Projets de recherche

Code: 14C35JP

Blood transfusion is a prohibited method in sport. The currently applied method to detect homologous blood transfusion is based on the detection of cell surface blood group antigens that would be different between donors. There is no method available to detect autologous blood transfusion. However, there is evidence that changes occur in red blood cells (RBCs) stored ex-vivo (e.g. in a blood bag or as a frozen concentrate) that do not occur in a normal RBC population. The present proposal intends to empirically select antibodies from a large phage display antibody library able to recognize those specific changes in stored RBCs. Using the novel technology of single cell selection, antibodies recognizing only stored cells will be selected and against antigens broadly occurring in stored cells and that do not show in naturally aging RBCs. Thus, the objectives of the project are:

1.- Selection of an antibody(s) clone from a phage display Library able to specifically identify red blood cells which have been stored in a blood bag or frozen and reconstituted as red blood cell concentrate.

2.- Study the kinetic evolution of the detected antigen along storage period or blood manipulation.

3.- Produce recombinant antibody formats containing the proper tag for optimal use for cell sorting technologies (MACS, FACS)

4.- Development of a Magnetic-activated cell sorting (MACS) procedure for single cell detection.

5.- Adaptation of the methodology to a Flow-Cytometry platform for higher throughput.

Main Findings: 

AIMS
The main objective of the project was to the develop of antibodies able to recognize changes in red blood cells (RBCs) related to ex-vivo storage to unambiguously identify the use of blood transfusion, both autologous blood transfusion (ABT) or homologous blood transfusion (HBT).

RESULTS
Eight promising clones (single domain antibodies fragments, sdAb) were selected showing potential application as a screening and/or confirmation test to selectively recognise stored RBCs.
On the other hand, flow cytometry has proven to be sensitive enough to detect 0.1% cells of a target population and, using a pre-concentration technique like cell sorting, this limit of detection can be greatly increased.
Once the sdAb fragments have been selected from the phage display library, they can be optimised by modifying each position in the CDR regions to increase specificity, optimise the tag sequences with which they are expressed, etc. so that the final instrumental set-up is optimised.

CONCLUSIONS
The overall results demonstrate that the selection of antibodies from a large phage display library is a feasible approach to develop a procedure to detect surface antigens produced on RBCs through ex-vivo blood storage. These antigens can be potentially detected using flow cytometry in a minute subpopulation of RBCs in a freshly drawn blood sample

Code: 14B11MT 

Key regulator of skeletal muscle mass in developing embryonic and adult skeletal muscle is the growth factor myostatin. In animal models, both myostatin deficiency and inhibition resulted in an increased muscle mass and function due to an elevated volume and/or number of skeletal muscle fibers. Consequently, myostatin inhibitors may not only provide a therapeutic approach for the treatment of muscular diseases but also be misused as performance-enhancing agents in sports. 
The aim of the planned project is to establish a detection method for MYO-029 (Stamulumab), a recombinant human myostatin antibody which was developed by Wyeth Pharmaceuticals in 2005. MYO-029 specifically blocks myostatin signaling by preventing the interaction between the growth factor and its target receptor. Mice treated with such a neutralizing antibody showed an enhanced muscularity and a first clinical trial on humans yielded a good tolerance and safety of the drug. Within this project, two different immunological approaches will be used for the detection of MYO-029 misuse in cheating athletes. Both the western blot and ELISA will use recombinant human myostatin as bait to capture MYO-029 potentially present in plasma/serum samples. Antibody specifically bound to the immobilized antigen can then be detected by using an enzyme-coupled secondary antibody direct against its species-specific portion. Additionally, an antibody-based internal standard will be implemented by using different species-specific secondary antibodies conjugated to distinct fluorophores. Following method optimization, both approaches will be thoroughly characterized to ensure their reliability, sensitivity, and transferability. They will serve as proof of principle for the detection of myostatin inhibitors and antibody-based pharmaceuticals in doping control samples.

