Kolbowski Lars, Belsom Adam, Pérez-López Ana M, Ly Tony, Rappsilber Juri
Chair of Bioanalytics, Technische Universität Berlin, 10623 Berlin, Germany.
Wellcome Centre for Cell Biology, University of Edinburgh, Edinburgh EH9 3BF, U.K.
JACS Au. 2023 Aug 17;3(8):2123-2130. doi: 10.1021/jacsau.3c00199. eCollection 2023 Aug 28.
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 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. Data are available via ProteomeXchange with identifier PXD040267.
交联质谱法提供了有关蛋白质结构和相互作用的关键信息。可被质谱裂解的交联剂被视为分析复杂混合物的基石。然而，它们在与肽相似的条件下发生裂解，导致交联剂和肽的混合裂解图谱。这妨碍了选择单个肽段进行独立鉴定。在此，我们引入了利用紫外光解离（UVPD）的正交裂解方法，以增加交联剂相对于肽段的裂解。我们设计并合成了一种交联剂，它可以在商用质谱仪配置下于213 nm处裂解。在对交联裂解物的分析中，交联剂与肽段的碎片强度比从传统可裂解交联剂的近1增加到了UVPD可裂解交联剂的5。这大大提高了为MS3选择单个肽段的灵敏度，在采用改进的双峰检测算法时更是如此。数据可通过ProteomeXchange获得，标识符为PXD040267。
