Light-induced orthogonal fragmentation of crosslinked peptides

Crosslinking mass spectrometry provides pivotal information on the structure and interaction of proteins. MS-cleavable crosslinkers are regarded as a cornerstone for the analysis of complex mixtures. Yet they fragment under similar conditions as peptides, leading to mixed fragmentation spectra of the crosslinker and peptide. This hampers selecting individual peptides for their independent identification. Here we introduce orthogonal cleavage, using ultraviolet photodissociation (UVPD) to increase crosslinker over peptide fragmentation. We designed and synthesized a crosslinker that can be cleaved at 213 nm in a commercial mass spectrometer configuration. In an analysis of crosslinked E. coli lysate, the crosslinker-to-peptide fragment intensity ratio increases from nearly 1 for a conventionally cleavable crosslinker to 5 for the UVPD cleavable crosslinker. This largely increased the sensitivity of selecting the individual peptides for MS3, even more so with an improved doublet detection algorithm.

交联质谱（Crosslinking mass spectrometry）可提供蛋白质结构与相互作用的关键信息。质谱可裂解交联剂（MS-cleavable crosslinkers）被视为复杂混合物分析的核心基石。然而这类交联剂会在与肽段相近的条件下发生裂解，导致交联剂与肽段的裂解谱图相互混杂，进而阻碍了单条肽段独立鉴定的筛选流程。为此本研究提出正交裂解策略：借助紫外光解离（ultraviolet photodissociation, UVPD）技术，提升交联剂相较于肽段的裂解偏好性。我们设计并合成了一款可在商用质谱仪配置下，通过213 nm波长实现特异性裂解的交联剂。在对交联大肠杆菌（E. coli）裂解物的分析中，常规可裂解交联剂的交联剂-肽段片段强度比仅接近1，而本研究开发的UVPD裂解型交联剂则将该比值提升至5。这大幅提升了用于MS³（三级质谱）分析的单条肽段筛选灵敏度，搭配经过优化的双肽段检测算法时，灵敏度还可得到进一步增强。