title: High-throughput ultra-high-performance liquid chromatography/tandem mass spectrometry quantitation of insulin-like growth factor-I and leucine-rich alpha-2-glycoprotein in serum as biomarkers of recombinant human growth hormone administration.
abstract: Insulin-like growth factor-I (IGF-I) is a known biomarker of recombinant human growth hormone (rhGH) abuse, and is also used clinically to confirm acromegaly. The protein leucine-rich alpha-2-glycoprotein (LRG) was recently identified as a putative biomarker of rhGH administration. The combination of an ACN depletion method and a 5-min ultra-high-performance liquid chromatography/tandem mass spectrometry (uHPLC/MS/MS)-based selected reaction monitoring (SRM) assay detected both IGF-I and LRG at endogenous concentrations. Four eight-point standard addition curves of IGF-I (16-2000 ng/mL) demonstrated good linearity (r(2) = 0.9991 and coefficients of variance (CVs) <13%). Serum samples from two rhGH administrations were extracted and their uHPLC/MS/MS-derived IGF-I concentrations correlated well against immunochemistry-derived values. Combining IGF-I and LRG data improved the separation of treated and placebo states compared with IGF-I alone, further strengthening the hypothesis that LRG is a biomarker of rhGH administration. Artificial neural networks (ANNs) analysis of the LRG and IGF-I data demonstrated an improved model over that developed using IGF-I alone, with a predictive accuracy of 97%, specificity of 96% and sensitivity of 100%. Receiver operator characteristic (ROC) analysis gave an AUC value of 0.98. This study demonstrates the first large scale and high throughput uHPLC/MS/MS-based quantitation of a medium abundance protein (IGF-I) in human serum. Furthermore, the data we have presented for the quantitative analysis of IGF-I suggest that, in this case, monitoring a single SRM transition to a trypsin peptide surrogate is a valid approach to protein quantitation by LC/MS/MS.
authors.affiliation: Drug Development Services, Quotient Bioresearch Ltd., Fordham CB7 5WW, UK. richard.kay@quotientbioresearch.com
authors.forename: R G
authors.initials: RG
authors.lastname: Kay
authors.forename: C
authors.initials: C
authors.lastname: Barton
authors.forename: C P
authors.initials: CP
authors.lastname: Velloso
authors.forename: P R
authors.initials: PR
authors.lastname: Brown
authors.forename: C
authors.initials: C
authors.lastname: Bartlett
authors.forename: A J
authors.initials: AJ
authors.lastname: Blazevich
authors.forename: R J
authors.initials: RJ
authors.lastname: Godfrey
authors.forename: G
authors.initials: G
authors.lastname: Goldspink
authors.forename: R
authors.initials: R
authors.lastname: Rees
authors.forename: G R
authors.initials: GR
authors.lastname: Ball
authors.forename: D A
authors.initials: DA
authors.lastname: Cowan
authors.forename: S D
authors.initials: SD
authors.lastname: Harridge
authors.forename: J
authors.initials: J
authors.lastname: Roberts
authors.forename: P
authors.initials: P
authors.lastname: Teale
authors.forename: C S
authors.initials: CS
authors.lastname: Creaser
mesh_terms: D000328:Adult; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D016903:Drug Monitoring / Q000379:methods*; D006023:Glycoproteins / Q000097:blood / Q000737:chemistry*; D019382:Human Growth Hormone / Q000008:administration & dosage*; D006801:Humans; D007334:Insulin-Like Growth Factor I / Q000737:chemistry*; D008297:Male; D053719:Tandem Mass Spectrometry / Q000379:methods*; D055815:Young Adult
chemical_list: D006023:Glycoproteins; C015246:LRG1 protein, human; D019382:Human Growth Hormone; D007334:Insulin-Like Growth Factor I