title: Online high-flow peptide immunoaffinity enrichment and nanoflow LC-MS/MS: assay development for total salivary pepsin/pepsinogen.
abstract: 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.
authors.affiliation: Pharmacokinetics Dynamics & Metabolism and Biotherapeutics & Translational Research, Pfizer Limited, Kent, UK. hendrik.neubert@pfizer.com
authors.forename: Hendrik
authors.initials: H
authors.lastname: Neubert
authors.forename: Jeremy
authors.initials: J
authors.lastname: Gale
authors.forename: David
authors.initials: D
authors.lastname: Muirhead
mesh_terms: 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*
chemical_list: D020116:Pepsinogen A; D010434:Pepsin A