An integrated workflow for crosslinking mass spectrometry.

Marta L Mendes,Francis J O'Reilly,Christian Speck,Sven H Giese,Marta Barbon,Martin Graham,Colin W Combe,Michael Bohlke‐Schneider,Zhuo A Chen,Wolfgang Baumeister,Lutz Fischer,Adam Belsom,Juri Rappsilber,Markus R Eisele,Therese Dau

doi:10.15252/msb.20198994

Abstract

We present a concise workflow to enhance the mass spectrometric detection of crosslinked peptides by introducing sequential digestion and the crosslink identification software xiSEARCH. Sequential digestion enhances peptide detection by selective shortening of long tryptic peptides. We demonstrate our simple 12‐fraction protocol for crosslinked multi‐protein complexes and cell lysates, quantitative analysis, and high‐density crosslinking, without requiring specific crosslinker features. This overall approach reveals dynamic protein–protein interaction sites, which are accessible, have fundamental functional relevance and are therefore ideally suited for the development of small molecule inhibitors.

Highlights

Crosslinking mass spectrometry (CLMS) has become a standard tool for the topological analysis of multi-protein complexes and has begun delivering high-density information on protein structures, insights into structural changes and the wiring of interaction networks in situ (O’Reilly & Rappsilber, 2018)
This novel approach expands the detectable structure space in proteins, allowing it to capture dynamic regions in protein complexes that are mechanistically important and a priori druggable, that hitherto have remained undisclosed by cryo-electron microscopy (cryo-EM) due to their flexible nature
The results of this protocol for our standard proteins were compared to a parallel digestion using the same four enzymes and using trypsin alone in four replica, maintaining the analytical effort comparable in all three cases (SEC fractionation, 12 injections)

Summary

Introduction

Crosslinking mass spectrometry (CLMS) has become a standard tool for the topological analysis of multi-protein complexes and has begun delivering high-density information on protein structures, insights into structural changes and the wiring of interaction networks in situ (O’Reilly & Rappsilber, 2018). Tryptic digestion generates crosslinked peptides of considerable size, a quality that has been exploited with their enrichment by SEC (Leitner et al, 2012b), but one that poses as a potential problem regarding their detection. We followed trypsin digestion with subsequent digestion by alternative proteases and developed xiSEARCH, a database search engine, allowing the search of multiple datasets resulting from the application of our protocol. This novel approach expands the detectable structure space in proteins, allowing it to capture dynamic regions in protein complexes that are mechanistically important and a priori druggable, that hitherto have remained undisclosed by cryo-EM due to their flexible nature

Methods

Results

Conclusion