the use of the metalloendopeptidase Lys-N and strong cation exchange with mass spectrometric analysis. The approach described here allows one to separate peptides with different functional groups. The peptides we are able to isolate are N-terminal peptides, phosphorylated peptides with a single lysine, peptides with a single basic residue (lysine), and peptides with multiply basic residues. When this separation strategy is combined with tandem mass spectrometry that involves both collision-induced dissociation and electron transfer dissociation, one can achieve an optimal target... Springer protocols feed by Protein Science news http://www.medworm.com/rss/comments.php?id=4870521 Fri, 27 May 2011 22:08:30 +0100 4870521 http://www.medworm.com/index.php?rid=4870520&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-148-2_10 In recent years the array of mass spectrometry (MS) applications to address questions in molecular and cellular biology has greatly expanded and continues to grow. Modern mass spectrometers allow for identification, characterization, as well as quantification of protein compositions and their modifications in complex biological samples. Prior to MS analysis any biological sample needs to be properly prepared for the experiment. Here we present a protocol that combines pre-separation of proteins by 1D gel electrophoresis followed by analysis of in situ digested protein products by tandem mass spectrometry (MS/MS). All steps of the sample preparation are explained in detail, and the procedure is compatible with downstream analysis on any mass spectrometer available. With minor adjustments th... Springer protocols feed by Protein Science news http://www.medworm.com/rss/comments.php?id=4870520 Fri, 27 May 2011 22:08:30 +0100 4870520 http://www.medworm.com/index.php?rid=4703361&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-068-3_23 PeptideAtlas is a web-accessible database of LC–MS/MS shotgun proteomics results from hundreds of experiments conducted in diverse laboratories, with all data processed via a uniform analysis pipeline. A total of 91 experiments on human plasma and serum are included. Using the PeptideAtlas web interface, users can browse and search the Human Plasma PeptideAtlas for identified peptides and identified proteins, view spectra, and select proteotypic peptides. Users can easily view supporting information such as chromosomal mapping, estimated abundances, and sequence alignments. Herein, the reader is instructed in the use of the Human Plasma PeptideAtlas through an illustrated exploration of cytokine receptors in plasma. (Source: Springer protocols feed by Protein Science) Springer protocols feed by Protein Science news http://www.medworm.com/rss/comments.php?id=4703361 Wed, 13 Apr 2011 04:14:52 +0100 4703361 http://www.medworm.com/index.php?rid=4703360&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-068-3_22 Mass spectrometry has become the method of choice for studying proteins in complex mixtures in a qualitative and quantitative fashion. The application of mass spectrometry-based proteomics analyses on plasma has correspondingly been established as an important method for disease-associated biomarker discovery and validation. Yet despite being a readily available human sample, plasma poses several important challenges to the proteomics researcher. With a focus on bioinformatics aspects, this chapter will discuss the problems involved in analyzing plasma proteomics data, along with the scope of solutions available through specialised tools and sophisticated analysis methods. (Source: Springer protocols feed by Protein Science) Springer protocols feed by Protein Science news http://www.medworm.com/rss/comments.php?id=4703360 Wed, 13 Apr 2011 04:14:52 +0100 4703360 http://www.medworm.com/index.php?rid=4703359&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-068-3_21 Mass spectrometry-based proteomics has made the transition from the analysis of a single or only a few proteins per experiment to a high-throughput platform for discovery/validation that can analyze several hundreds to thousands of proteins in a single experiment. This increase in analytical capability hinged on four main components: (1) innovative methodologies, (2) improved instruments, (3) ready availability of comprehensive protein sequence databases, and (4) development of sophisticated software algorithms to match fragmentation mass spectra against these databases. But as the throughput of the approach increased, so did the necessity to manage and automate the data processing workflow. Indeed, modern instruments generate tens of thousands of fragmentation spectra per hour, providing ... Springer protocols feed by Protein Science news http://www.medworm.com/rss/comments.php?id=4703359 Wed, 13 Apr 2011 04:14:51 +0100 4703359 http://www.medworm.com/index.php?rid=4703358&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-068-3_20 This chapter presents a case study, which applies statistical design and analysis to an LC–MS-based ­investigation of subjects with coronary artery disease. First, we discuss the principles of statistical ­experimental design, and the specification of an Analysis of Variance (ANOVA) model that describes the major sources of variation in the data. Second, we discuss procedures for detecting differentially abundant proteins, estimating protein abundance in individual samples, testing predefined groups of proteins for enrichment in differential abundance, and calculating sample size for a future experiment. The discussion is accompanied by examples of computer code implemented in the open-source statistical software R, which can be followed for an independent implementation of a... Springer protocols feed by Protein Science news http://www.medworm.com/rss/comments.php?id=4703358 Wed, 13 Apr 2011 04:14:51 +0100 4703358 http://www.medworm.com/index.php?rid=4703357&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-068-3_19 Blood platelets are key players standing at the crossroads between physiologically occurring hemostasis and pathologic thrombus formation. As these cellular particles lack a nucleus, intra- and intercellular processes involved in platelet activity and function are almost exclusively regulated on the protein level. In particular, posttranslational protein modification by phosphorylation, which allows for a quick and highly dynamic transduction of cellular signals, is discussed in this context. In addition, since platelet activation and aggregation usually require surface contact with the surrounding tissue, special interest focuses on this contacting region, and hence on the subproteome of the platelet plasma membrane. In this chapter, we present a mass spectrometry-driven approach capable ... Springer protocols feed by Protein Science news http://www.medworm.com/rss/comments.php?id=4703357 Wed, 13 Apr 2011 04:14:51 +0100 4703357 http://www.medworm.com/index.php?rid=4703356&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-068-3_18 Platelets are specialized blood cells that play central roles in physiologic and pathologic processes of hemostasis, wound healing, host defense, thrombosis, inflammation, and tumor metastasis. Activation of platelets is crucial for platelet function that includes a complex interplay of adhesion, signaling molecules, and release of bioactive factors. Transfusion of platelet concentrates is an important treatment component for thrombocytopenia and bleeding. Recent progress in high-throughput mRNA and protein profiling techniques has advanced the understanding of platelet biological functions toward identifying novel platelet-expressed and secreted proteins, analyzing functional changes between normal and pathologic states, and determining the effects of processing and storage on platelet co... Springer protocols feed by Protein Science news http://www.medworm.com/rss/comments.php?id=4703356 Wed, 13 Apr 2011 04:14:51 +0100 4703356 http://www.medworm.com/index.php?rid=4703355&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-068-3_17 Cryoprecipitate is a concentrate of high-molecular-weight plasma proteins that precipitate when frozen plasma is slowly thawed at 1–6°C. The concentrate contains factor VIII (antihemophilic factor), von Willebrand factor (vWF), fibrinogen, factor XIII, fibronectin, and small amounts of other plasma proteins. Clinical-grade preparations of cryoprecipitate are mainly used to treat fibrinogen deficiency caused by acute bleeding or functional abnormalities of the fibrinogen protein. In the past, cryoprecipitate was used to treat von Willebrand disease and hemophilia A (factor VIII deficiency), but the availability of more highly purified coagulation factor concentrates or recombinant protein preparations has superseded the use of cryoprecipitate for these coagulopathies. Cryodepleted... Springer protocols feed by Protein Science news http://www.medworm.com/rss/comments.php?id=4703355 Wed, 13 Apr 2011 04:14:51 +0100 4703355