Selective cross-linking of coinciding protein assemblies by in-gel cross-linking mass spectrometry.

Susanne Arnold,Matti F Pronker,Vojtech Franc,Johannes F Hevler,Alfredo Cabrera‐Orefice,Marie V Lukassen,Albert J R Heck

doi:10.15252/embj.2020106174

Abstract

Cross‐linking mass spectrometry has developed into an important method to study protein structures and interactions. The in‐solution cross‐linking workflows involve time and sample consuming steps and do not provide sensible solutions for differentiating cross‐links obtained from co‐occurring protein oligomers, complexes, or conformers. Here we developed a cross‐linking workflow combining blue native PAGE with in‐gel cross‐linking mass spectrometry (IGX‐MS). This workflow circumvents steps, such as buffer exchange and cross‐linker concentration optimization. Additionally, IGX‐MS enables the parallel analysis of co‐occurring protein complexes using only small amounts of sample. Another benefit of IGX‐MS, demonstrated by experiments on GroEL and purified bovine heart mitochondria, is the substantial reduction of undesired over‐length cross‐links compared to in‐solution cross‐linking. We next used IGX‐MS to investigate the complement components C5, C6, and their hetero‐dimeric C5b6 complex. The obtained cross‐links were used to generate a refined structural model of the complement component C6, resembling C6 in its inactivated state. This finding shows that IGX‐MS can provide new insights into the initial stages of the terminal complement pathway.

Highlights

Over the last decades, bimolecular mass spectrometry (MS), with its ability to analyze low amounts of samples with high speed and sensitivity, has evolved into a central pillar beneficial for integrative structural biology (Lossl et al, 2016; Kaur et al, 2019; Robinson, 2019; de Souza & Picotti, 2020)
10 μg of purified E. coli GroEL diluted in a Tris buffer was subjected to blue native polyacrylamide gel electrophoresis (BN-PAGE) as described previously (Wittig et al, 2006)
Bands corresponding to the native 14-mer GroEL (MW = 800 kDa) were excised from the BN-PAGE (Appendix Fig S1A) and further cut into small pieces and incubated with or without the cross-linker reagent DSS

Summary

Introduction

Bimolecular mass spectrometry (MS), with its ability to analyze low amounts of samples with high speed and sensitivity, has evolved into a central pillar beneficial for integrative structural biology (Lossl et al, 2016; Kaur et al, 2019; Robinson, 2019; de Souza & Picotti, 2020). With recent advances in instrumentation, sample preparation, and data analysis, especially XL-MS has started to fulfill its potential to complement wellestablished structural methods such as X-ray crystallography, nuclear magnetic resonance spectroscopy (NMR), and cryo-electron microscopy (cryo-EM; Leitner et al, 2016; Matthew Allen Bullock et al, 2016; Rappsilber, 2011). Recent advances in search engines for more efficient identification of cross-linked peptides allowed structural studies of purified proteins or protein complexes, as well as large-scale experiments with more complex samples like purified organelles or cell lysates, using buffer systems which aim to meet physiological relevant conditions (Klykov et al, 2018; Chen & Rappsilber, 2019; Gotze et al, 2019; Beveridge et al, 2020).

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