狙击法质谱学对于生物医学研究中的蛋白质鉴定和定量至关重要，但蛋白质亚型的特征化却具有挑战性，原因是由于蛋白质之间共享的肽段数量庞大，这妨碍了我们对蛋白质亚型调控及其在正常生物学和疾病生物学中的作用的理解。我们通过基于计算机模拟和实验数据系统评估了狙击法质谱学在蛋白质亚型特征化中面临的挑战和机遇，然后提出了一种基于图论的方法名为SEPepQuant，以最大化亚型的特征化。利用已发表的一项诱导多能干细胞研究和两项人类肝细胞癌研究的数据，我们证明了SEPepQuant在解决现有方法的关键限制方面的能力，提供了更全面的亚型水平特征化，鉴定了数百个亚型水平调控事件，并促进了跨研究的简化比较。我们的分析提供了坚实的证据，支持蛋白质亚型调控在正常和疾病过程中普遍存在的作用，并且SEPepQuant在生物学和转化研究中具有广泛的应用。© 2023. Springer Nature Limited.

Shotgun proteomics is essential for protein identification and quantification in biomedical research, but protein isoform characterization is challenging due to the extensive number of peptides shared across proteins, hindering our understanding of protein isoform regulation and their roles in normal and disease biology. We systematically assess the challenge and opportunities of shotgun proteomics-based protein isoform characterization using in silico and experimental data, and then present SEPepQuant, a graph theory-based approach to maximize isoform characterization. Using published data from one induced pluripotent stem cell study and two human hepatocellular carcinoma studies, we demonstrate the ability of SEPepQuant in addressing the key limitations of existing methods, providing more comprehensive isoform-level characterization, identifying hundreds of isoform-level regulation events, and facilitating streamlined cross-study comparisons. Our analysis provides solid evidence to support a widespread role of protein isoform regulation in normal and disease processes, and SEPepQuant has broad applications to biological and translational research.© 2023. Springer Nature Limited.