Characterization and Detection of Erythropoietin Fc Fusion Proteins Using Liquid Chromatography-Mass Spectrometry.

Erythropoietin Fc (EPO-Fc) fusion proteins are potential drug candidates that have been designed for the treatment of anemia in humans by stimulating erythrocyte production. Such compounds can be considered performance-enhancing agents that may be used by athletes in endurance sports. This study describes the primary structure of commercially available EPO-Fc based on comprehensive liquid chromatography coupled with mass spectrometry (LC-MS) analysis. A bottom-up approach and the intact molecular weight (MW) measurement of deglycosylated protein and its IdeS proteolytic fractions was used to determine the amino acid sequence of EPO-Fc. Using multiple proteases, peptides covering unknown fusion breakpoints (spacer peptides) were identified. We demonstrated that "spacer peptides" can be used in the determination of EPO-Fc fusion proteins in biological samples using common LC-tandem MS methods.

Comparison of various in vitro model systems of the metabolism of synthetic doping peptides: Proteolytic enzymes, human blood serum, liver and kidney microsomes and liver S9 fraction.

Small peptides with a molecular weight of <2kDa represent a performance-enhancing substances. However, in vivo studies with human volunteers are limited because most of these peptides are not approved for human consumption. Thus, relevant in vitro models are a basic tool to study their metabolism for anti-doping purposes. To choose the best in vitro model the biotransformation of growth hormone releasing peptides (GHRPs), Desmopressin and TB-500 was investigated using various in vitro systems. High metabolic activity was observed during incubation of GHRPs and TB-500 with human kidney microsomes (HKM) and liver S9 fraction. Peptides degraded through cleavage of all bonds regardless protective modifications in primary structure. HKM and liver S9 fraction demonstrated enzymatic deamidation activity removing C-terminal amide group from all GHRPs. Fewer metabolites were produced during incubation with human serum. The metabolite pattern obtained with commercially available proteases was poor and included nonspecific hydrolyzed compounds. Thus, the maximum diversity of metabolites was achieved with HKM and liver S9 fraction which makes them the most efficient in vitro model systems for peptides biotransformation study. BIOLOGICAL SIGNIFICANCE: Currently, >60 peptide medicines are FDA approved and marketed in the United States as biopharmaceutical products. Approximately 140 peptide drugs are in clinical trials and about 500 therapeutic peptides in preclinical development. There is an emerging interest in small peptides with a molecular weight of <2kDa, which can be used as doping in modern sport due a wide spectrum of their physiological activity. Most of peptide doping products are not yet approved for human use and some of them undergo preclinical or clinical trials, which complicates the study of metabolism in vivo. The investigation of the metabolism with in vitro methods is an alternative that does not require a human participation and an approval by the Ethics Committee.

Solid-phase extraction of small biologically active peptides on cartridges and microelution 96-well plates from human urine.

Currently liquid chromatography - mass spectrometry (LC-MS) analysis after solid-phase extraction (SPE) on weak cation-exchange cartridges is a method of choice for anti-doping analysis of small bioactive peptides such as growth hormone releasing peptides (GHRPs), desmoporessin, LHRH, and TB-500 short fragment. Dilution of urine samples with phosphate buffer for pH adjustment and SPE on weak cation exchange microelution plates was tested as a means to increase throughput of this analysis. Dilution using 200 mM phosphate buffer provides good buffering capacity without affecting the peptides recoveries. SPE on microelution plates was performed on Waters Positive Pressure-96 Processor with subsequent evaporation of eluates in nitrogen flow. Though the use of smaller sample volume decreases the pre-concentration factor and increases the limits of detection of 5 out of 17 detected peptides, the recovery, linearity, and reproducibility of the microelution extraction were comparable with cartridge SPE. The effectiveness of protocols was confirmed by analysis of urine samples containing ipamorelin, and GHRP-6 and its metabolites. SPE after urine sample dilution with buffer can be used for faster sample preparation. The use of microelution plates decreases consumption of solvents and allows processing of up to 96 samples simultaneously. Cartridge SPE with manual рН adjustment remains the best option for confirmation. Copyright © 2015 John Wiley & Sons, Ltd.

Fc-fragment removal allows the EPO-Fc fusion protein to be detected in blood samples by IEF-PAGE.

EPO-Fc proteins have been under investigation as a potential drug for treating anaemia and have shown larger half-life values than other erythropoiesis-stimulating agents (ESAs). Sodium dodecyl sulfate/sodium N-lauroylsarcosinate polyacrylamide gel electrophoresis (SDS/SAR-PAGE) methods and subsequent immunoblotting are used for routine anti-doping analysis. This paper reports that EPO-Fc fusion proteins can be detected in serum samples by isoelectric focusing-polyacrylamide gel electrophoresis (IEF-PAGE) in carrier ampholyte-based gels with a pH 2-6 gradient after removing the Fc part via site-specific IdeS protease cleavage. The IdeS-digested EPO-Fc protein yields three fragments: two Fc fragments and one dimeric EPO-hinge fragment. After IEF-PAGE was followed by double Western blotting with chemiluminescent detection, the dimeric EPO-hinge fragment showed a unique isoelectric pattern, which differed from those of any other currently known analogue of EPO. We observed that the removal of the Fc fragment from EPO-Fc reduced the apparent molecular weight of entire fusion protein and increased its electrophoretic mobility. As a result, the band for the EPO-hinge fragment was located in a region between the rEPO and NESP standards, at which lower amounts of serum proteins are present. Simple and selective protocols for determining the EPO-Fc protein in human serum were developed to extend the methodological anti-doping arsenal. This protocol has been characterized. The limit of detection (LOD) of the IEF-PAGE method was 20 pg, and that of SDS/SAR-PAGE was 15 pg.

