Engineered design of new nano-micro materials for proteomics and phosphoproteomics applications

• Autor(a) principal: Martins, Gonçalo Nuno Gouveia
• Data de Publicação: 2017
• Tipo de documento: Dissertação
• Idioma: eng
• Título da fonte: Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
• Texto Completo: http://hdl.handle.net/10362/25879
• topic: Domínio/Área Científica::Engenharia e Tecnologia::Engenharia Química

For a deep understanding of the biological mechanisms of the living organisms, a detailed study of phosphoproteins is vital. Digestion and pre-concentration of phosphoproteins are critical steps in phosphoproteomics. analysis. In this work, we used a new nano-sized system in both steps. For digestion of phosphoproteins the standard used trypsin enzyme was selected. This trypsin was immobilized in an iron core to avoid trypsin auto-lysis. This new system was found to be highly effective for protein digestion and when compared with the available commercial systems, it performed better. Thus, it was possible to identify four times more proteins than using the standard procedures. Phosphoproteins are estimated to be 30% of the entire proteome, however, exist some issues regarding its identification such as low levels of concentration. To overcome these obstacles, ion metal affinity chromatography, IMAC, is currently used. In this work, we synthesized new nano-sized IMACs from a polystyrene matrix. These polystyrene matrices were also synthesized using a 23 experimental design to unravel the conditions to create them in a certain range of size. Using a polystyrene matrix of 40nm, it was possible to create an IMAC of 250nm, in the nano scale range. Metals used for the IMACs were titanium and lanthanum. Both IMACs proved to be efficient on the phosphopeptide enrichment having superior capacity than others described in the literature. The number of phosphopeptides identified from a simple sample of α-casein was 99, five times more than the best results described in literature.