In proteogenomic studies, many genome-annotated events, for example, single amino acid variation (SAAV) and short INDEL, are often unobserved in shotgun proteomics. Therefore, we propose an analysis pipeline called LeTE-fusion (Le, peptide length; T, theoretical values; E, experimental data) to first investigate whether peptides with certain lengths are observed more often in mass spectrometry (MS)-based proteomics, which may hinder peptide identification causing difficulty in detecting genome-annotated events. By applying LeTE-fusion on different MS-based proteome data sets, we found peptides within 7–20 amino acids are more frequently identified, possibly attributed to MS-related factors instead of proteases. We then further extended the usage of LeTE-fusion on four variant-containing-sequence data sets (SAAV-only) with various sample complexity up to the whole human proteome scale, which yields theoretically ∼70% variants observable in an ideal shotgun proteomics. However, only ∼40% of variants might be detectable in real shotgun proteomic experiments when LeTE-fusion utilizes the experimentally observed variant-site-containing wild-type peptides in PeptideAtlas to estimate the expected observable coverage of variants. Finally, we conducted a case study on HEK293 cell line with variants reported at genomic level that were also identified in shotgun proteomics to demonstrate the efficacy of LeTE-fusion on estimating expected observable coverage of variants. To the best of our knowledge, this is the first study to systematically investigate the detection limits of genome-annotated events via shotgun proteomics using such analysis pipeline.

中文翻译：

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通过LeTE-Fusion Analysis Pipeline评估在Shot弹枪蛋白质组学中检测变异的可能性

在蛋白质组学研究中，通常在shot弹枪蛋白质组学中未观察到许多带有基因组注释的事件，例如单氨基酸变异（SAAV）和短INDEL。因此，我们提出了一个称为LeTE-fusion（Le，肽段长度； T，理论值； E，实验数据）的分析管道，以首先研究在基于质谱（MS）的蛋白质组学中是否更经常观察到某些长度的肽段，可能会阻碍多肽鉴定，从而导致难以检测基因组注释事件。通过将LeTE-fusion应用于不同的基于MS的蛋白质组数据集，我们发现7-20个氨基酸内的肽被更频繁地鉴定，可能归因于与MS相关的因素而不是蛋白酶。然后，我们进一步将LeTE-fusion在具有各种样本复杂性的四个包含变体的序列数据集（仅SAAV）上的使用范围扩展到整个人类蛋白质组规模，从理论上讲，在理想的shot弹枪蛋白质组学中可观察到约70％的变体。但是，当LeTE-fusion利用PeptideAtlas中实验观察到的含有变体位点的野生型肽来估计变体的预期可观察范围时，在真正的shot弹枪蛋白质组学实验中仅可检测到约40％的变体。最后，我们对HEK293细胞系进行了案例研究，其基因组水平报道了变异体，这些变异体在shot弹枪蛋白质组学中也得到了证实，以证明LeTE-fusion在估计变异体预期可观察性方面的功效。据我们所知，