A proteomic approach combining MS and bioinformatic analysis for the detection and identification of biomarkers of administration of exogenous human growth hormone in humans

Boateng, Joshua; Kay, Richard G.; Lancashire, Lee; Brown, Pamela R.; Velloso, Cristiana P.; Bouloux, Pierre; Teale, P.; Roberts, Jane D.; Rees, Robert C.; Harridge, Sdr; Goldspink, Geoffrey; Creaser, Colin S.

doi:10.1002/prca.200800190

Cited by 8 publications

(3 citation statements)

References 36 publications

(47 reference statements)

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“…Furthermore, rbST immunoassays were not capable to distinguish the almost identical recombinant and endogenous forms of bST [19], [20], [24], [25] and mass spectrometry-based methods on the other hand required very tedious sample preparation procedures [26], [27]. For screening of rbST in cattle, a few biomarker-based methods were developed, but focused on a single candidate biomarker only [4], [9], [28]–[30]. In a recent study, three candidate biomarkers were combined in one screening tool, but the <5% false-compliant rate target could not be achieved [31].…”

Section: Introductionmentioning

confidence: 99%

Multiple Protein Biomarker Assessment for Recombinant Bovine Somatotropin (rbST) Abuse in Cattle

Ludwig¹,

Smits²,

Veer³

et al. 2012

PLoS ONE

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Biomarker profiling, as a rapid screening approach for detection of hormone abuse, requires well selected candidate biomarkers and a thorough in vivo biomarker evaluation as previously done for detection of growth hormone doping in athletes. The bovine equivalent of growth hormone, called recombinant bovine somatotropin (rbST) is (il)legally administered to enhance milk production in dairy cows. In this study, first a generic sample pre-treatment and 4-plex flow cytometric immunoassay (FCIA) were developed for simultaneous measurement of four candidate biomarkers selected from literature: insulin-like growth factor 1 (IGF-1), its binding protein 2 (IGFBP2), osteocalcin and endogenously produced antibodies against rbST. Next, bovine serum samples from two extensive controlled rbST animal treatment studies were used for in vivo validation and biomarker evaluation. Finally, advanced statistic tools were tested for the assessment of biomarker combination quality aiming to correctly identify rbST-treated animals. The statistical prediction tool k-nearest neighbours using a combination of the biomarkers osteocalcin and endogenously produced antibodies against rbST proved to be very reliable and correctly predicted 95% of the treated samples starting from the second rbST injection until the end of the treatment period and even thereafter. With the same biomarker combination, only 12% of untreated animals appeared false-positive. This reliability meets the requirements of Commission Decision 2002/657/EC for screening methods in veterinary control. From the results of this multidisciplinary study, it is concluded that the osteocalcin – anti-rbST-antibodies combination represent fit-for-purpose biomarkers for screening of rbST abuse in dairy cattle and can be reliably measured in both the developed 4-plex FCIA as well as in a cost-effective 2-plex microsphere-based binding assay. This screening method can be incorporated in routine veterinary monitoring programmes: in the European Union for detection of rbST abuse and in the control of rbST-free dairy farms in the United States of America and other countries.

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Section: Introductionmentioning

confidence: 99%

Multiple Protein Biomarker Assessment for Recombinant Bovine Somatotropin (rbST) Abuse in Cattle

Ludwig¹,

Smits²,

Veer³

et al. 2012

PLoS ONE

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“…The ACN depletion method used in this study has been shown to enrich the serum concentrations of a number of low molecular weight proteins prior to nano‐LC/MS/MS analysis 21. The depletion technique (in combination with ANN analysis) was applied successfully to the identification of LRG as a biomarker to rhGH abuse in humans 14. We have further demonstrated that ACN depletion, in combination with tryptic digestion and uHPLC/MS/MS with SRM detection, may be used for the quantitative analysis of serum proteins in large sample cohorts.…”

Section: Discussionmentioning

confidence: 99%

“…A biomarker discovery study performed by our laboratories identified the protein leucine‐rich α‐2‐glycoprotein (LRG) as a putative biomarker of rhGH administration through the use of liquid chromatography/mass spectrometry (LC/MS) and artificial neural networks (ANNs) 14. To confirm LRG as a true biomarker of rhGH abuse, samples from additional administrations would need to be analysed.…”

mentioning

confidence: 99%

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

Kay

Barton

Velloso

et al. 2009

Rapid Comm Mass Spectrometry

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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.

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Current Awareness in Drug Testing and Analysis

2010

Drug Testing and Analysis

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Alcohol and Medicines (DRUID) program, and standardization of DUID research. Whereas OF testing is now routine in many monitoring programs, currently, the major shortcoming is the dearth of controlled drug administration studies published to guide interpretation. This review presents an overview of OF testing benefits and limits. Progress in OF that has occurred during the last 5 years in respect of collection, screening, confirmation, and interpretation of cannabinoids, opioids, amphetamines, cocaine, and benzodiazepines is reviewed. Methodology examined includes controlled drug administration studies, immunoassay and chromatographic methods, collection devices, point-of-collection testing device performance, and recent applications of OF testing. Substance Abuse and Mental Health Services Administration approval of OF testing has been delayed because questions about drug OF disposition had not yet been resolved, and performance of collection device and testing assays necessitated advancement. Unusual mass spectrometric dissociation pathway of protonated isoquinoline-3-carboxamides due to multiple reversible water adduct formation in the gas phase Collision-induced dissociation behavior of various substituted isoquinoline-3carboxamides, which are amongst a group of drug candidates for the treatment of anemic disorders (e.g., FG-2216), was studied and facilitated the formulation of the general mechanisms underlying the unusual fragmentation behavior 200

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A proteomic approach combining MS and bioinformatic analysis for the detection and identification of biomarkers of administration of exogenous human growth hormone in humans

Cited by 8 publications

References 36 publications

Multiple Protein Biomarker Assessment for Recombinant Bovine Somatotropin (rbST) Abuse in Cattle

Multiple Protein Biomarker Assessment for Recombinant Bovine Somatotropin (rbST) Abuse in Cattle

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

Current Awareness in Drug Testing and Analysis

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