Cross-linking and other structural proteomics techniques: how chemistry is enabling mass spectrometry applications in structural biology

Abstract

The biological function of proteins is heavily influenced by their structures and their organization into assemblies such as protein complexes and regulatory networks. Mass spectrometry (MS) has been a key enabling technology for high-throughput and comprehensive protein identification and quantification on a proteome-wide scale. Besides these essential contributions, MS can also be used to study higher-order structures of biomacromolecules in a variety of ways. In one approach, intact proteins or protein complexes may be directly probed in the mass spectrometer. Alternatively, various forms of solution-phase chemistry are used to introduce modifications in intact proteins and localizing these modifications by MS analysis at the peptide level is used to derive structural information. Here, I will put a spotlight on the central role of chemistry in such mass spectrometry-based methods that bridge proteomics and structural biology, with a particular emphasis on chemical cross-linking of protein complexes.

Fig. 1. Different levels of information that can be derived from MS-based proteomics experiments directly (black arrows) or indirectly (grey arrows). PEPTIDER refers to a hypothetical amino acid sequence of a peptide, the grey dot above the sequence symbolizes a modification. Structural analysis by MS is discussed in more detail in this perspective. Functional properties of proteins can by derived indirectly from MS data by obtaining data about subcellular localization, distribution between different cell types, abundance changes in response to biological perturbations, and involvement in protein–protein interactions, among others.

Fig. 2. Schematic illustration of the information derived from structural proteomics/structural MS techniques using a hypothetic binary complex.

Fig. 3. Experimental workflow for hydrogen/deuterium exchange/MS.

Fig. 4. Experimental workflow for covalent labeling/MS.

Fig. 5. Different levels of spatial information derived from cross-linking experiments. From left to right: the presence of cross-links puts subunit B in the proximity of subunit A; localization of the contact (cross-linking) sites on the two proteins narrows down the possible location of B relative to A; defining an upper bound of the distance between cross-linked residue as specified by cross-linker structure positions and orients the subunits according to their binding interface.

Fig. 6. Common chemistries to couple reactive residues in chemical cross-linking. DMTMM, 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride; EDC, N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride; NHS, N-hydroxysuccinimide.

Fig. 7. Experimental workflow for chemical cross-linking/MS.