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.