以蛋白質體學技術研究果蠅酪氨酸磷酸化及酪氨酸磷酸水解脢之受質

Abstract

Abstract To know the signaling pathways that protein tyrosine phosphatases (PTPs) are involved, it is important to identify the substrates of PTPs. However, prior strategies for identification of substrates of PTPs were suboptimal. Therefore establishment of a generic method for large scale screening of potential substrates of PTPs is necessary. In the first part of this thesis, substrate trapping was coupled with mass spectrometry to identify substrates of PTP61F, an ortholog of human PTP1B. The total lysate or enriched tyrosine proteins were used as input to perform substrate trapping. To reduce non specific associated proteins, which is the major cause of false positive identification, vanadate, a competitive inhibitor, was used to specifically elute the substrates from PTP-substrate complexes. More than 60 substrates were identified in our substrate trapping assay. The substrates identified in this study highlight a connection of PTP61F to cytosketal regulation through focal adhesion components. Subsequent genetic and biochemistry studies by others in our group have since demonstrated that several of the identified substrates are direct and physiological substrates in vivo. Moreover, this strategy has already been extended to other PTPs such as PTPmeg in Drosophila. The second part of this work aimed to develop a method to facilitate the quantitative analysis of multiple proteins in Drosophila in vivo. This quantitative strategy could be combined with other methods such as substrate trapping or RNAi interference to help us to investigate the functional PTPome. We have successfully established a viable SILAC fly approach for this aim. We showed that metabolic labeling with lysine was suboptimal for quantitative experiment in Drosophila in vivo because the conversion of heavy isotope labeled lysine to other unexpected amino acids affected quantitation accuracy. On the other hand, we have also investigated the alternative use of Arg10 in the applications of in vivo SILAC strategy. Although the conversion of arginine to other amino acid could be observed, the conversion rates seemed to be negligible except for proline. Thus upon a simple normalization of arginine to proline effcet, the systematic bias could be largely rectified. In our Arg0 to Arg10 1:1 test, 98% of protein ratios were within ±0.5, indicative of high accuracy. Additionally, we have also applied the SILAC flies using Arg10 labeling strategy for quantitative comparison of the expressed proteomes, between puparium formation and 3 h after puparium. Our data has shown similar changes in expression at the mRNA and protein levels. We have also successful used 5% SILAC yeast fly food to replace the diet with exclusive yeast. This makes SILAC flies inexpensive and more practical for quantitative experiments. Collectively we have established generic platforms incorporating shotgun and quantitative proteomic strategies to facilitate the study of functional PTPomics.