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

Abstract

Formaldehyde is a widely used fixative in biology and medicine. The current mechanism of formaldehyde cross-linking of proteins is the formation of a methylene bridge that incorporates one carbon atom into the link. Here, we present mass spectrometry data that largely refute this mechanism. Instead, the data reveal that cross-linking of structured proteins mainly involves a reaction that incorporates two carbon atoms into the link. Under MS/MS fragmentation, the link cleaves symmetrically to yield previously unrecognized fragments carrying a modification of one carbon atom. If these characteristics are considered, then formaldehyde cross-linking is readily applicable to the structural approach of cross-linking coupled to mass spectrometry. Using a cross-linked mixture of purified proteins, a suitable analysis identifies tens of cross-links that fit well with their atomic structures. A more elaborate in situ cross-linking of human cells in culture identified 469 intra-protein and 90 inter-protein cross-links, which also agreed with available atomic structures. Interestingly, many of these cross-links could not be mapped onto a known structure and thus provide new structural insights. For example, two cross-links involving the protein βNAC localize its binding site on the ribosome. Also of note are cross-links of actin with several auxiliary proteins for which the structure is unknown. Based on these findings we suggest a revised chemical reaction, which has relevance to the reactivity and toxicity of formaldehyde.

Low cell number proteomic analysis using in-cell protease digests reveals a robust signature for cell cycle state classification

“…We anticipate that the in-cell digest will be broadly applicable to characterise the proteomes of formaldehyde fixed cells. Recently published data suggest that formaldehyde crosslinks can be directly detected from MS data [42]. We anticipate the in-cell digest would enhance the sensitivity of crosslink detection and lead to an increase in identified protein-protein interactions.…”

The structural heterogeneity of α-synuclein is governed by several distinct subpopulations with interconversion times slower than milliseconds

“…We add the prepared BS 3 solution to α-syn to a final concentration of 1 mM and incubate at 4 °C for 1.5 h with shaking at 600 rpm. We quench the cross-linking reaction by the addition of 20 mM ammonium bicarbonate for 20 min 62,63 . We prepare samples for MS and MS data analysis as described by Slavin et al 64 .…”

Global analysis of protein-RNA interactions in SARS-CoV-2 infected cells reveals key regulators of infection

“…While these studies are important advances towards the understanding of viral ribonucleoproteins (RNPs), the choice of the crosslinking and RNA isolation approach leads to distinct depths and specificities. For example, while formaldehyde is a more efficient crosslinker than ultraviolet light (UV), it also promotes protein-protein crosslinks allowing the capture of indirect interactions through proteinprotein bridges (Tayri-Wilk et al, 2020). Despite their pros and cons, these studies discovered from dozens to hundreds of cellular proteins that engage with viral RNA in infected cells, highlighting that the viral RNA is a hub for complex host-virus interactions (Kim et al, 2020a;Knoener et al, 2017;LaPointe et al, 2018;Ooi et al, 2019;Phillips et al, 2016;Viktorovskaya et al, 2016).…”