Stability assessment of the human proteotypic peptides for proteomics quantification

Mass spectrometry coupled to liquid chromatography is one of the most powerful technologies for proteome quantification in a variety of biomedical samples, including cells, tissues and liquid biopsies. In peptide-centric workflows protein mixtures proteins are enzymatically digested to peptides prior their analysis. Experimental spectra are used for peptide identification, and extracted peptide areas, peak heights, or spectral counts are used to infer protein quantities using different heuristics. However, proteome-wide quantification studies rarely identify all potential peptides for any given protein while targeted proteomics experiments focus on a set of peptides for the proteins of interest. Consequently, proteomics methods rely on the use of a limited subset of all possible peptides as proxies for protein quantitation, which are specified either prior or after data acquisition. Several studies have evaluated the quantitative response of tryptic peptides and have described rules to guide peptide selection for protein quantification. However, beyond the effect of tryptic digestion and missed cleavages, short and mid-term peptide stability can also have a profound effect on the measurement reproducibility, specially when analysing proteomics samples from large cohorts of patients that expand for several days or weeks of measurement. In this project we assessed the stability of more than 100,000 peptides from the proteotypic peptide set of the Proteome Tools collection, and we used the obtained experimental data to train and validate a new peptide stability predictor for proteomics quantification.

液相色谱-质谱联用（liquid chromatography-mass spectrometry, LC-MS）技术是当前用于对细胞、组织与液体活检等多种生物医学样本开展蛋白质组定量的最强力技术之一。在以肽为中心的工作流程中，蛋白质混合物需先经酶解为肽段，再进行后续分析。实验质谱图可用于肽段鉴定，而通过提取肽段峰面积、峰高或谱图计数等数据，可借助多种启发式方法推断蛋白质的定量水平。
然而，全蛋白质组定量研究极少能鉴定出任一目标蛋白质的全部潜在肽段；而靶向蛋白质组学实验则仅针对目标蛋白质的一组特定肽段展开研究。因此，蛋白质组学方法通常仅选取全部潜在肽段中的有限子集作为蛋白质定量的替代标志物，这些肽段可在数据采集前或采集后预先指定。
已有多项研究评估了胰蛋白酶肽的定量响应特性，并总结出指导蛋白质定量用肽段选择的相关规则。但除胰蛋白酶酶解效率与漏切位点的影响外，肽段的中短期稳定性同样会对测量重现性产生显著影响，尤其是在分析来自大型患者队列的样本时——这类样本的整体检测周期往往长达数天乃至数周。
本项目针对Proteome Tools collection中的蛋白质组专属肽段（proteotypic peptide）集内的10万余条肽段开展了稳定性评估，并基于获取的实验数据训练并验证了一款全新的、用于蛋白质组定量的肽段稳定性预测器。