| Abstract [eng] |
Amino Acid Profiling of Yeast Grown in Stressful Conditions Using Isotope Ratio Mass Spectrometry Compound specific stable isotope analysis recently was one off the main applied tools in metabolomics studies. Amino acid (AA) specific isotope analysis of biological extracts may help to understand development or find specific markers for diseases or other stressors. One of these stressors, which is still widely researched, is oxidative stress. This study deals with development and application of methods needed to perform and master isotopic analysis with gas chromatography followed by mass spectroscopy or isotope ratio mass spectroscopy. Herein described experiments deals with development and application of AA extraction, purification, derivatization and gas chromatography methods. Standardized oxidative stress method was developed and used to collect affected yeast Saccharomyces cerevisiae cells. Stable isotope analysis were performed on AA extracts acquired from different yeast strains grown in elevated oxidative stress conditions. Evaluated different and strain – unique AA isotope ratio shifts in cells grown on stress. Results show that oxidative stress does greater impact on isotope ratio in those yeast strains which does not possess catalase type enzymes. Isotopic ratios of 15N/14N and 13C/12C are prone to change more in mentioned strains compared to those which did have catalases. AA specific isotope analysis were also performed on AA extracted from different AA domains. It was found out that AA undergo larger isotopic shift in those domains, which are more dynamic: free AA and AA of soluble proteome. Though AA which belong to insoluble proteome are more resilient to isotopic changes. |
