Characterization of homodimer interfaces with cross-linking mass spectrometry and isotopically labeled proteins

Abstract

Cross-linking coupled with mass spectrometry (XL-MS) has emerged as a powerful strategy for the identification of protein-protein interactions, characterization of interaction regions, and obtainment of structural information on proteins and protein complexes. In XL-MS, proteins or complexes are covalently stabilized with cross-linkers and digested, followed by identification of the cross-linked peptides by tandem mass spectrometry (MS/MS). This provides spatial constraints that enable modeling of protein (complex) structures and regions of interaction. However, most XL-MS approaches are not capable of differentiating intramolecular from intermolecular links in multimeric complexes, and therefore they cannot be used to study homodimer interfaces. We have recently developed an approach that overcomes this limitation by stable isotope-labeling of one of the two monomers, thereby creating a homodimer with one 'light' and one 'heavy' monomer. Here, we describe a step-by-step protocol for stable isotope-labeling, followed by controlled denaturation and refolding in the presence of the wild-type protein. The resulting light-heavy dimers are cross-linked, digested, and analyzed by mass spectrometry. We show how to quantitatively analyze the corresponding data with SIM-XL, an XL-MS software with a module tailored toward the MS/MS data from homodimers. In addition, we provide a video tutorial of the data analysis with this protocol. This protocol can be performed in ∼14 d, and requires basic biochemical and mass spectrometry skills.

Cross-linking/Mass Spectrometry: A Community-Wide, Comparative Study Towards Establishing Best Practice Guidelines

“…XL-MS is now increasingly being used for deriving protein-protein interaction maps, both in vitro and in vivo , where interacting proteins are covalently connected by the cross-linking reaction. 6,7,8,9,10…”

Human Stress-inducible Hsp70 Has a High Propensity to Form ATP-dependent Antiparallel Dimers That Are Differentially Regulated by Cochaperone Binding*

“…The reaction was stopped by adding Tris, pH 8 (80 mM final concentration), samples were supplemented with 2ϫ CSB loading buffer and analyzed by SDS-PAGE and Western blotting with Hsp70 antibody. by the error-prone intra-and inter-molecular cross-links differentiation because of the homo-dimeric nature of Hsp70 (61). Although the residues mediating NBD-SBD␣ contacts in the antiparallel dimer are conserved, the NBD-NBD interfaces show lower conservation, as documented by inspection of crystal structures and our homology-based Hsp70 dimer model (supplemental Fig.…”

π-Helix controls activity of oxygen-sensing diguanylate cyclases

“…Data analysis was performed using Spectrum Identification Machine (SIM-XL) [29,30]. SIM-XL uses a unique paradigm for search-space reduction and utilizes reporter ions for identifying tandem mass spectra derived from cross-linked peptides.…”

“…!P); enzyme C/N terminal -C-terminal; deconvoluted MS/MS -false; fragmentation method -HCD; number of isotopic possibilities -4; minimum amino acid residues per chain -4; intra-link max charge -4; maximum missed cleavages -3; peaks matched cutoff -0; minimum MH (linear peptide) -600; maximum MH (linear peptides) -6000. Identified cross-links were screened manually for cross-links with inter-link and intra-link scores of 2.0 or greater and fragmentation spectra peaks representing y (C-terminal fragment) and b (N-terminal fragment) ions that include the crosslinked residues as suggested by the developers of SIM-XL [30] ( Supplementary Figures S5 and S6).…”

“…BpeGReg contains 17 lysine residues throughout the 485 amino acid sequence (Figure 2). There were only two globin domain (loop between helices αG and αH)-middle domain (helix αB) cross-links identified using Spectrum Identification Machine [29,30]. after inspecting the cross-linking spectra for the tetramer form of BpeGReg (Figure 3).…”