High‐throughput ultra‐high‐performance liquid chromatography/tandem mass spectrometry quantitation of insulin‐like growth factor‐I and leucine‐rich α‐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.

Targeted mass spectrometry analysis of the proteins IGF1, IGF2, IBP2, IBP3 and A2GL by blood protein precipitation

“…The conditions for protein precipitation by organic solvents can be adjusted to preferentially precipitate proteins of medium and high molecular weight (MW) while recovering low mass proteins in the supernatant [19,[28][29][30][31][32][33]. We precipitated proteins by using 60% ACN as it provided the best compromise between efficient precipitation of medium and high mass proteins and sufficient protein recovery, which is important as it allows for reduction of the sample volume needed for the analysis.…”

“…The proteins of medium and low abundance insulin-like growth factor 1 (IGF1), insulin-like growth factor 2 (IGF2), insulin-like growth factor binding protein 2 (IBP2), insulin-like growth factor binding protein 3 (IBP3) and leucine-rich alpha-2-glycoprotein (A2GL) have been proposed as indirect biomarkers for the detection of GH administration [17][18][19], of interest in anti-doping analysis and for prognostic monitoring of GH-related disease treatments, and as putative biomarkers for breast cancer diagnosis [20][21][22][23][24]. Their quantification is typically achieved independently by classical methods such as ELISA or RIA assays [18], necessitating blood sample volumes that range between 50 and 200 μL for each assay.…”

“…Their quantification is typically achieved independently by classical methods such as ELISA or RIA assays [18], necessitating blood sample volumes that range between 50 and 200 μL for each assay. Specific LC-MS/MS methods have been recently developed for plasma/ serum samples for quantification of IGF1 [25] and IGF1 and A2GL [19] by protein precipitation, and of IGF1 by specific antibody-based purification [26], necessitating 100 μL, 30 μL and 40 μL, respectively. In this manuscript, we describe a sensitive and scalable method for the quantification of all five proteins by protein precipitation and selected reaction monitoring (SRM) LC-MS/MS analysis [27], requiring a total of 7 μL of plasma, and reaching a throughput of up to 80 analyses per day.…”

“…In this manuscript, we describe a sensitive and scalable method for the quantification of all five proteins by protein precipitation and selected reaction monitoring (SRM) LC-MS/MS analysis [27], requiring a total of 7 μL of plasma, and reaching a throughput of up to 80 analyses per day. This method includes an initial protein precipitation protocol optimised to extract the low mass proteins from blood samples [19,[28][29][30][31][32][33] for reduced signal suppression and increased sensitivity. We have benchmarked this method for the analysis of 40 human blood samples including 20 patients diagnosed with breast cancer.…”

Interlaboratory Agreement of Insulin-like Growth Factor 1 Concentrations Measured by Mass Spectrometry

“…The sample preparation method must also completely dissociate and remove high-affinity binding proteins that complex with IGF-1 in serum and interfere with quantification (20 ). Previous methods have used acetonitrile depletion of serum to measure IGF-1 peptides after trypsin digestion (21,22 ).…”