title: Targeted mass spectrometry analysis of the proteins IGF1, IGF2, IBP2, IBP3 and A2GL by blood protein precipitation.
abstract: Biomarker analysis of blood samples by liquid chromatography (LC) mass spectrometry (MS) is extremely challenging due to the high protein concentration range, characterised by abundant proteins that suppress and mask other proteins of lower abundance. This situation is further aggravated when using fast high-throughput methods, which are necessary for analysis of hundreds and thousands of samples in clinical laboratories. The blood proteins IGF1, IGF2, IBP2, IBP3 and A2GL have been proposed as indirect biomarkers for detection of GH administration and as putative biomarkers for breast cancer diagnosis. We describe a sensitive and scalable method to quantify these 5 proteins of medium and low abundance by selected reaction monitoring (SRM) LC-MS/MS analysis in blood samples. Our method requires 7μL of plasma and reaches a throughput of up to ca. 80 analyses per day. It includes an initial protein precipitation protocol optimised for extraction of low mass proteins from blood samples for reduced signal suppression and increased sensitivity in LC-MS/MS. We benchmarked this method for the analysis of 40 individual blood samples including 20 patients diagnosed with breast cancer.
  
  
  CONCLUSIONS
  The interest for MS-based biomarker analysis in body fluids is steadily increasing as proteomics methodology translates into clinical laboratories. We describe a method for detection of 5 distinct proteins of low mass and medium to low abundance, which are of interest in anti-doping and clinical analysis. The analytical setup is simple and robust and is suitable for high-throughput instrument configurations.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Southern Denmark, Campusvej 55, DK-5230 Odense M, Denmark.
authors.forename: Gerard
authors.initials: G
authors.lastname: Such-Sanmartín
authors.affiliation: Thermo Fisher Scientific, DK-5000 Odense C, Denmark.
authors.forename: Nicolai
authors.initials: N
authors.lastname: Bache
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Southern Denmark, Campusvej 55, DK-5230 Odense M, Denmark; Department of Biochemistry, Pharmacology and Genetics, Odense University Hospital, Odense, Denmark.
authors.forename: Anne K
authors.initials: AK
authors.lastname: Callesen
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Southern Denmark, Campusvej 55, DK-5230 Odense M, Denmark.
authors.forename: Adelina
authors.initials: A
authors.lastname: Rogowska-Wrzesinska
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Southern Denmark, Campusvej 55, DK-5230 Odense M, Denmark. Electronic address: jenseno@bmb.sdu.dk.
authors.forename: Ole N
authors.initials: ON
authors.lastname: Jensen
mesh_terms: D014408:Biomarkers, Tumor / Q000032:analysis / Q000097:blood*; D001943:Breast Neoplasms / Q000097:blood*; D005260:Female; D005590:Fractional Precipitation / Q000379:methods; D006023:Glycoproteins / Q000032:analysis / Q000097:blood*; D006801:Humans; D018971:Insulin-Like Growth Factor Binding Protein 2 / Q000032:analysis / Q000097:blood*; D018972:Insulin-Like Growth Factor Binding Protein 3 / Q000032:analysis / Q000097:blood*; D007334:Insulin-Like Growth Factor I / Q000032:analysis / Q000378:metabolism*; D007335:Insulin-Like Growth Factor II / Q000032:analysis / Q000378:metabolism*; D013058:Mass Spectrometry; D009363:Neoplasm Proteins / Q000032:analysis / Q000097:blood*
chemical_list: D014408:Biomarkers, Tumor; D006023:Glycoproteins; C583557:IGF1 protein, human; C035170:IGF2 protein, human; C577903:IGFBP2 protein, human; C515497:IGFBP3 protein, human; D018971:Insulin-Like Growth Factor Binding Protein 2; D018972:Insulin-Like Growth Factor Binding Protein 3; C015246:LRG1 protein, human; D009363:Neoplasm Proteins; D007334:Insulin-Like Growth Factor I; D007335:Insulin-Like Growth Factor II
keywords: Anti-doping; Blood samples; Insulin-like growth factor; LC-MS/MS; SRM; Targeted analysis