Quantitative proteomics using chemical probes

Chemical proteomics utilizes synthetic chemical probes to selectively capture and enrich particular classes of proteins from complex biological samples, enabling their characterization by proteomic techniques. The study focused on development of a robust chemical proteomics method to characterise and quantitate cellular protein targets in biological models using an in-house developed chemical probe that harbor's a potent protein kinase C inhibitor, bisindolylmaleimide (Bis) as the reactive group. The newly developed chemical proteomics method was applied to characterise the Bis-binding protein targets in basal breast cancer cell line (MDA-MB-231). Two protein kinases, serine/arginine-rich protein-specific kinase 1 (SRPK1) and interferon induced RNAdependent protein kinase (PKR) were identified that were not previously known to bind to Bis. Further, a SILAC based quantitation strategy was used to examine changes in Bisbinding proteins following phorbol ester (PMA) stimulation. This provided novel evidence for PMA regulation of the enzymes glyceraldehyde-3-phosphate dehydrogenase (GAPDH), nucleolar RNA helicase 2 (DDX21) and heterogenous ribonucleoprotein M (HNRPM). This cell culture based cancer model provided the foundation to expand the chemical proteomics study to a tissue based system using Bis-probes. The study focused on Bisbinding metabolic enzymes from the liver of mice bearing a C26 adenocarcinoma with accompanying high IL-6 levels that induced cancer cachexia (cachectic). Bis-binding enzymes were compared with mice bearing a C26 adenocarcinoma with low IL-6 levels that failed to induced cancer cachexia (non-cachectic) and control mice with no tumour. Spectral counting was used to quantify the relative abundance of the Bis-captured enzymes. In the cachectic model overall carbohydrate metabolism, the branched chain amino acid degradation and lipid metabolism was significantly reduced, while the hepatic acute phase protein synthesis was induced. Further, the marked reduction in the cytochrome P450 enzymes is consistent with the impaired xenobiotic metabolism and the subsequent chemotoxicity encountered in cancer cachexia patients. The Bis-probe was also applied in a human adipose derived stem cell (hADSC) model to characterise and quantitate the differential expression of Bis-binding proteins during chemical induction of neurogenic differentiation. Several key enzymes involved in the neuronal differentiation of stem cells were captured by the Bis-probe at different time points of chemical induction. In the final study, kinase active-site peptide enrichment analysis was carried out in phorbol ester stimulated and resting basal breast cancer cells using a newly commercialised desthiobiotin-ATP probe. This analysis showed more than two-thirds of the identified targets in both the PMA treated and the resting MDA-MB-231 cells as ATP binding proteins which included 23 protein kinases. Of the identified protein kinases16 were common to both the treated and resting groups and 7 protein kinases each were unique to the PMA treated samples and the resting cells. In summary, this thesis demonstrates the versatile use of quantitative chemical proteomics for biomedical investigations.