Cross-Linking Mass Spectrometry: An Emerging Technology for Interactomics and Structural Biology

Abstract

Figure 1

The General XL-MS analysis workflow.

Figure 2. MS analysis workflows for MS-cleavable (A–C) and non-cleavable cross-linked peptides (D)

(A) MSⁿ analysis of a DSSO cross-linked peptide (α-β). (B) MS²-based analysis of a DSSO cross-linked peptide with sequential CID and ETD fragmentation. (C) MS² analysis workflow of a DSBU cross-linked peptide with HCD. (D). MS² analysis of a DSS cross-linked peptide with HCD.

Figure 3. In vitro cross-linking strategies for protein complexes

(A) 1- and 2-step in-solution in vitro cross-linking protocols for protein complexes. (B) 1- and 2-step on-bead in vitro cross-linking protocols for affinity purified protein complexes. 2-step protocols in (A) and (B) involve in vitro mild FA cross-linking prior to the second cross-linking step. (C) 2-step cross-linking protocol by coupling in vivo mild FA prefixing with on-bead in vitro cross-linking of affinity purified protein complexes.

Figure 4. General scheme for integrative structural modeling

Integrative structural determination of the protein complexes proceeds through four main stages: (1) data is first gathered from various structural elucidation methodologies; (2) subunits are represented from high-resolution structures while other forms of low-resolution structural data are translated into spatial restraints; (3) configurational sampling produces an ensemble of structures that satisfies translated restraints; and (4) the ensemble structure is analyzed and validated. Adapted from Wang, et al (This research was originally published in J. Biol. Chem. Wang, X., Chemmama, I.E., Yu, C., Huszagh, A., Xu, Y., Viner, R., Block, S.A., Cimermancic, P., Rychnovsky, S.D., Ye, Y., Sali, A., Huang, L. The Proteasome-Interacting Ecm29 Protein Disassembles the 26S Proteasome in Response to Oxidative Stress. J Biol. Chem. 2017, 292(39):16310–16320. © the American Society for Biochemistry and Molecular Biology).

Figure 5. Workflows for proteome-level in vitro and in vivo QXL-MS experiments using MS-cleavable cross-linkers and MSⁿ-based acquisition

Pair-wise QXL-MS can be carried out using (A) cross-linking labeling with isotope-coded reagents and (B) SILAC-based metabolic labeling. Cross-linked peptides are identified in MS³, while corresponding parent ions are quantified in MS¹ based on their spectral abundance. (C) QMIX-based multiplexed QXL-MS strategy, in which each peptide digest is labeled with a distinct isobaric labeling reagent and then mixed equivalently prior to LC-MSⁿ analysis. Cross-linked peptides are both identified and quantified in MS³, based on sequencing ions and quantitative reporter ions released during HCD fragmentation. Note: Reporter ions in MS³ can be significantly enhanced using synchronous precursor selection (SPS) acquisition to increase quantitation sensitivity and accuracy.