biostudies-literatureUnknown36(2)Nagy KIn recent years, alternative enzymes with varied specificities have gained importance in MS-based bottom-up proteomics, offering orthogonal information about biological samples and advantages in certain applications. However, most mass spectrometric workflows are optimized for tryptic digests. This raises the questions of whether enzyme specificity impacts mass spectrometry and if current methods for nontryptic digests are suboptimal. The success of peptide and protein identifications relies on the information content of MS/MS spectra, influenced by collision energy in collision-induced dissociation. We investigated this by conducting LC-MS/MS measurements with different enzymes, including trypsin, Arg-C, Glu-C, Asp-N, and chymotrypsin, at varying collision energies. We analyzed peptide scJournal of the American Society for Mass Spectrometry299-308https://www.ebi.ac.uk/biostudies/studies/S-EPMC11808764biostudies-literatureThe Enzyme Effect: Broadening the Horizon of MS Optimization to Nontryptic Digestion in Proteomics.PMC11808764Nagy KRevesz ASandor PVekey KDrahos LfalseThe Enzyme Effect: Broadening the Horizon of MS Optimization to Nontryptic Digestion in Proteomics.In recent years, alternative enzymes with varied specificities have gained importance in MS-based bottom-up proteomics, offering orthogonal information about biological samples and advantages in certain applications. However, most mass spectrometric workflows are optimized for tryptic digests. This raises the questions of whether enzyme specificity impacts mass spectrometry and if current methods for nontryptic digests are suboptimal. The success of peptide and protein identifications relies on the information content of MS/MS spectra, influenced by collision energy in collision-induced dissociation. We investigated this by conducting LC-MS/MS measurements with different enzymes, including trypsin, Arg-C, Glu-C, Asp-N, and chymotrypsin, at varying collision energies. We analyzed peptide sc2025-01-01T00:00:00Z2025 Feb2025-04-26T16:35:15.77Z2025-04-06T15:13:43.909ZS-EPMC118087643980370310.1021/jasms.4c00396