Main Findings:

Myostatin is the key regulator of skeletal muscle mass and inhibition of its signaling pathway can result both in an increased muscle mass and function. Within this project, two complementary detection methods for the recombinant human anti-myostatin antibody MYO-029 were developed by using immunological approaches.  
First, a qualitative western blot-based assay was set up and comprehensively characterized. It was found to be highly specific, robust, and linear from 0.1 to 1 µg/mL. The precision was successfully specified at three different concentration levels and the recovery of the affinity purification was 58%. Consequently, in consideration of the World Anti-Doping Code International Standard for Laboratories (ISL) 2015, paragraph 6.2.4.3.1.3, the assay can be considered fit-for-purpose for an application in routine doping control analysis.  
Additionally, an ELISA capable of detecting MYO-029 in human serum was developed as initial testing procedure. The conducted experiments show that a very sensitive and reproducible detection of anti-myostatin antibodies in a microtiter plate is possible. However, further optimization and characterization e.g. to reduce non-specific background signals is advisable if such approaches will be considered for routine doping controls, especially when multiplexing for different antibodies relevant for doping controls is desirable. 

Code: 14A01RB 

Because of their performance-enhancing effects anabolic androgenic steroids (AAS) have been prohibited by the International Olympic Committee since 1976. However, they still belong to the most frequently detected substances in doping tests and improvement of the detection capabilities is one major aim in anti-doping research.  This includes a better understanding of their metabolism in the human body and the discovery of long-term metabolites. In this context cytochromes P450s (P450s), who contribute to the biotransformation and subsequent renal clearance of exogenous compounds as well as to the biosynthesis of endogenous steroids, are a promising target for the investigation of the metabolic pathways of exogenous steroids like AAS. While the metabolism of xenobiotics by liver P450s is well studied, their biotransformation by adrenal P450s is nearly unknown. Consequently, our aim is to study the metabolism of AAS by P450s from the human adrenal cortex, in order to provide new reference material for doping analysis. Adrenal P450s are involved in the biosynthesis of steroid hormones and only very few cases regarding the metabolism of xenobiotics by these P450s have been described so far. Therefore, our project aims to investigate the in vitro metabolism of AAS by these P450s employing the world-wide unique set of purified proteins available in our lab. 
The applicability of discovered novel metabolites as (long-term) markers in doping tests will be examined by comparison with the urinary excretion profile in a mass spectrometry analysis. For metabolites, that turn out to be useful biomarkers, a larger scale production by genetically modified bacterial cell factories will be developed to create a basis for the supply of these metabolites to other laboratories for incorporation in routine doping analysis. 

Main Findings: 

Anabolic androgenic steroids (AAS) are frequently misused for doping purposes due to their performance enhancing effects. In the human body, they are metabolized, as all other drugs, in the liver to facilitate their clearance via the urine. The detection of AAS metabolites in the urine of an athlete by doping controls can demonstrate a doping delict. Most metabolic reactions in the liver are carried out by a group of proteins, or enzymes, called cytochromes P450. Closely related enzymes, which also belong to the cytochrome P450 group, are responsible for the synthesis of natural steroid hormones in the adrenal, in reproductive and some other tissues. The question arises whether these steroid-synthesizing enzymes can also contribute to the metabolism of AAS and produce alternative target metabolites for doping controls. The six human steroid-synthesizing enzymes (CYP11A1, CYP11B1, CYP11B2, CYP17A1, CYP19A1 and CYP21A2) were produced in genetically modified bacteria (Escherichia coli) and purified to evaluate their capability to metabolize the AAS oral-turinabol (OT), stanozolol and oxandrolone. Three of the enzymes (CYP11A1, CYP11B1 and CYP11B2) were found to metabolize OT and the structures of the emerging metabolites were elucidated by different methods (nuclear magnetic resonance spectroscopy, liquid chromatography tandem mass spectrometry). We identified five new OT metabolites (11β- hydroxy-OT, 11β,18-dihydroxy-OT, 11β-hydroxy-OT-18-aldehyde, 2-hydroxy-OT and 2,16-dihydroxy-OT) that have not been described in the literature to date and therefore represented interesting candidates as target analytes for doping controls. However, when we analyzed urine from an OT administration study, none of the compounds could be detected. This suggests that the new OT metabolites are further metabolized by additional enzymes prior to their excretion with the urine, which hampered their detection. In conclusion, the new OT metabolites cannot be used themselves to demonstrate a doping delict, but can serve as intermediates for the synthesis of their downstream metabolites, that might be excreted with the urine.

