Thermo Fisher Scientific (Bremen) , 28199 Bremen, Germany.
J Am Chem Soc. 2016 Aug 31;138(34):10860-8. doi: 10.1021/jacs.6b05147. Epub 2016 Aug 16.
Tandem mass spectrometry can provide structural information on intact protein assemblies, generating mass fingerprints indicative of the stoichiometry and quaternary arrangement of the subunits. However, in such experiments, collision-induced dissociation yields restricted information due to simultaneous subunit unfolding, charge rearrangement, and subsequent ejection of a highly charged unfolded single subunit. Alternative fragmentation strategies can potentially overcome this and supply a deeper level of structural detail. Here, we implemented ultraviolet photodissociation (UVPD) on an Orbitrap mass spectrometer optimized for native MS and benchmark its performance to HCD fragmentation using various protein oligomers. We investigated dimeric β-lactoglobulin, dimeric superoxide dismutase, dimeric and tetrameric concanavalin A, and heptameric GroES and Gp31; ranging in molecular weight from 32 to 102 kDa. We find that, for the investigated systems, UVPD produces more symmetric charge partitioning than HCD. While HCD spectra show sporadic fragmentation over the full protein backbone sequence of the subunits with a bias toward fragmenting labile bonds, UVPD spectra provided higher sequence coverage. Taken together, we conclude that UVPD is a strong addition to the toolbox of fragmentation methods for top-down proteomics experiments, especially for native protein assemblies.
串联质谱可以提供完整蛋白质组装体的结构信息，产生质量指纹，指示亚基的化学计量和四级排列。然而，在这样的实验中，由于同时发生亚基展开、电荷重排以及随后高度展开的单个亚基的逐出，碰撞诱导解离会产生有限的信息。替代的碎片化策略可能会克服这一点，并提供更深入的结构细节。在这里，我们在优化用于天然 MS 的 Orbitrap 质谱仪上实施了紫外光解（UVPD），并使用各种蛋白质寡聚体对其性能与 HCD 碎片化进行了基准测试。我们研究了二聚体β-乳球蛋白、二聚体超氧化物歧化酶、二聚体和四聚体伴刀豆球蛋白 A 以及七聚体 GroES 和 Gp31；分子量范围从 32 到 102 kDa。我们发现，对于所研究的系统，UVPD 产生的电荷分布比 HCD 更对称。虽然 HCD 光谱显示亚基的整个蛋白质骨架序列中有零星的碎片化，并且偏向于断裂不稳定的键，但 UVPD 光谱提供了更高的序列覆盖率。总的来说，我们得出结论，UVPD 是一种强大的碎片化方法，可用于自上而下的蛋白质组学实验，特别是用于天然蛋白质组装体。
