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© Research
Publication : Nature protocols

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

Scientific Fields
Diseases
Organisms
Applications
Technique

Published in Nature protocols - 01 Feb 2018

Lima DB, Melchior JT, Morris J, Barbosa VC, Chamot-Rooke J, Fioramonte M, Souza TACB, Fischer JSG, Gozzo FC, Carvalho PC, Davidson WS

Link to Pubmed [PMID] – 29388937

Nat Protoc 2018 Mar;13(3):431-458

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.

https://www.ncbi.nlm.nih.gov/pubmed/29388937