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.

Keywords: crosslinking mass spectrometry (XL-MS); intereactome; structural biology; systems biology.

FIGURE 1

XL‐MS bridges the gap between structural biology and systems biology. Structural biology is illustrated in the left panel with representative protein 3‐D structures generated with traditional technologies such as cryo‐EM, X‐ray crystallography, and NMR. Middle panel shows schematic view of the basic XL‐MS process, which is elaborated in greater detail in XL‐MS workflow discussion and Figure 2. Crosslinkers with two reactive groups connected via a spacer can be applied to intact living cells. The information on crosslinked amino acid residues in protein complexes is extracted to build a network of XL constraints. These define distances between the crosslinked residue pairs based on the crosslinker spacer length. Integrative modeling is performed with the XL distance constraints among proteins and available protein 3‐D structures to assemble protein complexes. Right panel depicts how this information is used to illuminate systems‐level information on protein interactions and structures within living cells, which are represented with cartoons of various colors, shapes, and sizes to indicate different proteins and cellular compartments (cartoon picture is adapted from the artwork of David Goodsell. Proteome‐level XL constraints collected from living cells enables visualization of protein conformations and interactions as they exist within the crowded macromolecular environment, ultimately enabling a systems‐level structural biology view from inside the cell

FIGURE 2

General workflow of XL‐MS