Online High-Flow Peptide Immunoaffinity Enrichment and Nanoflow LC-MS/MS: Assay Development for Total Salivary Pepsin/Pepsinogen

BACKGROUND: Detection limit challenges associated with measuring low-abundance protein biomarkers can be addressed with hybrid immunoaffinity-mass spectrometric assays, such as antipeptide antibody capture followed by liquid chromatography/tandem mass spectrometry (LC-MS/MS). Popular assay formats use magnetic bead-based immunoaffinity enrichment and nanoflow LC-MS/MS or high-flow immunoaffinity chromatography coupled online to conventional LCMS/MS. As a proof of principle, we describe a novel online immunoaffinity LC-MS/MS configuration that combines high-flow peptide immunoaffinity enrichment and nanoflow LC-MS/MS. METHODS: We configured and validated an assay for the measurement of total pepsin/pepsinogen from human saliva that uses a pepsinogen standard. Saliva was heat-inactivated to quench residual enzymatic activity and then digested with endoproteinase AspN. Online immunoaffinity enrichment using an antipeptide antibody directed against the pepsin C-terminal sequence, DRANNQVGLAPVA, was linked to nanoflow liquid chromatography and selected reaction monitoring mass spectrometry. We used the assay to measure pepsin/pepsinogen concentrations in human saliva from presumed healthy volunteers. RESULTS: Heat inactivation at 100 degrees C for 25 min stabilized the target peptide. The final assay had < 15% interassay relative error and < 15% interassay CV across a range of 4.08-2980 pmol/L human pepsinogen (0.165-120 mu g/L). Low but quantifiable signals were observed in some samples from presumed normal healthy volunteers ranging from 4.3 to 16.6 pmol/L (0.17-0.67 mu g/L) total salivary pepsin/pepsinogen. CONCLUSIONS: This assay approach provides a high-sensitivity platform for protein bioanalysis in the low picomolar range. It bears the potential to deliver additional data on the salivary occurrence of pepsin/pepsinogen with greater confidence than previously. (C) 2010 American Association for Clinical Chemistry