| The separation and detection of proteins and peptides are crucial in elucidating disease processes and biomarker discovery. The following research describes the development of various liquid separation schemes for pre-fractionation of complex biological mixtures prior to analysis by mass spectrometry (MS). Proteomic profiling methods were developed for analysis of different types of biological samples including cells, extracellular fractions, and urine. The complex and unique nature of each sample type present a significant analytical challenge and has stimulated interest in approaches to reduce sample complexity prior to identification by mass (MS) or tandem (MS/MS) mass spectrometry. The approaches in this dissertation were developed to investigate human cancers, particularly, human breast malignancy, an isogenic metastatic model, and bladder cancer.; The first study demonstrates a preparative liquid isoelectric focusing and mass mapping technique to study a xenograft model of progressive proliferative breast disease consisting of one normal (MCF10A) and two malignant cell lines (MCFIOACA1a and MCF10ACA1d). Of the 40 proteins identified, 22 were found to be highly expressed in the malignant cells.; In a second part of research, chromatofocusing/mass mapping technique was used for the study of the isogenic metastasis model of MDA-MB-435 cell lines. The approach was used to analyze proteins secreted from metastasic M4A4 and nonmetastatic NM2C5 cells as well as the protein contents collected from the cell lysates. A total of 72 differentially expressed proteins were identified in this model. Interestingly, many of the expressed proteins in this model were found to be modified.; The third part of research focused on the development of high throughput methods. Monolithic high performance liquid chromatography-MS/MS was performed for rapid protein identification and resulted in the identification of large number of proteins in fully malignant human breast cancer cells. In addition, a rapid, high-sensitivity technique, Concanavalin A affinity chromatography/nanoflow liquid chromatography-MS/MS, was developed to investigate the profiling of N-linked glycoproteins in human urine specimens obtained from bladder cancer patients and normal individuals. A total of 186 urinary proteins were identified with high confidence and the method was applicable to as little as 10 mL of urine. |
