Thermo Fisher Scientific , 28199 Bremen, Germany.
Anal Chem. 2013 Dec 3;85(23):11163-73. doi: 10.1021/ac4029328. Epub 2013 Nov 20.
Native mass spectrometry (MS) is becoming an important integral part of structural proteomics and system biology research. The approach holds great promise for elucidating higher levels of protein structure: from primary to quaternary. This requires the most efficient use of tandem MS, which is the cornerstone of MS-based approaches. In this work, we advance a two-step fragmentation approach, or (pseudo)-MS(3), from native protein complexes to a set of constituent fragment ions. Using an efficient desolvation approach and quadrupole selection in the extended mass-to-charge (m/z) range, we have accomplished sequential dissociation of large protein complexes, such as phosporylase B (194 kDa), pyruvate kinase (232 kDa), and GroEL (801 kDa), to highly charged monomers which were then dissociated to a set of multiply charged fragmentation products. Fragment ion signals were acquired with a high resolution, high mass accuracy Orbitrap instrument that enabled highly confident identifications of the precursor monomer subunits. The developed approach is expected to enable characterization of stoichiometry and composition of endogenous native protein complexes at an unprecedented level of detail.
天然质谱（MS）正成为结构蛋白质组学和系统生物学研究的一个重要组成部分。该方法有望阐明更高层次的蛋白质结构：从一级到四级。这需要最有效地利用串联 MS，这是基于 MS 的方法的基石。在这项工作中，我们提出了一种两步碎裂方法，或（伪）MS(3)，从天然蛋白质复合物到一组组成的片段离子。使用高效的去溶剂化方法和在扩展质荷比（m/z）范围内的四极选择，我们已经成功地对大蛋白质复合物进行了顺序解离，例如磷酸化酶 B（194 kDa）、丙酮酸激酶（232 kDa）和 GroEL（801 kDa），将其解离成一组带有多个电荷的片段产物。利用高分辨率、高质量精度的 Orbitrap 仪器获得了片段离子信号，这使得对前体单体亚基进行高度置信的鉴定成为可能。所开发的方法有望以前所未有的详细程度来表征内源性天然蛋白质复合物的化学计量和组成。
