Current trends in mass spectrometry of peptides and proteins: Application to veterinary and sports‐doping control

To detect doping with growth hormone (GH), GH isoform and biomarkers tests are available. Both methods use population-based decision limits. Future testing in anti-doping is progressing toward individual-based reference ranges, and it is possible that with such an approach the sensitivity to detect GH doping may increase. In addition to monitoring different proteins, the use of miRNAs as future GH biomarkers has been discussed. Here we have longitudinally studied the serum concentrations of IGF-I, P-III-NP and the different GH isoforms in nine healthy men prior to, during and after two weeks' administration with low doses (1 and 4 IU/day) of recGH. Moreover, three putative miRNAs were analyzed. The results show that 80% of the participants were identified as atypical findings using the GH isoform test. However, the participants were only positive 1.5-3 hours directly after an injection. Only one of the participants reached a GH-2000 score indicative of doping when a population-based decision limit was applied. When IGF-I and P-III-NP were longitudinally monitored, 88% of the participants were identified above an individual upper threshold arbitrarily calculated as three standard deviations above the mean values of four baseline samples. The miRNA levels displayed large intra-subject variations that did not change in relation to recGH administration. Our results show that the GH isoform test is very sensitive in detecting low doses of recGH but with a short detection window. Moreover, longitudinally monitoring of IGF-I and P-III-NP may be a promising future approach to detect GH doping.

Section: Hours and 3 Hours) After An Injection This Results Is In Agrmentioning

confidence: 99%

Longitudinally monitoring of P‐III‐NP, IGF‐I, and GH‐2000 score increases the probability of detecting two weeks’ administration of low‐dose recombinant growth hormone compared to GH‐2000 decision limit and GH isoform test and micro RNA markers

Lehtihet

Bhuiyan

Dalby

et al. 2018

“…[1][2][3][4] Additionally, growth hormone releasing peptides (GHRPs) trigger the complete growth hormone cascade and thus, all growth-hormone-related activities, accelerating recovery from injuries and stimulating muscle growth. [5][6][7] Gonadotropin-releasing hormone (GnRH), also known as luteinizing hormone-releasing hormone (LH-RH) or gonadorelin, regulates the secretion of gonadotropins. Because of its activity to stimulate gonadotropin and testosterone secretion, GnRH is regarded as a growth promoting agent.…”

Section: Introductionmentioning

confidence: 99%

“…Because of its activity to stimulate gonadotropin and testosterone secretion, GnRH is regarded as a growth promoting agent. 6 Due to their pharmacological effects, the peptides mentioned can be potentially misused in sports to illicitly alter performance. In fact, dermorphin was detected and confirmed in official post-competition equine plasma and urine samples in North America, 8 and GHRP-2 and GHRP-6 were identified in urine samples from human athletes.…”

Section: Introductionmentioning

confidence: 99%

A comprehensive approach to detecting multitudinous bioactive peptides in equine plasma and urine using hydrophilic interaction liquid chromatography coupled to high resolution mass spectrometry

Guan

You

et al. 2019

Bioactive peptides possess pharmacological effects and can be illicitly used in sports. To deter such misuse, an untargeted method using high resolution mass spectrometry (HRMS) has been developed for comprehensive detection of multitudinous exogenous peptides in equine plasma and urine. Forty‐four peptides were extracted using mixed‐mode solid‐phase extraction (SPE) from plasma and urine, separated with a hydrophilic interaction liquid chromatography (HILIC) column, and detected on an HRMS instrument. Ammonium formate as a mobile phase additive had effects on HILIC retention and charge state distribution of the peptides. The acetonitrile percentage in the reconstitution solution affected the solubility of peptide neat standards and peptides in plasma and urine extracts differently. The stability of the peptides in plasma at ambient temperature was assessed. The limit of detection (LOD) was 10–50 pg/mL for most of the peptides in plasma, and ≤ 500 pg/mL for the remaining. LOD was 100–400 pg/mL for the majority of the analytes in urine, and ≤ 4000 pg/mL for the others. The method was used successfully to analyze incurred plasma and urine samples from research horses administered dermorphin. Even in the absence of reference standards, dermorphin metabolites (aFGYPS‐NH2, YaFG, and YaF) were identified. These results demonstrate that data generated with this method can be retrospectively reviewed for peptides that are unknown at the time of sample analysis without requiring re‐analysis of the sample. This method provides a powerful novel tool for detection of numerous bioactive peptides and their metabolites in equine plasma and urine for doping control.

“…1 Currently, 2 different strategies are available for detection of exogenous administration, the first one directly measuring the hormone itself, the second one referring to the GH-responsive biomarkers insulin-like growth factor-I (IGF-I) and procollagen III N-terminal propeptide (P-III-NP). 2,3 A challenge with direct GH-measurement it is that, owing to pulsatile secretion, elevated serum levels are not conclusive as to illicit intake; an aberrant isoform composition of serum GH needs to be detected instead. To this end, with the presently approved 'differential' immunoassay, the amounts of GH measured by two types of antibodies of different specificity are compared: One of them preferentially binds to 22 kDa-GH ('rec GH'), the molecular form evidently used as doping agent; the second one ('pit GH'), in contrast, has been designed to recognize a variety of GH isoforms.…”

Section: Introductionmentioning

confidence: 99%

Growth hormone isoform‐differential mass spectrometry for doping control purposes

Arsene

Schulze

Röthke

et al. 2018

Mass spectrometry (MS) allows for monitoring growth hormone (GH) isoform compositions at high specificity. It is demonstrated that this capability can be used to reliably detect alterations as elicited by (putative) doping with 22 kDa-GH. Sample treatment consists of enzymatic protein cleavage, followed by 2-step liquid chromatography clean-up, prior to analysis by MS. The protocol does not depend on antibodies for analyte extraction at any stage. Therefore, MS opens an opportunity for independent confirmation, if combined with the antibody-based isoform differential test presently used in practice. To check the fitness-for-purpose of this concept, GH-free serum was spiked with pure 22 kDa- and 20 kDa-GH covering a representative range of concentrations (0.5-9.4 μg/L), while the 22kDa fraction was within a range of 80%-85%, or at 100%, the latter simulating an administration of 22 kDa-GH. Mean deviation of 22 kDa-fractions found was within less than 3% for samples with total GH>= 1 μg/L . Beyond this, results by antibody-free isoform-differential MS, as described, were in line with those of the World Anti-Doping Agency-approved antibody-based test for 18 native sera and 3 positive controls. In this context, relating 22 kDa-GH to total-GH rather than 22 kDa+20 kDa was considered as an alternative strategy to earlier approaches. However, 20 kDa-GH as an additional measurand, next to 22 kDa- and total-GH, provides useful extra information, as it directly indicates the presence or absence of a non-22 kDa-GH form.