In this dissertation, efforts were focused on the development of targeted proteomic assays to elucidate differences in protein profiles present in diseases and their correlation with other molecular markers (proteins or microRNA).
In Chapter 2, a high-sensitivity TFA-free LC-MS method is described. The analysis of proteins by reversed-phase liquid chromatography (RPLC) commonly involves the use of TFA as an ion-pairing agent, even though it forms adducts and suppresses sensitivity. The presence of adducts can complicate protein molecular weight assignment especially when protein isoforms coelute as in the case of histones. To mitigate the complicating effects of TFA adducts in protein LC-MS, TFA-free methods for protein separation optimized. Protein standards and histones were used to evaluate TFA-free separations using capillary (0.3 mm id) and nanoscale (0.1 mm id) C8 columns with the ion-pairing agents, formic acid or acetic acid. The optimized method was then used to examine the applicability of the approach for histone characterization in human cancer cell lines and primary tumor cells from chronic lymphocytic leukemia patients.
In chapter 3, a targeted mass spectrometry approach was used to examine nitration and nitrosylation of tyrosine residues on tropomyosin. A highly versatile target-driven MS/MS strategy was developed to facilitate identification and quantification of especially low abundance protein post-translational modifications. The LC-MS/MS analysis was carried out on a LTQ-Orbitrap mass spectrometer to take advantage of its high mass resolution and high mass accuracy. A recursive process was used to discover, verify and quantify all the possible nitrated and nitrosylated peptides. Measurement of nitrotyrosine and nitrosyltyrosine on Tm highlights the utility of this approach for discovering and characterizing the challenging low abundant post-translational modifications.
In Chapter 4, phosphotyrosine (pTyr) protein enrichment was used to assist the identification of new potentially druggable targets in FLT3 internal tandem duplication (FLT3 ITD) driven acute myeloid leukemia (AML). The MV4-11 cell line was used in this study because it carries the FLT3 ITD activating mutation. pTyr protein enrichment was used to characterize protein extracts from MV4-11 cells before and after treated with the tyrosine kinases inhibitor PKC412. The use of 4G10 anti-pTyr conjugated beads yielded high quality pTyr enrichment with low background. New FLT3 downstream signaling effectors were expected to be identified from pTyr immunopurified protein complex by LC-MS3 analysis, and serve as novel therapeutic targets in AML.
In Chapter 5, DICER-associated or DGCR8-associated proteins were studied by LC-MS/MS following immunoprecipitation with Flag-DICER or Flag-DGCR8. Among the identified proteins, nucleolin (NCL), a major nucleolar protein often up-regulated in cancer, was detected and confirmed as a component of the DROSHA-DGCR8 complex by coimmunoprecipitation experiments, as previously reported. Further research was focused on this RNA-binding protein NCL to characterize its role in miRNA biogenesis. Experimental data showed NCL regulates the biogenesis of a specific cohort of miRNAs. Since miRNA level modulation as a therapeutic approach has been considered challenging so far, these findings could have a strong clinical impact on the development of future miRNA-based anti-cancer therapies.