Quantitative Phosphoprotein Analysis in Adipose Tissue

Abstract

Background: Mass spectrometry-based proteomics has increasingly been the choice of method for the global analysis of the composition, modification and dynamics of proteins. Quantitative analysis of the proteome with tandem mass tag is a technique for calculating the relative abundance of the proteome in tissues and organelles. In this thesis, present a novel bottom-up method for the quantitative analysis of the phospho proteome in adipose tissue. Materials and methods: The approach employed in this thesis is a bottom-up based method. The samples were enriched with TiO2 using the enrichment protocols published by Dickhut et al. to determine which protocol yielded the highest number of phosphopeptides. The next step was to validate the procedure by running the procedure on HeLa cells. The last step in procedure was to determine the labeling of the samples (before or after enrichment). The post-processing of data output was done on PD, Peaks and PeptideShaker to determine the most compatible search engine. Results and discussion: Mascot in PD identified the highest number of phosphopeptides with 252 confident identifications compared to Sequest HT (146), Peaks (239) and PeptideShaker (197). Mascot was unique in 51 phosphopeptides compared to Peaks’ 33 unique phosphopeptide identifications. PD was also successful in interpreting the fragment spectra giving relatively good information about the fragments compared to Peaks and PeptideShaker. Micro column protocol was more successful in terms of number of identification (252) compared to the batch mode protocol (147 identified). PhosSTOP had a negative effect with the results and was therefore excluded from further experiments, as the phosphopeptides identified with PhosSTOP were lower and less confident. The procedure was validated using HeLa cell with 1068 phosphopeptides confidently identified. The labeling step was determined to be after enrichment based on the number of confident identification (147 phosphopeptides labeled before enrichment versus 3 phosphopeptides labeled after enrichment). Conclusion: In this this study, a method for quantifying the phospho proteome in adipose tissue has been presented. The method conducts the micro column protocol, which has proven to be more successful than the batch mode protocol. The procedure was validated using HeLa cells, which worked successfully. The labeling of samples was determined to be conducted before enrichment due to low yield when labeling after enrichment. Mascot search engine in PD was conducted due to the higher compatibility with the data compared to PeptideShaker and Peaks.