Code: 13D15GP

The use of anabolic androgenic steroids (AAS) is widespread within both competitive and recreational sports in which large, strong muscles are an advantage or the primary goal (bodybuilding). To build more muscle mass and to enhance muscle force and power, AAS are particularly effective. In this study, we want to investigate the long-term effects in the skeletal muscles that in particular are related to nuclei of the muscle fibers. Because the nucleus of a cell controls the different cellular-processes, including adaptations to training, long-term or irreversible changes in the number of nuclei in a muscle fiber will potentially give a person using AAS an advantage also after he/she stops using the drugs. Athletes caught as drug users are quarantined for at most 2 years for a first time offence, however, this should be reevaluated if the effects of AAS are shown to last many years after the athlete stops using the drugs. These research aims for a human study are directly derived from a recent animal study conducted by some of the co-investigators in this application. In this human study we will recruit males (20-40 years) with no strength training experience. The volunteers will be administrated a testosterone preparation or placebo (saline) by intramuscular injections during a 3 month period. Concomitantly they will follow a heavy strength training program. After the initial 3 months the participants will refrain from strength training for 9 months, but then re-train their muscle for 3 months without any drugs. Muscle tissue samples, measures of muscle mass and strength will be collected and assessed before and after each period. The main question is if the volunteers that received testosterone during the initial 3 months will re-build their muscle faster than the participants that received placebo.
The use of anabolic steroids has in an animal study been shown to give
long-term effects in skeletal muscle, by adding nuclei (DNA) to the muscle fibres and accelerating re-training. This means that the advantage of anabolic steroid use may be present in years after the athlete has stopped using the drugs. This study aims to test the hypothesis that the results from this animal study applies for humans.

Code: 13D26RV 

The misuse of testosterone and other endogenous anabolic androgenic steroids is detected through alterations in the urinary steroid profile. The steroid profile is composed of concentrations and ratios of endogenous steroid hormones, their precursors and metabolites, and it is measured in the glucuronide metabolic fraction. Glucuronidation of testosterone and other androgens is catalyzed by UDP-glucuronosyltransferases (UGTs). Green and white tea extracts inhibit the isoenzyme UGT2B17 in "in vitro" studies and, therefore, glucuronidation of testosterone and other androgens. Due to structural similarities, it is possible that tea extracts also alter the activity of other isoenzymes involved in the glucuronidation of androgens.
Tea is the most widely consumed beverage in the world next to water and, for this reason, the relevance of the inhibition of UGT isoenzymes by tea constituents in the "in vivo" metabolism of all androgens included in the steroid profile deserves to be studied. The consumption of tea may produce alterations in the steroid profile leading to misinterpretations on the longitudinal studies and/or masking the exogenous administration of some endogenous steroids.
The aim of the present research project is to investigate the effect of green and white tea on the urinary steroid profile in healthy volunteers. The effect of an acute exposition to one of the main flavonoids of tea, epigallocatechin-3-gallate, and the effect of regular tea consumption on the steroid profile will be evaluated in Caucasian population.

Main Findings: 

The steroid profile (SP) has been included in the athlete’s biological passport to detect the misuse of endogenous anabolic androgenic steroids in sports. The SP is composed of concentrations of testosterone (T) and related metabolites (epitestosterone, androsterone, etiocholanolone, 5α-androstane-3α,17β-diol and 5β-androstane-3α,17β-diol) and the ratios between them. Green tea (GT), along with its flavonol epigallocatechin-3-gallate (EGCG), has been shown in in vitro studies to inhibit the UGT2B17 isoenzyme, which is involved in the glucuronidation of T and related metabolites included in SP. Therefore, GT consumption could alter the SP leading to misunderstandings in doping controls. The aim of the present work was to study the effect of GT consumption on the SP.
A clinical study was developed with 29 male volunteers, covering a wide range of T/E ratio values (arm 1, 0.12±0.02, n=12; arm 2, 1.64±0.90, n=17). The clinical protocol was designed to evaluate the effects after repetitive consumption of high doses of EGCG. For this reason, participants were asked to consume 5 GT beverages along the whole day for 6 consecutive days and, in day 7, they consumed 9 GT beverages. Urine samples were collected before and during tea consumption. The SP metabolites were measured using gas chromatography-mass spectrometry.  
The excretion rates of the SP metabolites did not change during and after GT consumption.  Stable excretion profiles were obtained in daily periods as well as in excretion rates over the day for all metabolites included in the SP. Moreover, the individual evaluation of the subject’s steroidal biological passport resulted in normal sequences. The results obtained show that GT consumption does not distort the establishment of normal ranges of SP parameters. Therefore, GT administration does not need to be considered a confounding factor in the SP evaluation.

