Quantitative mass spectrometric investigations of protein biomarkers: Serum thymidine kinase 1 and human osteopontin

Mass spectrometry is being increasingly used in biomarker research mainly due to its ability to achieve high selectivity coupled with high sensitivity. This dissertation focuses on quantitative mass spectrometric investigations of two protein biomarkers i.e. serum thymidine kinase 1 (TK1) and human osteopontin (hOPN).;A liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) method for measuring the activity of TK1 in serum by monitoring the conversion of a TK1 specific exogenous substrate, 3'-deoxy-3'-fluorothymidine (FLT), to its mono-phosphorylated form 3'-deoxy-3'-fluorothymidine monophosphate (FLT-MP). A method to quantify FLT-MP on LC-MS/MS was developed and validated. The method was linear over the range of 2.5-2000 ng/mL with a mean correlation coefficient of 0.9935. The applicability of the developed method was demonstrated by measuring TK1 activity in serum from hepatocellular carcinoma (HCC) patients and age-matched controls.;Another method was developed and validated for quantifying hOPN from plasma using immunoaffinity isolations coupled with microflow LC-MS/MS. A biologically relevant tryptic peptide `GDSVVYGLR' was used as a signature peptide. The method was validated over a range of 25-600 ng/mL. A stable isotope labeled (SIL) peptide GDSVVYGLR* and an extended SIL peptide TYDGRGDSVV*YGLRSKSKKF' were evaluated as internal standards (IS) to account for digestion variability. In the digestion variability studies, the use of extended SIL peptide as internal standard limited the total variability within +/-30% in comparison to +/-70% when the SIL-peptide was used. The applicability of the validated method was demonstrated by analyzing plasma samples obtained from 10 healthy individuals and 10 breast cancer patients.;In a proof of concept investigation, a SIL-peptide was evaluated as an internal standard to compensate for immunocapture variability during quantification of hOPN by immunoaffinity coupled LC-MS/MS. Immunocapture variability was induced by varying the antibody amount per well. The use of SIL-peptide reduced the immunocapture variability from +/-81% to +/-37% immunocapture variability. In addition, an attempt was made to develop a cell based system to evaluate tobacco products for cardiovascular risk based on the LC-MS/MS measurement of secreted osteopontin and MMP-3 cleaved osteopontin fragments. However, in our preliminary investigations did not yield detectable levels of the secreted osteopontin concentrations in the cell culture studies and hence this study was terminated.