Gam Lay Harn
Universiti Sains Malaysia, Malaysia
Posters & Accepted Abstracts: J Chromatogr Sep Tech
Quantitative analysis for protein remains as a challenge. This is because protein is a macromolecule and most of the proteins undergo post translational modification such as glycosylation and phosphorylation. These modifications cause variable detectability of the proteins when analyzed using mass spectrometer. Although, quantitative analysis of proteins is commonly carried out using bioassay, due to limitation in the sensitivity of bioassays, proteins which are present in the minute quantities in biological fluids cannot be determined accurately. Furthermore, interferences from the matrix will also suppress the sensitivity of bioassays. We have developed a method to simultaneously identify and quantify hCG (human chorionic gonadotropin); this hormone is misused by male athlete to enhance their performances in sport by inducing the secretion of endogenous testosterone. The hormone can be found in trace quantity in the urine of doped male athletes. Besides sensitivity, the use of bioassay for quantification of hCG is being challenged as antibodies for hCG cross react with LH (luteinizing hormone) that resembles hCG. Sample preparation plays a critical role in getting good analysis data. We used immune affinity clean up and enrichment self-packed column to isolate hCG in urine. The isolated hormone was then subjected to reduction, alkylation and digestion with trypsin enzyme. A tryptic peptide that is unique to hCG in its amino acid sequence was selected to be the quantifying marker. The marker peptide detected in the full scan MS was isolated and excited to collision induced dissociation (CID) in the MS/MS scan. The MS/MS data generated gave unique identification for hCG; furthermore, three fragment ions of the marker peptide were used for quantification of hCG. Using such an approach, we eliminated the background interferences and thus increase the sensitivity of the method.
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