Multiaspect Examinations of Possible Alternative Mappings of Identified Variant Peptides: A Case Study on the HEK293 Cell Line

Adopting proteogenomics
approach to validate single nucleotide
variation events by identifying corresponding single amino acid variant
peptides from mass spectrometry (MS)-based proteomics data facilitates
translational and clinical research. Although variant peptides are
usually identified from MS data with a stringent false discovery rate
(FDR), FDR control could fail to eliminate dubious results caused
by several issues; thus, postexamination to eliminate dubious results
is required. However, comprehensive postexaminations of identification
results are still lacking. Therefore, we propose a framework of three
bottom-up levels, peptide–spectrum match, peptide, and variant
event levels, that consists of rigorous 11-aspect examinations from
the MS perspective to further confirm the reliability of variant events.
As a proof of concept and showing feasibility, we demonstrate 11 examinations
on the identified variant peptides from an HEK293 cell line data set,
where various database search strategies were applied to maximize
the number of identified variant PSMs with an FDR <1% for postexaminations.
The results showed that only FDR criterion is insufficient to validate
identified variant peptides and the 11 postexaminations can reveal
low-confidence variant events detected by shotgun proteomics experiments.
Therefore, we suggest that postexaminations of identified variant
events based on the proposed framework are necessary for proteogenomics
studies.

采用蛋白质基因组学（proteogenomics）策略，通过基于质谱（MS）的蛋白质组学数据中鉴定对应的单氨基酸变异肽段以验证单核苷酸变异事件，可推动转化医学与临床研究。尽管通常会以严格的错误发现率（false discovery rate, FDR）控制从质谱数据中鉴定变异肽段，但FDR控制仍无法消除由多种问题引发的可疑结果，因此需要开展事后核查以剔除此类结果。然而，目前仍缺乏针对鉴定结果的全面事后核查方案。为此，我们提出一个涵盖三个自下而上层级的框架：肽段-谱匹配（peptide–spectrum match）、肽段及变异事件层级，该框架包含从质谱视角出发的11项严谨核查维度，以进一步确认变异事件的可靠性。作为概念验证并展示其可行性，我们针对某HEK293细胞系数据集所鉴定的变异肽段开展了11项核查：该数据集采用多种数据库搜索策略，以最大化鉴定出FDR<1%的变异肽段-谱匹配结果用于后续核查。结果表明，仅依靠FDR标准不足以验证所鉴定的变异肽段，而这11项事后核查可揭示鸟枪法蛋白质组学实验中检测到的低置信度变异事件。因此，我们建议在蛋白质基因组学研究中，基于所提出的框架对已鉴定的变异事件开展事后核查是十分必要的。