Multi-User Equipment related to grant 201308617-7 - HPLC coupled with mass spectrometry

• Grant number: 15/02482-8
• Support Opportunities: Multi-user Equipment Program
• Start date: June 01, 2015
• End date: May 31, 2022
• Field of knowledge: Biological Sciences - Microbiology - Applied Microbiology
• Principal Investigator: Adalberto Pessoa Junior
• Grantee: Adalberto Pessoa Junior
• Host Institution: Faculdade de Ciências Farmacêuticas (FCF). Universidade de São Paulo (USP). São Paulo , SP, Brazil
• City of the host institution: São Paulo
• Associated research grant: 13/08617-7 - Production of extracellular L-asparaginase: from bioprospecting to the engineering of an antileukemic biopharmaceutical, AP.TEM
• EMU web page: Página do Equipamento Multiusuário não informada
• Type of equipment: Caracterização de Materiais - Análises Químicas - Cromatografia líquida acoplada a espectrômetro de massa
• Manufacturer: Fabricante não informado
• Model: Modelo não informado

Abstract

This LC / MS system will aim to analyze the degree of oligomerization of the different isoforms of ASNases produced by size exclusion chromatography, since it is assumed that the active form of these enzymes are only tetramers. Moreover, this technique will be used to characterize the products of these isoforms exposure to human cysteine protease (commercially available from Sigma-Aldrich Company), such as cathepsin B and asparagine endopeptidase (AEP). These enzymes are considered the main enzymes involved in ASNases degradation, leading to clearance of the same, as well as the production of immunogenic peptides, critical factors for allergic response to treatment with the biopharmaceutical. Another important analysis will be the proteolytic degradation arising from human plasma by yet unknown factors. For this, ASNases produced will be incubated with human plasma (commercially available - Sigma-Aldrich) for further evaluation whether or not occured degradation and what were the products, allowing identify vulnerable sites of the enzyme that could guide the engineering of proteins through site directed mutations in search of greater stability in vivo. Furthermore, ASNases resulting from fungal origin in many cases does not always have its amino acid sequence already known, which require characterization by proteolysis and mass spectrometry (peptide mass fingerprint) for its isolation, characterization and subsequent cloning of the gene. The equipment is extremely versatile and current, allowing multiple analyzes of proteins (as noted above), and proteomics, metabolomics, among other important applications in pharmacy area. (AU)