Does trypsin cut before proline?

Trypsin is the most commonly used enzyme in mass spectrometry for protein digestion with high substrate specificity. Many peptide identification algorithms incorporate these specificity rules as filtering criteria. A generally accepted "Keil rule" is that trypsin cleaves next to arginine or lysine, but not before proline. Since this rule was derived two decades ago based on a small number of experimentally confirmed cleavages, we decided to re-examine it using 14.5 million tandem spectra (2 orders of magnitude increase in the number of observed tryptic cleavages). Our analysis revealed a surprisingly large number of cleavages before proline. We examine several hypotheses to explain these cleavages and argue that trypsin specificity rules used in peptide identification algorithms should be modified to "legitimatize" cleavages before proline. Our approach can be applied to analyze any protease, and we further argue that specificity rules for other enzymes should also be re-evaluated based on statistical evidence derived from large MS/MS data sets.