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.
Bibliographical noteFunding Information:
This work was supported by the Israel Science Foundation grant number 1768/15. M.S. was supported by the FSHD Global Foundation grant number 41. We thank Uri Raviv for his help and advice in various stages of this work. We thank David Morgenstern and Dina Schneidman for critical reading of the manuscript.
© 2020, The Author(s).