Mass spectrometry reveals the chemistry of formaldehyde cross-linking in structured proteins

Whole-cell cross-linking coupled to mass spectrometry is one of the few tools that can probe protein–protein interactions in intact cells. A very attractive reagent for this purpose is formaldehyde, a small molecule which is known to rapidly penetrate into all cellular compartments and to preserve the protein structure. In light of these benefits, it is surprising that identification of formaldehyde cross-links by mass spectrometry has so far been unsuccessful. Here we report mass spectrometry data that reveal formaldehyde cross-links to be the dimerization product of two formaldehyde-induced amino acid modifications. By integrating the revised mechanism into a customized search algorithm, we identify hundreds of cross-links from in situ formaldehyde fixation of human cells. Interestingly, many of the cross-links could not be mapped onto known atomic structures, and thus provide new structural insights. These findings enhance the use of formaldehyde cross-linking and mass spectrometry for structural studies. Formaldehyde (FA) is a popular cross-linking reagent, but applying it for cross-linking mass spectrometry (XLMS) has been largely unsuccessful. Here, the authors show that cross-links in structured proteins are the product of two FA molecules and identify hundreds of FA cross-links by XLMS in vitro and in situ.