Code: T13M04CM 

Blood transfusion is the most effective means to increase the number of red blood cells, and enhance athletic performance in endurance events. Due to the complicated procedures with transfusion, the use of Epo has been the dominating method for blood doping, but improved detection technologies have forced cheating athletes again to use autologous blood doping. To date no reliable detection method for autologous blood transfusions exists, and the need for a direct detecting method for autologous blood transfusion is obvious and urgent.
It is known that during storage in the blood bags, RBCs are degraded, the degraded proteins (the Ådegradome‚) in circulation will be proportionally higher in doped athlete post-transfusion compared to pre-transfusion or non-transfused athlete.
We have develop an assay/procedure that can discriminate doped from non-doped athletes with a probability of >96%.
Work Plan for current project includes:
A) Verify selected biomarkers/procedure by a repeat autologous blood transfusion study B) Establish the sensitivity and specificity of the procedure to detect autologous blood doping C) Investigate three possible confounding factors: 1) elite endurance training 2) Differences between Males and Females 3) High altitude and low oxygen training.

Main Findings: 

Blood transfusion remains one of the most effective means to increase the number of red blood cells ((RBCs; i.e. hemoglobin mass) in any athlete, and thereby enhance athletic performance in endurance events. The non-medical use of this process is banned by the World Anti-Doping Agency (WADA).
We reported that RBCs change when stored (Malm et al. WADA Grant 08C06CM), and that these changes can be detected by global proteomic methods, and thus be used to develop a ÅDoped‚ profile for autologous blood transfusion. 
In the present study, we address challenges attributed to three possible confounding factors: 1) exercice training at high altitude 2) high intensity exercise training 3) sex (male or female). The test subjects analyzed were correctly placed into the respective category, with no false positives or false negative samples using the global proteomics profile modelled using OPLS-DA statistical analysis. The disclosed results support the viability of the test method to accurately detect autologous blood transfusion. The confounding factors (high altitude, exercise intensity or athletesƒ sex) did not affect the test to detect autologous blood transfusion in the subjects comprised of both elite and recreational athletes. Hematological variables analysis in our hands, did not separate groups investigated as robustly as the proteomic profile analysis test using the same statistical approach. These results support continued protocol development of the test method for enhancement of the current blood passport or athlete biological passport. This work is conducted in collaboration with the doping control laboratory in Huddinge, Sweden. Our team is now focused on reducing the complexity of instrumentation required, amending protocols to existing infrastructure, and expand the scope of the analysis to address possible confounding influence related to genetic variations.

Code: 13A10PV 

Because of the advances in analytical methodology, it has become possible to Screen for compounds in complex biological matrices at very low concentrations. As a result, during the last decade, the role of minor steroid metabolites have gained importance in steroid detection due to their more specific nature. In this light, our group developed an analytical screening method that encompasses minor androgenic steroid metabolites. Some of these minor steroid metabolites are promising new biomarkers in steroid profiling and can contribute substantially to the evidence of misuse with various endogenous steroids. The endogenous concentrations of these minor metabolites are very low and a strong increase in concentration is obtained when anabolic steroids are administered. This makes these minor metabolites, theoretically, excellent compounds to confirm on IRMS because the endogenous dilution is very small. During this project, a method will be developed that is able to analyze some of these minor metabolites on IRMS. Urinary concentrations beneath a certain concentration threshold are considered as endogenous, urine samples with a higher concentration are suspicious and will be forwarded to IRMS for confirmation. This approach is complementary to the present classic markers (of which the T/E ratio is the most important one) and will reduce the number of false negatives on two levels. It has been shown in the past that the T/E ratio does not always increase with the administration of testosterone. In such cases, minor metabolites might trigger an IRMS confirmation, whereas the classic markers do not. On a second level, elevated values of classic markers might still lead to a negative IRMS result if the endogenous dilution is too big. For minor metabolites this effect can almost be neglected.

