Native mass spectrometry (MS) continues to enjoy growing popularity as a means of providing a wealth of information on noncovalent biopolymer assemblies ranging from composition and binding stoichiometry to characterization of the topology of these assemblies. The latter frequently relies on supplementing MS measurements with limited fragmentation of the noncovalent complexes in the gas phase to identify the pairs of neighboring subunits. While this approach has met with much success in the past two decades, its implementation remains difficult (and the success record relatively modest) within one class of noncovalent assemblies: protein complexes in which at least one binding partner has multiple subunits cross-linked by disulfide bonds. We approach this problem by inducing chemical reduction of disulfide bonds under nondenaturing conditions in solution followed by native MS analysis with online buffer exchange to remove unconsumed reagents that are incompatible with the electrospray ionization process. While this approach works well with systems comprised of thiol-linked subunits that remain stable upon reduction of the disulfide bridges (such as immunoglobulins), chemical reduction frequently gives rise to species that are unstable (prone to aggregation). This problem is circumvented by taking advantage of the recently introduced cross-path reactive chromatography platform (XPRC), which allows the disulfide reduction to be carried out in-line, thereby minimizing the loss of metastable protein subunits and their noncovalent complexes with the binding partners prior to MS analysis. The feasibility of this approach is demonstrated using hemoglobin complexes with haptoglobin 1-1, a glycoprotein consisting of four polypeptide chains cross-linked by disulfide bonds.

中文翻译：

---

通过在线二硫键还原和天然 MS 分析探索多亚基蛋白质复合物的结构


天然质谱 (MS) 作为提供非共价生物聚合物组装体丰富信息的一种手段，其范围从组成和结合化学计量到这些组装体的拓扑表征，继续受到越来越多的欢迎。后者经常依靠气相中非共价复合物的有限碎片来补充 MS 测量，以识别相邻亚基对。虽然这种方法在过去二十年中取得了很大成功，但在一类非共价组装中实施仍然很困难（并且成功记录相对有限）：其中至少一个结合配偶体具有通过二硫键交联的多个亚基的蛋白质复合物债券。我们通过在溶液中的非变性条件下诱导二硫键的化学还原来解决这个问题，然后通过在线缓冲液交换进行本机 MS 分析，以去除与电喷雾电离过程不相容的未消耗的试剂。虽然这种方法适用于由硫醇连接的亚基组成的系统，这些亚基在二硫键还原后保持稳定（例如免疫球蛋白），但化学还原经常会产生不稳定的物质（易于聚集）。通过利用最近引入的交叉路径反应层析平台 (XPRC) 可以避免这个问题，该平台允许在线进行二硫键还原，从而最大限度地减少亚稳蛋白亚基及其与结合伴侣的非共价复合物的损失MS 分析之前。使用血红蛋白与触珠蛋白 1-1 的复合物证明了该方法的可行性，触珠蛋白 1-1 是一种由四条通过二硫键交联的多肽链组成的糖蛋白。