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

Abstract

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.

Open Search Strategy for Inferring the Masses of Cross-Link Adducts on Proteins

“…This value is very close to 24.000 Da, which was recently shown to be the adduct mass of formaldehyde cross-links. 23 The absolute error in the formaldehyde case is 0.0054 Da, which corresponds to a relative error of 2.2 parts per million.…”

“…This value is very close to 12.000 Da, which is the well-known mass for the Schiff base modification induced by formaldehyde. 23 …”

“…We next tested in turn each of the three masses (32, 64, and 128 Da) in a dedicated search application for cross-links 23 ( Methods ). For the 32 and 128 Da masses, the application identified zero and seven cross-links above the score threshold, respectively.…”

“…This is similar to the fragmentation behavior of formaldehyde that also exhibits complete cleavage. 23 However, unlike formaldehyde, the glutaraldehyde adduct breaks in such a way that the entire 64 Da moiety is carried off on either of the peptides.…”

Targetedin situcross-linking mass spectrometry and integrative modeling reveal the architectures of Nsp1, Nsp2, and Nucleocapsid proteins from SARS-CoV-2

“…Because a link between two residues reports on their structural proximity, the list of identified links is a rich resource for modeling protein structures and interactions [8][9][10][11][12] . In situ CLMS had progressed significantly in recent years, with successful applications on isolated organelles [13][14][15][16][17] , bacteria 18,19 , human cells [20][21][22] , and heart tissue 23 .…”