Comparative proteomics in search of cancer disease markers
Abstract
This study demonstrates the methodology development for search of cancer disease markers with proteomic tools. The first portion of the research investigated the affinity selection using Lotus tetragonolobus lectin (LTA), which is specific for fucose α(1,6) linked to the inner core GlcNAc. The study with the model protein laetoferrin revealed the selectivity of the synthesized LTA column. The study was extended to more complicated human serum. The glycopeptide selected was deglycosylated in 18O containing medium to elucidate the structure of the glycan binding site. The second portion of the work was focused on high throughput peptide separations with monolithic based reversed-phase columns, which have relatively low backpressure at high mobile phase linear velocity. The analyte recovery, resolution, and concentration of the eluted peptides were studied from 1.0 ml/min to 10.0 ml/min with sequential increment. The results indicated that the analyte recovery and column efficiency remained constant from low to high flow rate. The third portion dealt with the quantification of the protein expression level with GIST reagent. Tryptic digests from horse cytochrome C was labeled with 1H5 and 2H5-forms of succinimidyl propionate, however, the separation between the heavy and light isotopic forms were significant. Different sets of isotopic codings, 12C 3 and 13C3-propionate were used to derivatize the bovine fetuin glycopeptide. The resolution between 12C and 13C-coded forms was decreased 10 fold. The affinity selection and GIST quantification technologies were applied to the search of cancer markers in lymphoma dogs. The serum from the lymphosarcoma dogs with and without chemotherapy were tryptic digested and GIST reagent labeled individually. LTA lectin affinity column selected fucose-containing glycopeptides. The differential display of selected protein expression map was generated based on isotope ratios, while peptides with significant concentration changes were sequenced with MS/MS to find the parent proteins.
Degree
Ph.D.
Advisors
Regnier, Purdue University.
Subject Area
Analytical chemistry|Oncology
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