Gevaert K, Goethals M, Martens L, Van Damme J, Staes A, Thomas GR et al.. Exploring proteomes and analyzing protein processing by mass spectrometric identification of sorted N-terminal peptides. Nat Biotechnol 21: 566-569

Department of Medical Protein Research, Flanders Interuniversity Institute for Biotechnology, Ghent University, A. Baertsoenkaai 3, B-9000 Ghent, Belgium.
Nature Biotechnology (Impact Factor: 41.51). 06/2003; 21(5):566-9. DOI: 10.1038/nbt810
Source: PubMed


Current non-gel techniques for analyzing proteomes rely heavily on mass spectrometric analysis of enzymatically digested protein mixtures. Prior to analysis, a highly complex peptide mixture is either separated on a multidimensional chromatographic system or it is first reduced in complexity by isolating sets of representative peptides. Recently, we developed a peptide isolation procedure based on diagonal electrophoresis and diagonal chromatography. We call it combined fractional diagonal chromatography (COFRADIC). In previous experiments, we used COFRADIC to identify more than 800 Escherichia coli proteins by tandem mass spectrometric (MS/MS) analysis of isolated methionine-containing peptides. Here, we describe a diagonal method to isolate N-terminal peptides. This reduces the complexity of the peptide sample, because each protein has one N terminus and is thus represented by only one peptide. In this new procedure, free amino groups in proteins are first blocked by acetylation and then digested with trypsin. After reverse-phase (RP) chromatographic fractionation of the generated peptide mixture, internal peptides are blocked using 2,4,6-trinitrobenzenesulfonic acid (TNBS); they display a strong hydrophobic shift and therefore segregate from the unaltered N-terminal peptides during a second identical separation step. N-terminal peptides can thereby be specifically collected for further liquid chromatography (LC)-MS/MS analysis. Omitting the acetylation step results in the isolation of non-lysine-containing N-terminal peptides from in vivo blocked proteins.

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Available from: An Staes, Feb 19, 2015
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    • "It should be noted, however, that all these modified methodologies are based on the key assumption that a protein can be identified based on the sequence of a single or multiple tryptic peptides originating from this protein or that only peptides with a certain amino acid are isolated (Amado et al. 2013). These modifications include isotope labeling (Gygi et al. 1999), COFRADIC—or combined fractional diagonal chromatography (Gevaert and Vandekerckhove 2004), Nteromics (Gevaert et al. 2003), and combinatorial peptidomics (or peptide arrays) (Soloviev et al. 2003). Other strategies are also available, including the emerging technology of protein chips (Fung et al. 2001). "
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    • "Recently, we introduced charge-based fractional diagonal chromatography (ChaFRADIC) [48], which is based on the same principle as CO(mbined)FRADIC [44] developed by Gevaert et al., however, makes use of strong cation exchange (SCX) instead of reversed phase chromatography. Notably, these FRADIC methods combine two-dimensional diagonal A. Saskia Venne et al. "
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    ABSTRACT: We applied an extended charge-based fractional diagonal chromatography (ChaFRADIC) workflow to analyze the N-terminal proteome of Arabidopsis thaliana seedlings. Using iTRAQ protein labeling and a multi-enzyme digestion approach including trypsin, GluC, and subtilisin, a total of 200 μg per enzyme, and measuring only 1/3 of each ChaFRADIC-enriched fraction by LC-MS, we quantified a total of 2791 unique N-terminal peptides corresponding to 2249 different unique N-termini from 1270 Arabidopsis proteins. Our data indicate the power, reproducibility, and sensitivity of the applied strategy that might be applicable to quantify proteolytic events from as little as 20 μg of protein per condition across up to eight different samples. Furthermore, our data clearly reflect the Methionine excision dogma as well as the N-end rule degradation pathway (NERP) discriminating into a stabilizing or destabilizing function of N-terminal amino acid residues. We found bona fide NERP destabilizing residues underrepresented, and the list of neo N-termini from wild type samples may represent a helpful resource during the evaluation of NERP substrate candidates. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.
    Proteomics 05/2015; 15(14). DOI:10.1002/pmic.201500014 · 3.81 Impact Factor
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    • "A major challenge in these procedures is to find a chemistry that favors α-amines on protein N termini over ε-amino groups in lysine side chains that are almost identical in reactivity against amine reactive labels [43] [44]. In contrast to these techniques for positive selection of N-terminal peptides, negative selection procedures, such as COmbined FRActional DIagonal Chromatography (COFRADIC) and Terminal Amine Isotopic Labeling of Substrates (TAILS) ignore the lysine problem but selectively remove internal peptides upon whole protein amine labeling and tryptic digest [45] [46]. Current positive and negative enrichment approaches for N-and C-terminal peptides have been recently comprehensively covered in excellent reviews [47] [48]. "
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