Large Scale Chemical Cross-linking Mass Spectrometry Perspectives

Abstract

The spectacular heterogeneity of a complex protein mixture from biological samples becomes even more difficult to tackle when one’s attention is shifted towards different protein complex topologies, transient interactions, or localization of PPIs. Meticulous protein-by-protein affinity pull-downs and yeast-two-hybrid screens are the two approaches currently used to decipher proteome-wide interaction networks. Another method is to employ chemical cross-linking, which gives not only identities of interactors, but could also provide information on the sites of interactions and interaction interfaces. Despite significant advances in mass spectrometry instrumentation over the last decade, mapping Protein-Protein Interactions (PPIs) using chemical cross-linking remains time consuming and requires substantial expertise, even in the simplest of systems. While robust methodologies and software exist for the analysis of binary PPIs and also for the single protein structure refinement using cross-linking-derived constraints, undertaking a proteome-wide cross-linking study is highly complex. Difficulties include i) identifying cross-linkers of the right length and selectivity that could capture interactions of interest; ii) enrichment of the cross-linked species; iii) identification and validation of the cross-linked peptides and cross-linked sites. In this review we examine existing literature aimed at the large-scale protein cross-linking and discuss possible paths for improvement. We also discuss short-length cross-linkers of broad specificity such as formaldehyde and diazirine-based photo-cross-linkers. These cross-linkers could potentially capture many types of interactions, without strict requirement for a particular amino-acid to be present at a given protein-protein interface. How these shortlength, broad specificity cross-linkers be applied to proteome-wide studies? We will suggest specific advances in methodology, instrumentation and software that are needed to make such a leap.

Probing structures of large protein complexes using zero-length cross-linking

“…This was effectively demonstrated by an in silico analysis that showed that substantially fewer zero-length cross-links were required to accurately reproduce a known protein structure via a modeling experiment, and to discard erroneous structures generated by modeling [23]. Moreover, because the reaction can only occur between residues that are roughly within salt bridge distance of each other, residue interactions captured by this method are more likely to describe direct contact sites rather than simply sites that are in close proximity [37]. In addition, zero-length cross-linking reagents are less likely to generate “dead-end” products, which occur when only one of the reactive groups is able to react with a site on a protein and the second site reacts either with water or a quenching reagent, compared to other cross-linkers.…”

“…This substantially complicates analysis of non-zero-length cross-link experiment data. Zero-length cross-link experiments are also less likely to generate “self-linked” products that occur when interacting reactive groups are on the same peptide [37], as both reactive groups would have to be on the same peptide and directly interact in a salt bridge. Dead-end and self-linked products of non-zero-length cross-linkers are problematic because they must be accounted for in database searches.…”

Host-Microbe Protein Interactions during Bacterial Infection

“…Relationships between two peptides from the same protein constituted the majority of identifications (intraprotein PPIs). Two residues within a single protein have a high likelihood of being in close physical contact within a cell, are therefore more frequently cross-linked, and make up a higher proportion of interactions in crosslinked datasets[22, 34]. Importantly, intraprotein interactions define proximal residues within a protein, and thereby provide valuable structural coordinates for identified proteins even when no known structure exists for this protein (Figure 2).…”

Protein-protein interaction analysis for functional characterization of helicases

“…Typically, chemical cross-links survive protein denaturation and digestion, and cross-linked peptide pairs, along with the cross-linking sites can be identified in a mass spectrometer. We recently reviewed this topic extensively in [36]. We also recommend earlier review by Trakselis et al, which discusses chemical cross-linking for structural analysis of protein complexes [37].…”

“…As a consequence, in [36] we suggested that long-length cross-linkers, selective for frequently occurring amino acids, are useful for establishing identities of interacting partners; while shorter, broadly selective cross-linkers are better suited for analyzing structure of PPI interfaaces. An affinity handle, such as biotinyl, or alkynyl for conjugation using click-chemistry; and MS 2 -labile reporter groups can be introduced into a long-length cross-linker [39, 40].…”