Comparison of a stable isotope labeled (SIL) peptide and an extended SIL peptide as internal standards to track digestion variability of an unstable signature peptide during quantification of a cancer biomarker, human osteopontin, from plasma using capillary microflow LC-MS/MS.

Human osteopontin (hOPN) is a secreted plasma protein which is elevated in various cancers and is indicative of poor prognosis. Here we describe investigations involving an extended peptide internal standard to track an unstable signature peptide followed by further method development and validation for quantitative measurement of hOPN from plasma using microflow liquid chromatography and tandem mass spectrometry (MFLC-MS/MS). A biologically relevant tryptic peptide 'GDSVVYGLR' was used as a signature peptide for this method. The optimized method involved immunocapture of the analyte protein using hOPN specific antibodies followed by trypsin digestion to obtain the signature peptide. Analysis was carried out on a Waters IonKey/MS system using a flow rate of 2.5μL/min. Immunocapture buffer was used as a surrogate matrix for the validation studies. The method was validated over a range of 25-600ng/mL. Intra-assay and inter-assay accuracies were within ±13%. Intra-assay and inter-assay precision were within 17%. A stable isotope labeled (SIL) peptide GDSVVYGLR* and an extended SIL peptide TYDGRGDSVV*YGLRSKSKKF were evaluated as internal standards (IS) to account for signature peptide digestion instability and variability. Inherent digestion variability i.e., under controlled conditions, was within ±20% with both IS peptides. In digestion variability studies, where trypsin activity was varied (20-180%), the use of the extended SIL peptide as an internal standard limited the variability to within ±30% of the normalized response. Alternatively, when the SIL peptide was used as the internal standard, the variability ranged from -67.4% to 50.6% of the normalized response. The applicability of the validated method was demonstrated by quantification of OPN from plasma samples obtained from 10 healthy individuals and 10 breast cancer patients. The plasma OPN concentrations in healthy individuals ranged from 38 to 85ng/mL with a mean concentration of 55.4±15.3ng/mL. A 1.5-12 fold increase in OPN concentrations, ranging from 85 to 637ng/mL, was seen in breast cancer patient samples.