Table_8_Mirror proteases of Ac-Trypsin and Ac-LysargiNase precisely improve novel event identifications in Mycolicibacterium smegmatis MC2 155 by proteogenomic analysis.XLSX

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 b/y 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 Mycolicibacterium smegmatis MC2 155 for proteogenomic analysis. The mirror proteases accurately identified 368 novel peptides, exhibiting 75–80% b and y ion coverages against 65–68% y or b ion coverages of Ac-Trypsin (38.9% b and 68.3% y) or Ac-LysargiNase (65.5% b and 39.6% y) as annotated peptides from M. smegmatis MC2 155. The complementary b and y ion series largely increased the reliability of overlapped sequences derived from novel peptides. Among these novel peptides, 311 peptides were annotated in other public M. smegmatis strains, and 57 novel peptides with more continuous b and y 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.

由于大规模蛋白质基因组学（proteogenomics）研究中缺乏统一的评价标准，精准识别新型肽段仍颇具挑战。胰蛋白酶（trypsin）与赖精氨酸酶（lysargiNase）的镜像蛋白酶可生成互补的b/y离子序列，无需借助不同假发现率（false discovery rate，FDR）排序筛选潜在靶标，即可在实验中高效评估真实的新型肽段。本研究以耻垢分枝杆菌（Mycolicibacterium smegmatis）MC2 155为研究对象，使用一对自研的乙酰化镜像蛋白酶——乙酰化胰蛋白酶（Ac-Trypsin）与乙酰化赖精氨酸酶（Ac-LysargiNase）开展蛋白质基因组学分析。该镜像蛋白酶可精准识别368条新型肽段，其b、y离子覆盖率可达75%~80%；而以耻垢分枝杆菌MC2 155的注释肽段为参照时，单独使用乙酰化胰蛋白酶仅能实现38.9%的b离子覆盖率与68.3%的y离子覆盖率，单独使用乙酰化赖精氨酸酶则仅能实现65.5%的b离子覆盖率与39.6%的y离子覆盖率，二者的单酶覆盖率均仅为65%~68%。互补的b/y离子序列大幅提升了新型肽段重叠序列的可信度。在上述新型肽段中，311条可在其他公开的耻垢分枝杆菌菌株中被注释，经光谱质量评估后，还筛选出57条拥有更连续b/y离子对的新型肽段用于后续分析。这使得镜像蛋白酶可成功修正6个已注释蛋白的N端，并检测到17个新的编码开放阅读框（open reading frame，ORF）。我们认为，镜像蛋白酶将成为原核与真核生物蛋白质基因组学研究中用于新型肽段检测的有效策略。