School of Chemistry and Physics, The University of Adelaide, Adelaide, SA, 5005, Australia.
J Am Soc Mass Spectrom. 2012 Aug;23(8):1364-75. doi: 10.1007/s13361-012-0407-x. Epub 2012 May 30.
Chemical cross-linking combined with mass spectrometry (MS) is an analytical tool used to elucidate the topologies of proteins and protein complexes. However, identification of the low abundance cross-linked peptides and modification sites amongst a large quantity of proteolytic fragments remains challenging. In this work, we present a strategy to identify cross-linked peptides by negative ion MS for the first time. This approach is based around the facile cleavages of disulfide bonds in the negative mode, and allows identification of cross-linked products based on their characteristic fragmentations. MS(3) analysis of the cross-linked peptides allows for their sequencing and identification, with residue specific location of cross-linking sites. We demonstrate the applicability of the commercially available cystine based cross-linking reagent dithiobis(succinimidyl) propionate (DSP) and identify cross-linked peptides from ubiquitin. In each instance, the characteristic fragmentation behavior of the cross-linked species is described. The data presented here indicate that this negative ion approach may be a useful tool to characterize the structures of proteins and protein complexes, and provides the basis for the development of high throughput negative ion MS chemical cross-linking strategies.
化学交联结合质谱（MS）是一种用于阐明蛋白质和蛋白质复合物拓扑结构的分析工具。然而，在大量蛋白水解片段中鉴定低丰度交联肽和修饰位点仍然具有挑战性。在这项工作中，我们首次提出了一种通过负离子 MS 鉴定交联肽的策略。该方法基于在负离子模式下二硫键的易断裂性，并允许根据其特征片段鉴定交联产物。交联肽的 MS(3)分析允许对其进行测序和鉴定，并确定交联位点的残基特异性位置。我们展示了市售的基于半胱氨酸的交联试剂二硫代双（琥珀酰亚胺基）丙酸酯（DSP）的适用性，并从泛素中鉴定出交联肽。在每种情况下，都描述了交联物种的特征碎裂行为。这里呈现的数据表明，这种负离子方法可能是一种有用的工具，可用于表征蛋白质和蛋白质复合物的结构，并为高通量负离子 MS 化学交联策略的发展提供了基础。
