Division of Chemistry I, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm 17177, Sweden.
Chemical Proteomics Unit, Science for Life Laboratory (SciLifeLab), Stockholm 17165, Sweden.
Anal Chem. 2024 Aug 20;96(33):13533-13541. doi: 10.1021/acs.analchem.4c02051. Epub 2024 Aug 7.
Here, we present a high-throughput virtual top-down proteomics approach that restores the molecular weight (MW) information in shotgun proteomics and demonstrates its utility in studying proteolytic events in programmed cell death. With gel-assisted proteome position integral shift (GAPPIS), we quantified over 7000 proteins in staurosporine-induced apoptotic HeLa cells and identified 84 proteins exhibiting in a statistically significant manner at least two of the following features: (i) a negative MW shift; (ii) an elevated ratio in a pair of a semitryptic and tryptic peptide, (iii) a negative shift in the standard deviation of MW estimated for different peptides, and (iv) a negative shift in skewness of the same data. Of these proteins, 58 molecules were previously unreported caspase 3 substrates. Further analysis identified the preferred cleavage sites consistent with the known caspase cleavages after the DXXD motif. As a powerful tool for high-throughput MW analysis simultaneously with the conventional expression analysis, the GAPPIS assay can prove useful in studying a broad range of biological processes involving proteolytic events.
在这里，我们提出了一种高通量的虚拟自上而下的蛋白质组学方法，该方法恢复了 shotgun 蛋白质组学中的分子量 (MW) 信息，并证明了其在研究程序性细胞死亡中的蛋白水解事件中的实用性。通过凝胶辅助的蛋白质组位置积分位移 (GAPPIS)，我们定量了 staurosporine 诱导的凋亡 HeLa 细胞中的超过 7000 种蛋白质，并鉴定了 84 种以统计学上显著的方式表现出至少以下两个特征的蛋白质：(i)MW 负向位移；(ii)一对半肽和肽的比值升高；(iii)MW 标准差的负向位移，以及 (iv)相同数据的偏度的负向位移。在这些蛋白质中，有 58 种分子是以前未报道过的 caspase 3 底物。进一步的分析确定了与 DXXD 基序后的已知半胱天冬酶切割一致的优先切割位点。作为一种与传统表达分析同时进行高通量 MW 分析的强大工具，GAPPIS 测定法可用于研究涉及蛋白水解事件的广泛的生物学过程。
