Online capillary weak cation exchange enrichment hyphenated to nanospray mass spectrometry for quantitation of a basic pegvisomant derived peptide.
A multidimensional LC-MS/MS configuration is described for targeted quantitation of a highly basic peptide chemically derived from pegvisomant as a surrogate for the intact protein. The method developed for an immunogenicity assay employed orthogonal separation of the target peptide commencing with flowing microgram quantities of total digests from a protein G extraction of human serum through a weak cation exchange (WCX) monolithic trap column. The basic peptide of interest was retained on the WCX column. Following a washing procedure, peptides were eluted with acetic acid and retained on a down-stream reverse phase trap compatible with online nanoflow LC-MS/MS. Such a LC configuration including the use of other sorbents such as strong cation exchange media is proposed as an enabling tool in assay development for quantitative protein bioanalysis by LC-MS/MS.
Neubert
Hendrik
H
Pfizer Global Research and Development, Ramsgate Road, Sandwich, Kent, UK. hendrik.neubert@pfizer.com
James
Ian
I
D053801:Capillary Electrochromatography; D002411:Cation Exchange Resins; D002853:Chromatography, Liquid / Q000295:instrumentation*; D003488:Cyanogen Bromide; D019382:Human Growth Hormone / Q000031:analogs & derivatives* / Q000737:chemistry / Q000276:immunology; D006801:Humans; D018189:Immunomagnetic Separation; D009419:Nerve Tissue Proteins / Q000276:immunology; D010455:Peptides / Q000097:blood / Q000302:isolation & purification*; D021241:Spectrometry, Mass, Electrospray Ionization; D053719:Tandem Mass Spectrometry
D002411:Cation Exchange Resins; C029152:G-substrate; D009419:Nerve Tissue Proteins; D010455:Peptides; D019382:Human Growth Hormone; C406545:pegvisomant; D003488:Cyanogen Bromide