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 LC-MS/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 microg/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 microg/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.
Neubert
Hendrik
H
Pharmacokinetics Dynamics & Metabolism and Biotherapeutics & Translational Research, Pfizer Limited, Kent, UK. hendrik.neubert@pfizer.com
Gale
Jeremy
J
Muirhead
David
D
D002138:Calibration; D002853:Chromatography, Liquid; D006801:Humans; D007118:Immunoassay; D013058:Mass Spectrometry; D046210:Microfluidic Analytical Techniques; D036103:Nanotechnology; D010434:Pepsin A / Q000032:analysis*; D020116:Pepsinogen A / Q000032:analysis* / Q000592:standards; D012015:Reference Standards; D012463:Saliva / Q000737:chemistry*
D020116:Pepsinogen A; D010434:Pepsin A