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Doctor of Philosophy
Secreted proteins play important roles in many cellular functions and molecular processes. Because secreted proteins potentially enter the blood stream, they can serve as valuable measures of health and disease useful for disease diagnosis and prognosis, therapeutic target identification, and patient stratification in personalized medicine. Consequently, significant interest exists in secreted protein analysis within complex biospecimens, particularly blood but significant bioanalytical challenges including the wide protein dynamic range >10 orders of magnitude remain. The cellular secretome therefore represents a viable alternative to direct biomarker discovery in biofluids. Finally, cellular systems are amenable to labeling for the production of intact stable isotope labeled (SIL) proteins that can be used as global internal standards for quantitative proteomics. In this dissertation, two secretome-focused studies were undertaken.
The first study involving candidate biomarker discovery in radiation-induced autophagy utilized the p53-null and inducible H1299 non-small cell lung cancer (NSCLC) secretome. The study identified 364 secreted proteins that were mainly associated with exosomes (N=224) and chaperone activity (N=21). CHGB and SCG2 were identified as potential population-based biomarkers (for patient stratification) due to their consistent overexpression in p53-null H1299 cell secretomes compared to p53-wt cells before and after radiation. FAM3C, CANX, EIF5A, GPI, and TXNRD1 were identified as candidate biomarkers for patient prognosis following radiotherapy due to their differential expression only in response to radiation treatment.
In the second study, a comprehensive glycoproteomics characterization of the SILAC-labeled HepG2 secretome was undertaken. 1635 SIL proteins, 492 of which were major plasma proteins including 192 cancer biomarkers were identified with high sequence coverage spanning six orders of magnitude. EDTA plasma spiked with the SIL secretomes yielded 63 proteins that were quantified with H/L ratios in all samples out of 1405 total proteins identified. Additionally, LC-MS/MS analysis of the Con A and WGA enriched 72h secretome:plasma sample afforded an opportunity to clearly distinguish between glycoproteins in plasma and the HepG2 secretome that share/differ in N-glycan structures.
Collectively, the two studies reveal the suitability of the H1299 cancer cell secretome as an experimental model for biomarker studies and support the HepG2 secretome as a viable platform for producing SIL glycoproteins.
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