Exploring proteomes and analyzing protein processing by mass spectrometric identification of sorted N-terminal peptides

• Published in: Nature biotechnology 2003-05, Vol.21 (5), p.566-569
• Main Authors: Goethals, Marc, Thomas, Grégoire R, Gevaert, Kris, Vandekerckhove, Joël, Martens, Lennart, Van Damme, Jozef, Staes, An
• Format: Article
• Language: English
• Subjects: Agriculture; Bioinformatics; Biological and medical sciences; Biomedical and Life Sciences; Biomedical Engineering/Biotechnology; Biomedicine; Biotechnology; Blood Platelets - chemistry; Blood Platelets - metabolism; Cell Membrane - metabolism; Cytosol - chemistry; E coli; Electrophoresis; Fractionation; Fundamental and applied biological sciences. Psychology; Humans; Life Sciences; Liquid chromatography; Mass Spectrometry - methods; Peptides; Peptides - analysis; Peptides - chemistry; Peptides - metabolism; Proteins; Proteome - analysis; Proteome - chemistry; Proteome - metabolism; technical-report
• ISSN: 1087-0156; 1546-1696
• DOI: 10.1038/nbt810

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 1 , 2 or it is first reduced in complexity by isolating sets of representative peptides 3 , 4 , 5 , 6 , 7 , 8 . Recently, we developed a peptide isolation procedure based on diagonal electrophoresis 9 and diagonal chromatography 10 . 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 11 . 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 12 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) 13 , 14 ; 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.