A new label-free approach for the determination of reaction rates in oxidative footprinting experiments

Autor(es)

Pilau, Eduardo J. ^{[1, 2, 3]} ; Iglesias, Amadeu H. ^{[1, 3]} ; Gozzo, Fabio C. ^{[1, 3]}

Afiliação do(s) autor(es)

^[1] Univ Estadual Campinas, Inst Chem, BR-13083970 Campinas, SP - Brazil

^[2] Univ Estadual Maringa, Dept Quim, BR-87020900 Maringa, Parana - Brazil

^[3] Inst Nacl Ciencia & Tecnol Bioanalit, BR-13083970 Campinas, SP - Brazil

• Tipo de documento: Artigo Científico
• Fonte: ANALYTICAL AND BIOANALYTICAL CHEMISTRY; v. 405, n. 24, p. 7679-7686, SEP 2013.
• Citações Web of Science: 2

Resumo

Hydroxyl radical-mediated oxidative footprinting coupled to mass spectrometric analysis is an attractive technique for protein surface mapping, conformational changes monitoring, and protein-ligand interfaces mapping in solution. In this technique, a protein is oxidized by in situ-generated hydroxy radicals and the site and rate of oxidation can be determined by proteolysis followed by mass spectrometric analysis. Changes in peptide oxidation rate can then be correlated to the changes in solvent exposure, and information about conformational changes or interaction domains can be obtained. The method relies, therefore, on the accurate measurements of peptide oxidation rate. Here, we describe a new label-free method to determine the oxidation rate of peptides that is based on the consumption of the unoxidized peptide instead of measuring the formation of oxidized peptides. The reaction rate thus obtained presents a better linearity and lower variation when compared to the traditional method. The label-free method is also simpler to implement and automation can be achieved through label-free quantitation software. (AU)

• Processo FAPESP: 04/14846-0 - Rede de proteoma do estado de São Paulo
• Beneficiário: Fabio Cesar Gozzo
• Modalidade de apoio: Auxílio à Pesquisa - Programa GENOMA
• Processo FAPESP: 08/57805-2 - Instituto de Bioanalítica
• Beneficiário: Lauro Tatsuo Kubota
• Modalidade de apoio: Auxílio à Pesquisa - Temático