Department of Biochemistry and Structural Biology, Center for Molecular Protein Science, Lund University, SE-22100 Lund, Sweden.
Protein Sci. 2011 Oct;20(10):1682-91. doi: 10.1002/pro.699. Epub 2011 Aug 18.
The lysine-specific crosslinker 3,3'-dithiobis(sulfosuccinimidylpropionate) (DTSSP) is commonly used in the structural characterization of proteins by chemical crosslinking and mass spectrometry and we here describe an efficient two-step LC-MALDI-TOF/TOF procedure to detect crosslinked peptides. First MS data are acquired, and the properties of isotope-labeled DTSSP are used in data analysis to identify candidate crosslinks. MSMS data are then acquired for a restricted number of precursor ions per spot for final crosslink identification. We show that the thiol-catalyzed exchange between crosslinked peptides, which is due to the disulfide bond in DTSSP and known to possibly obscure data, can be precisely quantified using isotope-labeled DTSSP. Crosslinked peptides are recognized as 8 Da doublet peaks and a new isotopic peak with twice the intensity appears in the middle of the doublet as a consequence of the thiol-exchange. False-positive crosslinks, formed exclusively by thiol-exchange, yield a 1:2:1 isotope pattern, whereas true crosslinks, formed by two lysine residues within crosslinkable distance in the native protein structure, yield a 1:0:1 isotope pattern. Peaks with a 1:X:1 isotope pattern, where 0 < X < 2, can be trusted as true crosslinks, with a defined proportion of the signal [2X/(2 + X)] being noise from the thiol-exchange. The thiol-exchange was correlated with the protein cysteine content and was minimized by shortening the trypsin incubation time, and for two molecular chaperone proteins with known structure all crosslinks fitted well to the structure data. The thiol-exchange can thus be controlled and isotope-labeled DTSSP safely used to detect true crosslinks between lysine residues in proteins.
赖氨酸特异性交联剂 3,3'-二硫代双（磺基琥珀酰亚胺基丙酸酯）（DTSSP）常用于通过化学交联和质谱法对蛋白质进行结构表征，我们在这里描述了一种有效的两步 LC-MALDI-TOF/TOF 程序来检测交联肽。首先获取 MS 数据，并在数据分析中使用同位素标记的 DTSSP 的性质来识别候选交联。然后，针对每个斑点的有限数量的前体离子获取 MSMS 数据，以进行最终的交联识别。我们表明，由于 DTSSP 中的二硫键，交联肽之间的硫醇催化交换可能会掩盖数据，但是可以使用同位素标记的 DTSSP 进行精确定量。交联肽被识别为 8 Da 双峰峰，由于硫醇交换，在双峰中间出现了一个新的同位素峰，其强度是双峰的两倍。仅由硫醇交换形成的假阳性交联产生 1:2:1 的同位素模式，而由天然蛋白质结构中交联距离内的两个赖氨酸残基形成的真正交联产生 1:0:1 的同位素模式。具有 1:X:1 同位素模式的峰（其中 0 < X < 2）可以被认为是真正的交联，信号的[2X/(2 + X)]部分是来自硫醇交换的噪声。硫醇交换与蛋白质半胱氨酸含量相关，并通过缩短胰蛋白酶孵育时间来最小化，对于具有已知结构的两种分子伴侣蛋白，所有交联都很好地符合结构数据。因此，可以控制硫醇交换并安全地使用同位素标记的 DTSSP 来检测蛋白质中赖氨酸残基之间的真实交联。
