Efficient and robust proteome-wide approaches for cross-linking mass spectrometry
Efficient and robust proteome-wide approaches for cross-linking mass spectrometry
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DOI：
10.1038/s41596-018-0074-x
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发表时间：
2018-12-01
期刊：
影响因子：
14.8
通讯作者：
Scheltema, Richard A.
中科院分区：
文献类型：
--
作者：
Klykov, Oleg;Steigenberger, Barbara;Scheltema, Richard A.
交联质谱（XL-MS）由于其在复杂蛋白质混合物中以无偏方式研究蛋白质-蛋白质相互作用（PPI）的潜力而受到相当大的关注。最近的发展已经能够从一个实验中检测数千个PPI。XL-MS的一个独特的优势，与其他方法相比，用于确定PPI，是它提供了直接的空间信息检测到的相互作用。这是通过使用双功能交联分子来实现的，所述双功能交联分子用共价键将两个氨基酸紧密连接。在蛋白水解消化后，这导致两个新连接的肽，这是可识别的MS。XL-MS已经收到了所需的提升，以处理更复杂的样品与交联化学与MS可裂解或基于交联剂和更快，更灵敏，更通用的MS平台的最新进展。该方案提供了我们的全蛋白质组天然蛋白质制备，然后使用气相可裂解的交联试剂二琥珀酰亚胺亚砜（DSSO）交联的优化条件的详细描述。交联后，我们展示了广泛的样品分馏和大大简化的数据分析与XlinkX在蛋白质组发现，以及随后的蛋白质结构研究与DisVis和HADDOCK。该方案产生高置信度的数据，可以在10天内进行，包括结构调查。
Cross-linking mass spectrometry (XL-MS) has received considerable interest, owing to its potential to investigate protein-protein interactions (PPIs) in an unbiased fashion in complex protein mixtures. Recent developments have enabled the detection of thousands of PPIs from a single experiment. A unique strength of XL-MS, in comparison with other methods for determining PPIs, is that it provides direct spatial information for the detected interactions. This is accomplished by the use of bifunctional cross-linking molecules that link two amino acids in close proximity with a covalent bond. Upon proteolytic digestion, this results in two newly linked peptides, which are identifiable by MS. XL-MS has received the required boost to tackle more-complex samples with recent advances in cross-linking chemistry with MS-cleavable or reporter-based cross-linkers and faster, more sensitive and more versatile MS platforms. This protocol provides a detailed description of our optimized conditions for a full-proteome native protein preparation followed by cross-linking using the gas-phase cleavable cross-linking reagent disuccinimidyl sulfoxide (DSSO). Following cross-linking, we demonstrate extensive sample fractionation and substantially simplified data analysis with XlinkX in Proteome Discoverer, as well as subsequent protein structure investigations with DisVis and HADDOCK. This protocol produces data of high confidence and can be performed within similar to 10 d, including structural investigations.