Identification and sequence determination by mass spectrometry have become routine analyses for soluble proteins. Membrane proteins, however, remain challenging targets due to their hydrophobicity and poor annotation. In particular small membrane proteins often remain unnoticed as they are largely inaccessible to Bottom-Up proteomics. Recent advances in structural biology, though, have led to multiple membrane protein complex structures being determined at sufficiently high resolution to detect uncharacterized, small subunits. In this work we offer a guide for the mass spectrometric characterization of solvent extraction-based purifications of small membrane proteins isolated from protein complexes and cellular membranes. We first demonstrate our Top-Down MALDI-MS/MS approach on a Photosystem II preparation, analyzing target protein masses between 2.5 and 9 kDa with high accuracy and sensitivity. Then we apply our technique to purify and sequence the mycobacterial ATP synthase c subunit, the molecular target of the antibiotic drug bedaquiline. We show that our approach can be used to directly track and pinpoint single amino acid mutations that lead to antibiotic resistance in only 4 h. While not applicable as a high-throughput pipeline, our MALDI-MS/MS and ISD-based approach can identify and provide valuable sequence information on small membrane proteins, which are inaccessible to conventional Bottom-Up techniques. We show that our approach can be used to unambiguously identify single-point mutations leading to antibiotic resistance in mycobacteria.

中文翻译：

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使用 MALDI-MS/MS 对小膜蛋白进行自上而下的鉴定和序列分析

质谱鉴定和序列测定已成为可溶性蛋白质的常规分析。然而，由于其疏水性和较差的注释，膜蛋白仍然是具有挑战性的目标。特别是小膜蛋白通常不会被注意到，因为自下而上的蛋白质组学在很大程度上无法接近它们。然而，结构生物学的最新进展已经导致以足够高的分辨率确定多个膜蛋白复杂结构，以检测未表征的小亚基。在这项工作中，我们为从蛋白质复合物和细胞膜中分离的小膜蛋白的基于溶剂萃取的纯化的质谱表征提供了指南。我们首先在 Photosystem II 制备上展示了我们的自上而下 MALDI-MS/MS 方法，以高精度和灵敏度分析 2.5 和 9 kDa 之间的目标蛋白质质量。然后我们应用我们的技术对分枝杆菌 ATP 合酶进行纯化和测序c亚基，抗生素药物贝达喹啉的分子靶点。我们表明，我们的方法可用于在 4 小时内直接跟踪和查明导致抗生素耐药性的单个氨基酸突变。虽然不适用于高通量管道，但我们的 MALDI-MS/MS 和基于 ISD 的方法可以识别并提供有关小膜蛋白的有价值的序列信息，这些信息是传统自下而上技术无法获得的。我们表明，我们的方法可用于明确识别导致分枝杆菌抗生素耐药性的单点突变。