TY - JOUR

T1 - Trypsin cleaves exclusively C-terminal to arginine and lysine residues

AU - Olsen, Jesper Velgaard

AU - Ong, Shao-En

AU - Mann, Matthias

N1 - Keywords: Animals; Arginine; Chromatography, High Pressure Liquid; Liver; Lysine; Male; Mass Spectrometry; Mice; Peptide Fragments; Peptide Mapping; Proteomics; Sequence Analysis, Protein; Spectroscopy, Fourier Transform Infrared; Trypsin

PY - 2004

Y1 - 2004

N2 - Almost all large-scale projects in mass spectrometry-based proteomics use trypsin to convert protein mixtures into more readily analyzable peptide populations. When searching peptide fragmentation spectra against sequence databases, potentially matching peptide sequences can be required to conform to tryptic specificity, namely, cleavage exclusively C-terminal to arginine or lysine. In many published reports, however, significant numbers of proteins are identified by non-tryptic peptides. Here we use the sub-parts per million mass accuracy of a new ion trap Fourier transform mass spectrometer to achieve more than a 100-fold increased confidence in peptide identification compared with typical ion trap experiments and show that trypsin cleaves solely C-terminal to arginine and lysine. We find that non-tryptic peptides occur only as the C-terminal peptides of proteins and as breakup products of fully tryptic peptides N-terminal to an internal proline. Simulating lower mass accuracy led to a large number of proteins erroneously identified with non-tryptic peptide hits. Our results indicate that such peptide hits in previous studies should be re-examined and that peptide identification should be based on strict trypsin specificity.

AB - Almost all large-scale projects in mass spectrometry-based proteomics use trypsin to convert protein mixtures into more readily analyzable peptide populations. When searching peptide fragmentation spectra against sequence databases, potentially matching peptide sequences can be required to conform to tryptic specificity, namely, cleavage exclusively C-terminal to arginine or lysine. In many published reports, however, significant numbers of proteins are identified by non-tryptic peptides. Here we use the sub-parts per million mass accuracy of a new ion trap Fourier transform mass spectrometer to achieve more than a 100-fold increased confidence in peptide identification compared with typical ion trap experiments and show that trypsin cleaves solely C-terminal to arginine and lysine. We find that non-tryptic peptides occur only as the C-terminal peptides of proteins and as breakup products of fully tryptic peptides N-terminal to an internal proline. Simulating lower mass accuracy led to a large number of proteins erroneously identified with non-tryptic peptide hits. Our results indicate that such peptide hits in previous studies should be re-examined and that peptide identification should be based on strict trypsin specificity.

U2 - 10.1074/mcp.T400003-MCP200

DO - 10.1074/mcp.T400003-MCP200

M3 - Journal article

C2 - 15034119

VL - 3

SP - 608

EP - 614

JO - Molecular and Cellular Proteomics

JF - Molecular and Cellular Proteomics

SN - 1535-9476

IS - 6

ER -