The human complement hetero-trimeric C8αβγ (C8) protein assembly (~ 150 kDa) is an important component of the membrane attack complex (MAC). C8 initiates membrane penetration and coordinates MAC pore formation. Here, we charted in detail the structural micro-heterogeneity within C8, purified from human plasma, combining high-resolution native mass spectrometry and (glyco)peptide-centric proteomics. The intact C8 proteoform profile revealed at least ~ 20 co-occurring MS signals. Additionally, we employed ion exchange chromatography to separate purified C8 into four distinct fractions. Their native MS analysis revealed even more detailed structural micro-heterogeneity on C8. Subsequent peptide-centric analysis, by proteolytic digestion of C8 and LC-MS/MS, provided site-specific quantitative profiles of different types of C8 glycosylation. Combining all this data provides a detailed specification of co-occurring C8 proteoforms, including experimental evidence on N-glycosylation, C-mannosylation, and O-glycosylation. In addition to the known N-glycosylation sites, two more N-glycosylation sites were detected on C8. Additionally, we elucidated the stoichiometry of all C-mannosylation sites in all the thrombospondin-like (TSP) domains of C8α and C8β. Lastly, our data contain the first experimental evidence of O-linked glycans located on C8γ. Albeit low abundant, these O-glycans are the first PTMs ever detected on this subunit. By placing the observed PTMs in structural models of free C8 and C8 embedded in the MAC, it may be speculated that some of the newly identified modifications may play a role in the MAC formation.

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人补体异源三聚体C8αβγ（C8）蛋白组装体（〜150 kDa）是膜攻击复合物（MAC）的重要组成部分。C8启动膜渗透并协调MAC孔的形成。在这里，我们详细绘制了从人类血浆中纯化的C8内的结构微异质性，并结合了高分辨率天然质谱和以（糖）肽为中心的蛋白质组学。完整的C8蛋白形谱显示至少〜20个同时出现的MS信号。此外，我们采用离子交换色谱法将纯化的C8分离为四个不同的馏分。他们的原生MS分析揭示了C8上更详细的结构微观异质性。随后的以肽为中心的分析，通过C8和LC-MS / MS的蛋白水解消化，提供了不同类型的C8糖基化的位点特异性定量分析。结合所有这些数据可提供共生C8蛋白形式的详细说明，包括有关N-糖基化，C-甘露糖基化和O-糖基化的实验证据。除了已知的N-糖基化位点外，在C8上还检测到另外两个N-糖基化位点。此外，我们阐明了C8α和C8β的所有血小板反应蛋白样（TSP）域中所有C-甘露糖基化位点的化学计量。最后，我们的数据包含位于C8γ上的O-连接聚糖的第一个实验证据。尽管O-聚糖含量低，但它们是该亚基上首次检测到的PTM。通过将观察到的PTM放置在MAC中嵌入的游离C8和C8的结构模型中，可以推测出一些新发现的修饰可能在MAC形成中起作用。

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