biostudies-literatureMichael ARMCanadian Network for Research and Innovation in Machining Technology, Natural Sciences and Engineering Research Council of Canada (NSERC Canadian Network for Research and Innovation in Machining Technology)8537https://www.ebi.ac.uk/biostudies/studies/S-EPMC11447256biostudies-literatureUnknown15(1)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 situNature communicationsCell fixation improves performance of in situ crosslinking mass spectrometry while preserving cellular ultrastructure.PMC11447256RGPIN 2017-04879Michael ARMAmaral BCEiriksson KHBall KLSchriemer DCfalseCell fixation improves performance of in situ crosslinking mass spectrometry while preserving cellular ultrastructure.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 situ2024-01-01T00:00:00Z2024 Oct2026-06-01T07:31:38.334Z2025-04-04T01:37:16.384ZS-EPMC114472563935838010.1038/s41467-024-52844-y