Cell fixation improves performance of in situ crosslinking mass spectrometry while preserving cellular ultrastructure.
Journal
Nature communications
ISSN: 2041-1723
Titre abrégé: Nat Commun
Pays: England
ID NLM: 101528555
Informations de publication
Date de publication:
02 Oct 2024
02 Oct 2024
Historique:
received:
15
04
2024
accepted:
24
09
2024
medline:
3
10
2024
pubmed:
3
10
2024
entrez:
2
10
2024
Statut:
epublish
Résumé
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.
Identifiants
pubmed: 39358380
doi: 10.1038/s41467-024-52844-y
pii: 10.1038/s41467-024-52844-y
doi:
Substances chimiques
Formaldehyde
1HG84L3525
Cross-Linking Reagents
0
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Pagination
8537Subventions
Organisme : Canadian 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)
ID : RGPIN 2017-04879
Informations de copyright
© 2024. The Author(s).
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