New advances in cross-linking mass spectrometry toward structural systems biology

Abstract

Elucidating protein-protein interaction (PPI) networks and their structural features within cells is central to understanding fundamental biology and associations of cell phenotypes with human pathologies. Owing to technological advancements during the last decade, cross-linking mass spectrometry (XL-MS) has become an enabling technology for delineating interaction landscapes of proteomes as they exist in living systems. XL-MS is unique due to its capability to simultaneously capture PPIs from native environments and uncover interaction contacts though identification of cross-linked peptides, thereby permitting the determination of both identity and connectivity of PPIs in cells. In combination with high resolution structural tools such as cryo-electron microscopy and AI-assisted prediction, XL-MS has contributed significantly to elucidating architectures of large protein assemblies. This review highlights the latest developments in XL-MS technologies and their applications in proteome-wide analysis to advance structural systems biology.

Keywords: Cross-linking mass spectrometry; Integrative structural analysis; Protein complexes; Protein–protein interaction; Structural proteomics; Structural systems biology.

Figure 1.. General XL-MS workflow.

Various sample types can be cross-linked, ranging in complexity from protein complexes to tissues and organs. Both ends of the cross-linker may target the same or different residues, while the spacer arm that connects the functional groups can be either MS-cleavable or not. To reduce complexity, proteins can be separated prior to or after cross-linking by subcellular or complex-centric fractionation, or affinity purification by tagged proteins or proximity labeling (PL). Following digestion, cross-links can be enriched by affinity purification or peptide fractionation. Cross-linked peptides can be purified if they contain a biotin or “click-able” site for appending biotin (B), phosphonic acid (P), or if an antibody recognizing the spacer arm of a cross-linker is used. Various chromatographic methods such as size-exclusion (SEC), strong-cation exchange (SCX), and high-PH reverse phase (bRP) can be used to reduce the complexity of cross-linked peptide samples prior to LC-MS analysis. Depending on the MS acquisition type (MS/MS or MSⁿ) and the type of cross-linker used (non-cleavable or MS-cleavable), various database search software are available to identify cross-linked peptides. Resulting cross-links can be used to generate 2-D XL-maps and XL-MS derived PPI, compartmental, pathway, and protein complex networks. Finally, cross-links can be used as distance restraints for integrative structure modeling or alongside AI-based structure prediction such as AlphaFold2 for protein structural elucidation.

Figure 2.. Selected cross-linkers discussed in this review.

Molecular structures for each cross-linker are shown alongside their corresponding references. Cross-linkers grouped and color-coded based on their targeted residues. Green: lysine-to-lysine, red: cysteine-to-cysteine, blue: lysine-to-any amino acid (nonspecific), yellow: lysine-to-cysteine, grey: lysine-to-acidic residue, orange: lysine/hydroxyl residue-to-lysine/hydroxyl residue, purple: acidic residue-to-acidic residue (requires coupling reagent such as DMTMM). The border of each group designates whether cross-linkers are MS-cleavable and/or enrichable. No border: non-cleavable and non-enrichable, thin dashed border: MS-cleavable but non-enrichable, solid border: non-cleavable but enrichable, and thick dashed border: MS-cleavable and enrichable.