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

Autor(es):Mostrar menos -

Lima, Diogo B. ^[1] ; Melchior, John T. ^[2] ; Morris, Jamie ^[2] ; Barbosa, Valmir C. ^[3] ; Chamot-Rooke, Julia ^[1] ; Fioramonte, Mariana ^[4] ; Souza, Tatiana A. C. B. ^[5] ; Fischer, Juliana S. G. ^[5] ; Gozzo, Fabio C. ^[4] ; Carvalho, Paulo C. ^{[5, 6]} ; Davidson, W. Sean ^[2]

Afiliação do(s) autor(es)

^[1] Inst Pasteur, Mass Spectrometry Biol Unit, CNRS USR 2000, Paris 2000 - France

^[2] Univ Cincinnati, Pathol & Lab Med, Cincinnati, OH 45220 - USA

^[3] Univ Fed Rio de Janeiro, Syst Engn & Comp Sci Program, Rio De Janeiro - Brazil

^[4] Univ Estadual Campinas, Dalton Mass Spectrometry Lab, Sao Paulo - Brazil

^[5] Fiocruz MS, Carlos Chagas Inst, Grp Computat Mass Spectrometry & Prote, Curitiba, Parana - Brazil

^[6] Fiocruz MS, Lab Toxinol, Rio De Janeiro - Brazil

• Tipo de documento: Artigo Científico
• Fonte: Nature Protocols; v. 13, n. 3, p. 431-458, MAR 2018.
• Citações Web of Science: 11

Resumo

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 `tight' 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 similar to 14 d, and requires basic biochemical and mass spectrometry skills. (AU)

• Processo FAPESP: 14/17264-3 - Novas fronteiras em proteômica estrutural: caracterizando estruturas de proteínas e complexos proteicos por espectrometria de massas
• Beneficiário: Fabio Cesar Gozzo
• Modalidade de apoio: Auxílio à Pesquisa - Temático
• Processo FAPESP: 12/10862-7 - Proteômica Estrutural por Ligação Cruzada Acoplada a Espectrometria de Massas: Desenvolvimentos, Estudos Fundamentais e Aplicações.
• Beneficiário: Mariana Fioramonte
• Modalidade de apoio: Bolsas no Brasil - Doutorado