Assess of catechol-O-methyltransferase soluble isoform by multiple reaction monitoring mass spectrometry

• Autor(a) principal: Diogo, Joana Margarida Santos
• Data de Publicação: 2016
• Tipo de documento: Dissertação
• Idioma: eng
• Título da fonte: Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
• Texto Completo: http://hdl.handle.net/10400.6/6788
• topic: Catechol-O-Metiltransferase Membranar; Catechol-O-Metiltransferase Solúvel; Multiple Reaction Monitoring; Pichia-Pastoris; Quantificação; Domínio/Área Científica::Engenharia e Tecnologia::Bioquímica

COMT is an enzyme that catalyses the transfer of the methyl group from S-adenosyl-L-methionine to the hydroxyl group of a large variety of catechols, including catechol-estrogens and the neurotransmitters dopamine and norepinephrine. The protein is expressed in both a soluble form (SCOMT) and a membrane-bound form with additional 50 residues at the N-terminal (MBCOMT). In most human tissues, the majority of COMT is present in soluble form and only a small fraction as MBCOMT. The human COMT gene has a common single-nucleotide polymorphism that results in substitution of methionine for valine at residue 108 of soluble protein and residue 158 of the membrane bound enzyme. The two alleles have been associated with several neurologic diseases, such as schizophrenia, anorexia nervosa and obsessive-compulsive disorder. Thus, due to the COMT's role in several mental disorders, accurate and selective measurements are essential requirements for improvement the COMT clinical investigation. Thus, in this work a specific and low-cost methodology is developed in order to measure COMT protein in Pichia pastoris lysates by liquid chromatography-multiple reaction monitoring (MRM) mass spectrometry. The method was validated according to international guidelines evaluating selectivity, linearity, precision and accuracy, limit of detection and limit of quantification, carry-over and matrix effects. All the evaluated parameters were found within the established confines. The method was able to quantify small amounts of both COMT isoforms in complex samples such as cell lysates, showing its applicability. The versatility of the analytical method proposed may allow its application in routine laboratory analysis of SCOMT and MBCOMT in various biological matrices such as blood, breast tissue, liver tissue or brain tissue.