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Abstract:
Proteomic analysis is used for in depth analysis of the expressed proteome of an organism. Nowadays, MS-based proteomic analysis is used for high throughput analysis of complex samples. The bottom up proteomic approach is based on tryptic digestion of proteins to peptides without previous protein extraction. These peptides are mass analyzed and used for ´bottom up` protein identification based on a reference database. Complex samples such as the complete digestive, water soluble fraction of the expressed proteome of selected cells can be analyzed with high sensitivity and mass accuracy. This study provides the first targeted proteomics approach by applying a combination of flow cytometry based cell sorting and shotgun proteomics. One of the main purposes was to test the parameter of this approach for sorting a minimum of fixed bacterial cells, labeled with stable isotopes, for a robust analysis of the expressed proteome. This targeted proteomic approach thus allows the calculation of in situ growth rates of marine microorganisms. Method variations like different types of cell fixation, varying trypsin concentrations and peptide separation either by chromatography or size exclusion were testes. The suggested standard protocol for peptide analysis and further protein identification was shown to be sufficient for the detection of isotopic mass shifts within a set of highly abundant proteins for samples with 2 x 106 cells. In addition, one possible application of proteomic analysis was shown exemplary. The grazing of the ciliate Uronema marinum on the stable isotope enriched marine Alphaproteobacterium Sphingomonas alaskensis was visualized over time by fluorescent microscopy and Nanoscale Secondary Ion – Mass spectrometry (NanoSIMS). Proteomic samples were taken for further bulk measurements of the incorporation of stable isotope enriched prey biomass into the predator proteome in order to calculate the grazing rate.
