Mirror proteases of Ac-Trypsin and Ac-LysargiNase precisely improve novel event identifications in MC 155 by proteogenomic analysis.

Accurate identification of novel peptides remains challenging because of the lack of evaluation criteria in large-scale proteogenomic studies. Mirror proteases of trypsin and lysargiNase can generate complementary / ion series, providing the opportunity to efficiently assess authentic novel peptides in experiments other than filter potential targets by different false discovery rates (FDRs) ranking. In this study, a pair of in-house developed acetylated mirror proteases, Ac-Trypsin and Ac-LysargiNase, were used in MC 155 for proteogenomic analysis. The mirror proteases accurately identified 368 novel peptides, exhibiting 75-80% and ion coverages against 65-68% or ion coverages of Ac-Trypsin (38.9% and 68.3% ) or Ac-LysargiNase (65.5% and 39.6% ) as annotated peptides from MC 155. The complementary and ion series largely increased the reliability of overlapped sequences derived from novel peptides. Among these novel peptides, 311 peptides were annotated in other public strains, and 57 novel peptides with more continuous and pairs were obtained for further analysis after spectral quality assessment. This enabled mirror proteases to successfully correct six annotated proteins' N-termini and detect 17 new coding open reading frames (ORFs). We believe that mirror proteases will be an effective strategy for novel peptide detection in both prokaryotic and eukaryotic proteogenomics.