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

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          Abstract

          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.

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          Author and article information

          Journal
          Mol Cell Proteomics
          Molecular & cellular proteomics : MCP
          American Society for Biochemistry & Molecular Biology (ASBMB)
          1535-9476
          1535-9476
          Jun 2004
          : 3
          : 6
          Affiliations
          [1 ] Center for Experimental BioInformatics, Department of Biochemistry and Molecular Biology, University of Southern Denmark, Campusvej 55, DK-5230 Odense M, Denmark.
          Article
          S1535-9476(20)33877-9
          10.1074/mcp.T400003-MCP200
          15034119
          d9c3398e-b0e5-4865-95a8-e3620a28b80a
          History

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