Michael Andrew R M, Amaral Bruno C, Ball Kallie L, Eiriksson Kristen H, Schriemer David C
Department of Biochemistry and Molecular Biology, University of Calgary, Calgary, Alberta, T2N-4N1, Canada.
Department of Chemistry, University of Calgary, Calgary, Alberta, T2N-4N1, Canada.
Nat Commun. 2024 Oct 2;15(1):8537. doi: 10.1038/s41467-024-52844-y.
Crosslinking mass spectrometry (XL-MS) has the potential to map the interactome of the cell with high resolution and depth of coverage. However, current in vivo XL-MS methods are hampered by crosslinkers that demonstrate low cell permeability and require long reaction times. Consequently, interactome sampling is not high and long incubation times can distort the cell, bringing into question the validity any protein interactions identified by the method. We address these issues with a fast formaldehyde-based fixation method applied prior to the introduction of secondary crosslinkers. Using human A549 cells and a range of reagents, we show that 4% formaldehyde fixation with membrane permeabilization preserves cellular ultrastructure and simultaneously improves reaction conditions for in situ XL-MS. Protein labeling yields can be increased even for nominally membrane-permeable reagents, and surprisingly, high-concentration formaldehyde does not compete with conventional amine-reactive crosslinking reagents. Prefixation with permeabilization uncouples cellular dynamics from crosslinker dynamics, enhancing control over crosslinking yield and permitting the use of any chemical crosslinker.
交联质谱法（XL-MS）有潜力以高分辨率和深度覆盖来绘制细胞的相互作用组图谱。然而，当前的体内XL-MS方法受到交联剂的限制，这些交联剂细胞通透性低且需要较长的反应时间。因此，相互作用组采样率不高，长时间孵育会使细胞变形，从而使该方法鉴定出的任何蛋白质相互作用的有效性受到质疑。我们通过在引入二级交联剂之前应用基于甲醛的快速固定方法来解决这些问题。使用人A549细胞和一系列试剂，我们表明4%甲醛固定并进行膜通透处理可保留细胞超微结构，同时改善原位XL-MS的反应条件。即使对于名义上可通透膜的试剂，蛋白质标记产率也能提高，而且令人惊讶的是，高浓度甲醛不会与传统的胺反应性交联剂竞争。固定并通透处理的预固定步骤将细胞动力学与交联剂动力学解耦，增强了对交联产率的控制，并允许使用任何化学交联剂。
