title: Detection and differentiation of 22 kDa and 20 kDa Growth Hormone proteoforms in human plasma by LC-MS/MS.
abstract: Human growth hormone (GH) is suspected to be widely and illegally used in sport to improve athletes' performance. For the detection of GH abuse, blood samples are screened for abnormal ratios between the 22 and 20 kDa GH proteoforms that demonstrate the administration of the synthetic hormone. Current detection methods are based on classical immunoassays as they provide sufficient sensitivity for the detection of GH proteoforms. These antibody based methods, however, suffer from unclear selectivity and potential cross-reactivity towards similar proteins. For unambiguous GH detection, we report a Mass Spectrometry ImmunoAssay (MSIA) that first enriches GH from plasma with an antibody of relatively low specificity, and subsequently quantifies the 22 and 20 kDa proteoforms by Selected Reaction Monitoring (SRM) LC-MS/MS analysis. This method proved superior to an antibody-free strategy based on GH purification by protein precipitation. Using GH-MSIA we successfully quantified the 22/20 kDa GH ratio in post-exercise capillary plasma extracted from two individuals. This GH-MSIA is applicable to anti-doping and GH-related disease analysis.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Southern Denmark, Campusvej 55, Odense M DK-5230, Denmark.
authors.forename: Gerard
authors.initials: G
authors.lastname: Such-Sanmartín
authors.affiliation: Thermo Scientific, Odense, Denmark.
authors.forename: Nicolai
authors.initials: N
authors.lastname: Bache
authors.affiliation: Bio-analysis Group, Neuroscience Research Program, Institut Hospital del Mar d'Invesigacions Mèdiques, Barcelona Biomedical Research Park, Spain.
authors.forename: Jaume
authors.initials: J
authors.lastname: Bosch
authors.affiliation: Bio-analysis Group, Neuroscience Research Program, Institut Hospital del Mar d'Invesigacions Mèdiques, Barcelona Biomedical Research Park, Spain; Department of Experimental and Health Sciences, Pompeu Fabra University, Barcelona Biomedical Research Park, Spain.
authors.forename: Ricardo
authors.initials: R
authors.lastname: Gutiérrez-Gallego
authors.affiliation: Bio-analysis Group, Neuroscience Research Program, Institut Hospital del Mar d'Invesigacions Mèdiques, Barcelona Biomedical Research Park, Spain; Department of Experimental and Health Sciences, Pompeu Fabra University, Barcelona Biomedical Research Park, Spain.
authors.forename: Jordi
authors.initials: J
authors.lastname: Segura
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Southern Denmark, Campusvej 55, Odense M DK-5230, Denmark. Electronic address: jenseno@bmb.sdu.dk.
authors.forename: Ole N
authors.initials: ON
authors.lastname: Jensen
mesh_terms: D000328:Adult; D001774:Blood Chemical Analysis / Q000379:methods; D002853:Chromatography, Liquid / Q000379:methods*; D015444:Exercise / Q000502:physiology; D005260:Female; D019382:Human Growth Hormone / Q000097:blood*; D006801:Humans; D007118:Immunoassay / Q000379:methods; D008297:Male; D008970:Molecular Weight; D020033:Protein Isoforms / Q000097:blood; D059748:Proteolysis; D012680:Sensitivity and Specificity; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D020033:Protein Isoforms; D019382:Human Growth Hormone
keywords: Antibody purification; Blood; Growth hormone; LC-MS/MS; Mass spectrometric immunoassay; Protein precipitation