Crosslinking mass spectrometry: A link between structural biology and systems biology

Abstract

Protein structure underpins functional roles in all biological processes; therefore, improved understanding of protein structures is of fundamental importance in nearly all biological and biomedical research areas. Traditional techniques such as X‐ray crystallography and more recently, cryo‐EM, can reveal structural features on isolated proteins/protein complexes at atomic resolution level and have become indispensable tools for structural biology. Crosslinking mass spectrometry (XL‐MS), on the other hand, is an emerging technique capable of capturing transient and dynamic information on protein interactions and assemblies in their native environment. The combination of XL‐MS with traditional techniques holds potential for bridging the gap between structural biology and systems biology approaches. Such a combination will enable visualization of protein structures and interactions within the crowded macromolecular environment in living systems that can dramatically increase understanding of biological functions. In this review, we first discuss general strategies of XL‐MS and then survey recent examples to show how qualitative and quantitative XL‐MS studies can be integrated with available protein structural data to better understand biological function at systems level.

Leveraging crosslinking mass spectrometry in structural and cell biology

“…The objective of crosslinking-MS is to provide distance restraints covering all relevant areas of a protein or complex and to capture the spatial and dynamic information on the system in solution. In a typical crosslinking-MS experiment, a soluble crosslinker is added to a sample ranging in complexity from a purified protein to a whole tissue (O'Reilly and Rappsilber, 2018;Sinz, 2018;Tang et al, 2021;Yu and Huang, 2018). By reacting with specific chemical moieties in the sample, crosslinkers introduce new covalent bonds, which report on the proximity of the chemical groups being probed.…”

Recent progress in mass spectrometry-based strategies for elucidating protein–protein interactions

“…PDB-MS suffers from false positives in the forms of high-abundance background proteins or artefacts from endogenous biotinylation The labelling time for different enzyme varies from 1 min to 24 h [12,13,16] XL-MS Crosslinking reagents can covalently connect two or more non-covalently interacting proteins, regardless of the duration and strength of the interaction. As such, even transient and weak PPIs can be preserved [45,46] The low efficiency (~ 1-5%) of crosslinking reagents, which often results in marginal crosslinks, where only the top 20-30% of proteins are detected When used in combination with X-ray crystallography, CryoEM, NMR and native MS, the spatial constraint data from XL-MS can guide molecular modelling, construct a connectivity map for determining subunit topology, and map the dynamic behaviour of the protein complex [49][50][51] The crosslinking reaction time may be relatively long (~ 30 min). Excessively long reaction time may result in large, crosslinked protein aggregates…”

“…the sum of the crosslinker spacer arm length and the side chain lengths of the two linked residues, can be calculated to impart an upper bound of the physical distance [48]. When used in combination with X-ray crystallography, CryoEM and native MS, such spatial constraint data can guide molecular modelling, construct connectivity map for determining subunit topology and map the dynamic behaviour of the protein complex [49][50][51].…”

Prospects and challenges of cancer systems medicine: from genes to disease networks

“…The major problem associated with these approaches is their restriction to identify highly stable PPIs. For the identification of more transient interactions in complex biological samples, another method termed cross-linking-MS (XL-MS), which also has the advantage of providing spatial information, is favored [ 167 ]. This method is based on covalently binding residues in two proteins through two reactive groups (usually amine-groups due to the prevalence of lysin residues in protein structures) that are connected via a spacer with a finite distance.…”