Determination of growth hormone releasing peptides metabolites in human urine after nasal administration of GHRP-1, GHRP-2, GHRP-6, Hexarelin, and Ipamorelin.

Growth hormone releasing peptides (GHRPs) stimulate secretion of endogenous growth hormone and are listed on the World Anti-Doping Agency (WADA) Prohibited List. To develop an effective method for GHRPs anti-doping control we have investigated metabolites of GHRP-1, GHRP-2, GHRP-6, Hexarelin, and Ipamorelin in urine after nasal administration. Each compound was administrated to one volunteer. Samples were collected for 2 days after administration, processed by solid-phase extraction on weak cation exchange cartridges and analyzed by means of nano-liquid chromatography - high resolution mass spectrometry. Six metabolites of GHRP-1 were identified. GHRP-1 in the parent form was not detected. GHRP-1 (2-4) free acid was detected in urine up to 27 h. GHRP-2, GHRP-2 free acid and GHRP-2 (1-3) free acid were detected in urine up to 47 h after administration. GHRP-6 was mostly excreted unchanged and detected in urine 23 h after administration, its metabolites were detectable for 12 h only. Hexarelin and Ipamorelin metabolized intensively and were excreted as a set of parent compounds with metabolites. Hexarelin (1-3) free acid and Ipamorelin (1-4) free acid were detected in urine samples after complete withdrawal of parent substances. GHRPs and their most prominent metabolites were included into routine ultra-pressure liquid chromatography-tandem mass spectrometry procedure. The method was fully validated, calibration curves of targeted analytes were obtained and excretion curves of GHRPs and their metabolites were plotted. Our results confirm that the detection window after GHRPs administration depends on individual metabolism, drug preparation form and the way of administration.

Possible cause of lack of positive samples on homologous blood transfusion.

Homologous blood transfusion is a prohibited method of blood manipulation that can be used to increase the number of erythrocytes circulating in the blood stream resulting in an increased oxygen transport capacity. In doping controls, homologous blood transfusions are determined by means of a procedure based on the detection of red blood cell phenotypes by flow cytometry. In the past six years, no adverse analytical findings concerning homologous blood transfusions were reported. One explanation for that phenomenon, assuming that athletes have not completely given up this kind of manipulation, would be a more careful selection of potential donors. If such a donor has the same set of minor erythrocyte antigens as the recipient, the established methodology to detect homologous transfusion would fail. We have hypothesized that any athlete can be a potential donor for teammates with the same RhD factor and AB0 blood group. Having analyzed the phenotype of erythrocytes of 535 Russian athletes in various endurance sports, several pairs of athletes with the same phenotype were observed. Based on the frequency of occurrence of red blood cell antigens, the theoretical probability of finding a donor within a team with exactly the same phenotype was calculated, and the existing number of occurrences where two individuals share the same phenotype in the same sport was in fact five times higher than the theoretical probability.

Anti-doping analyses at the Sochi Olympic and Paralympic Games 2014.

The laboratory anti-doping services during XXII Winter Olympic and XI Paralympic games in Sochi in 2014 were provided by a satellite laboratory facility located within the strictly secured Olympic Park. This laboratory, established and operated by the personnel of Antidoping Center, Moscow, has been authorized by the World Anti-Doping Agency (WADA) to conduct doping control analyses. The 4-floor building accommodated the most advanced analytical instrumentation and became a place of attraction for more than 50 Russian specialists and 25 foreign experts, including independent observers. In total, 2134 urine and 479 blood samples were delivered to the laboratory and analyzed during the Olympic Games (OG), and 403 urine and 108 blood samples - during the Paralympic Games (PG). The number of erythropoietin tests requested in urine was 946 and 166 at the OG and PG, respectively. Though included in the test distribution plan, a growth hormone analysis was cancelled by the Organizing Committee just before the Games. Several adverse analytical findings have been reported including pseudoephedrine (1 case), methylhexaneamine (4 cases), trimetazidine (1 case), dehydrochloromethyltestosterone (1 case), clostebol (1 case), and a designer stimulant N-ethyl-1-phenylbutan-2-amine (1 case).

Fluorescent immunoprecipitation analysis of cell surface proteins: a methodology compatible with mass-spectrometry.

Radiolabelling and biotinylation of cell proteins followed by immunoprecipitation is a common procedure for biochemical characterization of cell-surface antigens recognized by monoclonal antibodies. Here we present a new method of cell labelling with fluorescent dyes followed by immunoprecipitation and SDS-PAGE with subsequent detection of specific bands by fluorescence imaging devices. Fluorescent immunoprecipitation analysis (FIPA) of cell surface proteins is a fast and sensitive alternative to conventional immunoprecipitation methods, eliminating the need to employ radioactive or biotin labels. The proposed method is compatible with mass spectrometry analysis and permits the identification of immunoprecipitated proteins.