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.
Protein Data Bank
V.C.B. acknowledges a FAPERJ BBP grant, as well as support from CNPq and CAPES. P.C.C. acknowledges the Fundação Araucária Universal/Jovem Pesquisador Grant and support from PAPES VII and Universal CNPq. D.B.L. and J.C.-R. acknowledge the Centre National de la Recherche Scientifique (CNRS) for financial support. F.C.G. and M.F. acknowledge support from FAPESP (grants 2014/17264-3 and 2012/10862-7) and CNPq. The mass spectrometry methods were established in the UC Proteomics Laboratory on the Sciex 5600 + TripleTOF system, funded in part through an NIH-shared instrumentation grant (S10 RR027015-01; K.D. Greis, principal investigator). This work was also supported by an American Heart Association postdoctoral fellowship grant (16POST27710016 to J.T.M.) and by funding from the National Institutes of Health Heart, Lung and Blood Institute (R01 GM098458 and P01 HL128203 to W.S.D.).
Integrated supplementary information
Video tutorial for data analysis using SIM-XL.