Fig. 3: Workflow for formaldehyde pre-stabilization followed by in situ XL-MS. | Nature Communications

Fig. 3: Workflow for formaldehyde pre-stabilization followed by in situ XL-MS.

From: Cell fixation improves performance of in situ crosslinking mass spectrometry while preserving cellular ultrastructure

Fig. 3

Cells are fixed with 4% formaldehyde for 10 minutes. After fixation, excess formaldehyde is washed away prior to the introduction of the crosslinker. After secondary crosslinking, cells are collected, lysed, and formaldehyde linkages are reversed by boiling. Extracted protein is then cleaned up via a single-pot, solid-phase-enhanced sample-preparation (SP3)45 protocol and digested overnight with trypsin. Peptides then undergo high-pH fractionation for LC-MS/MS data acquisition. Data were then processed using pLink 217. Created with Biorender.com.

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