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首页> 外文期刊>Journal of mass spectrometry: JMS >Combining a NHS ester and glutaraldehyde improves crosslinking prior to MALDI MS analysis of intact protein complexes
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Combining a NHS ester and glutaraldehyde improves crosslinking prior to MALDI MS analysis of intact protein complexes

机译：在完整蛋白质复合物的MALDI MS分析之前，将NHS酯和戊二醛结合使用可改善交联

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摘要

Protein complexes play pivotal roles in cellular life. Nevertheless, their characterization remains a substantial challenge. Mass spectrometry (MS) is an emerging tool to study protein assemblies, and electrospray ionization (ESI) is often used because it preserves non-covalent interactions. Matrix-assisted laser desorption/ionization (MALDI) represents an important alternative to ESI because it is more tolerant to salts and detergents (e.g. necessary in the case of membrane complex analyses). Prior to MALDI-MS, the subunits should be crosslinked (XLed). Moreover, crosslinking (XLing) is useful when constraint distances are determined to obtain low-resolution structural information. Here we report a novel XLing approach to study protein complexes with MALDI-MS. We investigated two tetramers (i.e. alcohol dehydrogenase and aldolase) larger than 140kDa at two pH values (7.2 and 8.0). We tested two different crosslinkers (XLers) (i.e. BS3 and glutaraldehyde), used separately or in combination. We utilized gentle agitation and ultracentrifugation. Our data shows that the pH influenced the XLing when using a single XLer. Combining two XLers was demonstrated to be more efficient than using a reagent alone. In particular, the combination determined a higher degree of XLing and lower mass shift. This could suggest a ranking in target amino acid availability. First residues at specific distances are linked by BS3, then glutaraldehyde binds residues that are still available at larger distances. Ultracentrifugation and gentle agitation both provide similar degrees of XLing, but the former method determined a lower mass increment resulting from redundant XLing. To conclude, we present an efficient dual XLing approach for determining mass and stoichiometry of protein assemblies. Copyright (c) 2015 John Wiley & Sons, Ltd.
机译：蛋白质复合物在细胞生命中起关键作用。然而，它们的表征仍然是一个巨大的挑战。质谱（MS）是研究蛋白质组装的新兴工具，由于其保留了非共价相互作用，因此经常使用电喷雾电离（ESI）。基质辅助激光解吸/电离（MALDI）代表了ESI的重要替代方法，因为它更耐盐和去污剂（例如，在膜复合物分析中是必需的）。在MALDI-MS之前，应将亚基交联（XLed）。此外，当确定约束距离以获得低分辨率结构信息时，交联（XLing）很有用。在这里，我们报告了一种新型的XLing方法，用于使用MALDI-MS研究蛋白质复合物。我们研究了在两个pH值（7.2和8.0）下大于140kDa的两个四聚体（即醇脱氢酶和醛缩酶）。我们测试了两种不同的交联剂（XLers）（即BS3和戊二醛），分别使用或组合使用。我们利用了温和的搅拌和超速离心。我们的数据表明，使用单个XLer时，pH影响XLing。证明结合两个XLer比单独使用一种试剂更有效。特别地，该组合确定较高的XLing度和较低的质量偏移。这可能表明目标氨基酸的可用性排名。特定距离的第一个残基通过BS3连接，然后戊二醛结合仍在较大距离处可用的残基。超速离心和轻度搅动都提供相似的XLing程度，但是前一种方法确定了由于多余的XLing而导致的质量增加较低。总而言之，我们提出了一种有效的双重XLing方法，用于确定蛋白质装配的质量和化学计量。版权所有（c）2015 John Wiley＆Sons，Ltd.

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