Skinner Owen S, Haverland Nicole A, Fornelli Luca, Melani Rafael D, Do Vale Luis H F, Seckler Henrique S, Doubleday Peter F, Schachner Luis F, Srzentić Kristina, Kelleher Neil L, Compton Philip D
Department of Chemistry, Northwestern University, Evanston, Illinois, USA.
Proteomics Center of Excellence, Northwestern University, Evanston, Illinois, USA.
Nat Chem Biol. 2018 Jan;14(1):36-41. doi: 10.1038/nchembio.2515. Epub 2017 Nov 13.
Protein complexes exhibit great diversity in protein membership, post-translational modifications and noncovalent cofactors, enabling them to function as the actuators of many important biological processes. The exposition of these molecular features using current methods lacks either throughput or molecular specificity, ultimately limiting the use of protein complexes as direct analytical targets in a wide range of applications. Here, we apply native proteomics, enabled by a multistage tandem MS approach, to characterize 125 intact endogenous complexes and 217 distinct proteoforms derived from mouse heart and human cancer cell lines in discovery mode. The native conditions preserved soluble protein-protein interactions, high-stoichiometry noncovalent cofactors, covalent modifications to cysteines, and, remarkably, superoxide ligands bound to the metal cofactor of superoxide dismutase 2. These data enable precise compositional analysis of protein complexes as they exist in the cell and demonstrate a new approach that uses MS as a bridge to structural biology.
蛋白质复合物在蛋白质组成、翻译后修饰和非共价辅因子方面表现出极大的多样性，使其能够作为许多重要生物过程的驱动者发挥作用。使用当前方法揭示这些分子特征时，要么缺乏通量，要么缺乏分子特异性，最终限制了蛋白质复合物在广泛应用中作为直接分析靶点的使用。在这里，我们应用由多阶段串联质谱方法实现的天然蛋白质组学，以发现模式表征来自小鼠心脏和人类癌细胞系的125种完整内源性复合物和217种不同的蛋白质形式。天然条件保留了可溶性蛋白质-蛋白质相互作用、高化学计量的非共价辅因子、对半胱氨酸的共价修饰，并且值得注意的是，保留了与超氧化物歧化酶2的金属辅因子结合的超氧化物配体。这些数据能够对细胞中存在的蛋白质复合物进行精确的组成分析，并展示了一种将质谱用作通往结构生物学桥梁的新方法。
