Trypsin Cleaves Exclusively C-terminal to Arginine and Lysine Residues

TLDR

Mass spectrometry‑based proteomics routinely uses trypsin to generate peptides, relying on cleavage exclusively C‑terminal to arginine or lysine, yet many studies report non‑tryptic peptides. The authors employed a high‑accuracy ion trap Fourier transform mass spectrometer to dramatically increase peptide identification confidence and demonstrate that trypsin cleaves only C‑terminal to arginine and lysine. They compared the new instrument’s sub‑ppm mass accuracy to simulated lower‑accuracy data, revealing that reduced precision leads to many proteins being incorrectly assigned non‑tryptic peptide hits. Non‑tryptic peptides were found only as protein C‑terminal fragments or as breakup products of fully tryptic peptides N‑terminal to internal proline, indicating that previous identifications should be re‑examined and that strict trypsin specificity is essential.

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.