Institute of Chemistry, University of Campinas, CP 6154, 13083-970 Campinas, São Paulo, Brazil.
Anal Chem. 2010 Feb 1;82(3):909-16. doi: 10.1021/ac902051q.
Chemical cross-linking coupled to mass spectrometry analysis has become a realistic alternative to the study of proteins structure and interactions, especially when these systems are not amenable to high-resolution techniques such as protein crystallography or nuclear magnetic resonance. One of the main bottlenecks of this approach relies on the detection of cross-linked peptides, as they are usually present in substoichiometric amounts in complex samples. It was shown that one of the main fragmentation pathways of disuccinimidyl suberate (DSS) cross-linked peptides yields diagnostic ions, whose structure is composed of a rearranged lysine side chain and the spacer arm of the linker. In this report, we demonstrate the feasibility of detecting these modified peptides based on a precursor ion scan in a quadrupole time-of-flight (Q-TOF) instrument. It was shown that the fragmentation of nonmodified tryptic peptides hardly generates ions with the same nominal mass of the diagnostic ions, making the precursor ion scan very specific to N-hydroxysuccinimide (NHS)-based cross-linkers. Moreover, the experimental setup is the same as in the case of a regular cross-linking experiment, not demanding any additional experimental steps that would increase sample handling. The results obtained with protein samples allowed us to propose an algorithm that could be implemented in a software to process data from cross-linking experiments in an automated and high-throughput way.
化学交联结合质谱分析已成为研究蛋白质结构和相互作用的一种可行方法，尤其是当这些系统不适合高分辨率技术（如蛋白质晶体学或核磁共振）时。这种方法的主要瓶颈之一在于交联肽的检测，因为它们在复杂样品中通常以亚化学计量存在。已经表明，琥珀酰亚胺二琥珀酸酯（DSS）交联肽的主要碎裂途径之一会产生诊断离子，其结构由重排的赖氨酸侧链和连接子的间隔臂组成。在本报告中，我们展示了基于四极杆飞行时间（Q-TOF）仪器中的前体离子扫描来检测这些修饰肽的可行性。结果表明，非修饰胰蛋白酶肽的碎裂几乎不会产生与诊断离子相同名义质量的离子，使得前体离子扫描对基于 N-羟基琥珀酰亚胺（NHS）的交联剂非常特异。此外，实验设置与常规交联实验相同，不需要任何额外的实验步骤来增加样品处理。用蛋白质样品获得的结果允许我们提出一种算法，该算法可以在软件中实现，以自动化和高通量的方式处理交联实验的数据。
