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

Tamar Tayri-Wilk,Alex Motzik,Xue Sun,Deborah E Shalev,Ayelet Blass,Joanna Zamel,Oren Ram,Nir Kalisman,Shon Cohen,Moriya Slavin

doi:10.1038/s41467-020-16935-w

Tamar Tayri-Wilk, Alex Motzik + Show 8 more

Open Access

https://doi.org/10.1038/s41467-020-16935-w

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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.

Highlights

Whole-cell cross-linking coupled to mass spectrometry is one of the few tools that can probe protein–protein interactions in intact cells
We first surveyed the FA cross-linking products that occur within structured proteins by cross-linking a mixture of three purified proteins (bovine serum albumin (BSA), Ovotransferrin, and α-Amylase)
The mixture was incubated with FA for twenty minutes, and quenched, denatured, digested by trypsin into peptides, and analyzed by mass spectrometry (Fig. 1)

Summary

Introduction

Whole-cell cross-linking coupled to mass spectrometry is one of the few tools that can probe protein–protein interactions in intact cells. Mass spectrometry has confirmed this 12 Da addition to the masses of short linear peptides after FA incubation[5,9,10,11] These studies were not able to identify pairs of peptides that were linked via methylene bridges. It is unclear whether the observed 12 Da additions were bona fide cross-links or local modification of a single peptide. We conduct an unbiased mass-spectrometric search for the FA adduct that leads to a different reaction product with a mass of 24 Da and not the 12 Da expected This reaction only occurs in structured proteins (rather than peptides), perhaps explaining why earlier studies did not observe it

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