title: Development of high-throughput chemical extraction techniques and quantitative HPLC-MS/MS (SRM) assays for clinically relevant plasma proteins.
abstract: The clinical application of targeted plasma protein analysis by selective reaction monitoring of peptides using LC-MS/MS requires the development of robust, inexpensive protein extraction techniques with the potential for high-throughput applications. We present the development of a novel mixed-mode solid phase extraction (SPE) technique for the removal of high abundance and high molecular weight proteins from plasma. This technique, coupled with fused-core HPLC-MS/MS analysis is compared to a previously developed extraction method to study a range of proteins in plasma, including routinely measured biomarkers of growth hormone action. To further validate this technique, it was used for the quantification of insulin-like growth factor I (IGF-I) levels and compared to a state-of-the-art immunoassay on a fully automated analyzer. Clinical reference materials were applied for method development to allow for further interlaboratory comparisons. The LC-MS/MS approach quantified IGF-I in plasma with an accuracy that is within the guidelines for macromolecular assays in a regulated laboratory environment. Furthermore, IGF-I levels determined using the SPE and ACN methods with LC-MS/MS analysis correlated well with the immunoassay results. This demonstrates the applicability of mixed-mode SPE coupled with fused-core HPLC-MS/MS to quantify plasma proteins with results suitable for clinical applications.
authors.affiliation: Quotient Bioresearch Ltd, Newmarket Road, Fordham, Cambridgeshire CB7 5WW, United Kingdom.
authors.forename: Chris
authors.initials: C
authors.lastname: Barton
authors.forename: Richard G
authors.initials: RG
authors.lastname: Kay
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Gentzer
authors.forename: Frank
authors.initials: F
authors.lastname: Vitzthum
authors.forename: Steve
authors.initials: S
authors.lastname: Pleasance
mesh_terms: D000097:Acetonitriles / Q000737:chemistry*; D001798:Blood Proteins / Q000032:analysis*; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D006801:Humans; D007334:Insulin-Like Growth Factor I / Q000032:analysis; D015203:Reproducibility of Results; D052616:Solid Phase Extraction / Q000379:methods*; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D000097:Acetonitriles; D001798:Blood Proteins; D007334:Insulin-Like Growth Factor I; C032159:acetonitrile