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Quantitative Proteomic Analysis of ATP-binding Proteins

  • Author(s): Miao, Weili
  • Advisor(s): Wang, Yinsheng
  • et al.
Abstract

Targeted proteomics techniques, which rely on multiple-reaction monitoring (MRM) or parallel-reaction monitoring (PRM), have become extensively employed in quantitative proteomics studies. In this dissertation, we developed and employed targeted proteomics methods to the analyze comprehensively ATP-binding proteins, including kinases, heat shock proteins and helicases.

In Chapter 2, we expanded the kinome MRM library to include ~80% of the human kinome and employed this library, together with an ATP-affinity probe, for profiling comprehensively alterations of the kinases upon treatment with methylglyoxal. Our results led to the quantification of 328 unique kinases and the novel discovery of kinases involved in diabetes signaling pathways.

In Chapter 3, we developed a PRM-based targeted proteomic method to monitor the protein expression of ~ 80% of the human kinome. By employing this method, together with the method in Chapter 2, we assessed the alterations in protein expression and ATP binding affinities of over 300 kinases in cultured human cells elicited by three FDA-approved small-molecule kinase inhibitors (dabrafenib, vemurafenib and imatinib). We identified CHK1 and MAP2K5 as novel target kinases for imatinib and vemurafenib, respectively. We also employed the PRM-based targeted proteomic method to examine the reprogramming of the human kinome during colorectal cancer (CRC) metastasis in Chapter 4 and discovered phosphoribosyl pyrophosphate synthetase 2 (PRPS2) as a promoter for CRC metastasis.

In Chapter 5, we developed a PRM-based targeted proteomic method to quantitatively assess 70% of the human heat shock proteome and applied this method to analyze differential expression of heat shock proteins in three matched primary/metastatic pairs of melanoma cell lines. We were able to discover DNAJB4 as a suppressor for melanoma metastasis. We also found the expression of DNAJB4 was stimulated by three HSP90 inhibitors treatment in Chapter 6. We then demonstrated that the elevated expression of DNAJB4 occurs, in part, through an epitranscriptomic mechanism.

In Chapter 7, we developed a PRM-based targeted proteomic method that allows for the quantification of >80% of the human helicase proteome. By employing this method, together with a CRISPR-Cas9 genome editing method, we discovered that helicases may constitute an important group of client proteins for HSP90.

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