Main Findings: 

Screening: 
In most excretion studies, the minor metabolites had shorter detection times than at least one of the traditional screening markers that can trigger on IRMS confirmation (an elevated T/E ratio, elevated concentration of androsterone, etiocholanolone,…). This means that all samples that had elevated concentrations for a minor metabolite, would have been labelled ‘suspicious’ and forwarded to IRMS anyway because at least one other marker had a suspicious value. Nevertheless, when dealing with steroid profiles, one must always keep the large inter-individual differences in mind, meaning that this observation is not necessarily applicable for every individual. In the past, our lab received a sample with perfectly normal values for the traditional steroid profile parameters according to population based reference intervals. However, the 6αOH-ADION concentration was elevated, leading to an IRMS confirmation and consequently an adverse analytical finding was revealed. Based on the traditional parameters alone there would not have been an IRMS confirmation, illustrating the usefulness of monitoring 6αOH-ADION in the steroid profile.  
IRMS analysis 
In general, the IRMS detection time of the minor metabolites does not seem to outperform the traditional IRMS target compounds. There is a sharp concentration increase of minor metabolites after administration, but the concentrations also drop again relatively fast and this neutralizes the advantage of lower endogenous dilution. As a result, the traditional IRMS target compounds have a longer window in which their δ13C values remain in the exogenous range.

Code: T13M06AB

1. We propose to test several sample processing protocols following samples thawing (heating, mixing and digestion with a suitable restriction enzyme) to identify a procedure that ensures full homogeneity of thawed samples and their efficient amplification in PCR.

2. Once such protocol is found, we will use it to test if the presence of background nucleic acid in the RM formulation improves its stability at -20°C and -80°C.

3. In addition to these two main objectives, we propose to continue testing stability of the RM at two additional time points (fifteen and twenty four months).

4. Finally, as the best storage condition identified in our previous study was 4°C, we would like to test if RM ultrafiltration or addition to it of sodium azide as antibacterial measure for prolonged storage at 4°C affect its analysis by real-time PCR or digital PCR.

Main Findings

This study was a continuation of an earlier WADA-funded project on the development of a synthetic DNA reference material (RM) for use in testing for EPO gene doping. The main aim of the study was to investigate the factors that affect stability of the RM during storage at freezing temperatures. Firstly, we confirmed that the previously observed decrease in the concentration of the RM stored frozen was, indeed, the result of the RM degradation and not due to its inadequate reconstitution upon thawing or compromised amplification in polymerase chain reaction. Secondly, we demonstrated that addition to the RM formulation of background nucleic acid improves its stability at freezing temperatures. In the optimal formulation, the material was stable at 4°C, -20°C and -80°C for one year. Finally, we showed that conventional methods to prevent bacterial growth during prolonged storage of the RM at 4°C do not affect its intended application.

As the outcome, we developed optimal protocols for RM formulation, storage and preparation for use. This outcome is important for generating a fit-for-purpose RM for EPO gene doping detection, but also for future development of RMs for detecting other doping genes. Availability of such RMs will facilitate the development of routine tests for direct gene doping detection that are reliable, robust, and able to withstand legal scrutiny.

Code: 13A13MM 

When an athlete dopes the drug is changed by the body and excreted in the
urine. These changed drugs or drug metabolites must be processed by anti-doping laboratories to enable detection using a range of sophisticated techniques. A protein or enzyme called beta-glucuronidase, isolated from Escherichia coli (E. coli) bacteria, is routinely used by anti-doping labs to process samples prior to analysis. It has become an important basic tool used by analysts in the fight against doping. Unfortunately this E. coli beta-glucuronidase enzyme only works on some drug metabolites called glucuronides leaving others such as sulfates unprocessed, and this may mean that the doping goes undetected. Creating a new method to process sulfate metabolites would significantly improve anti-doping analysis.

In this project we will develop a separate bacterial protein isolated from Pseudomonas aeruginosa (P. aeruginosa) called a sulfatase that we have shown is able to process the sulfate metabolites that E. coli beta-glucuronidase cannot. However, the activity for some drug metabolites is low leading to inefficient hydrolysis. We will employ laboratory-based methods of rapid evolution to enhance the activity of the P. aeruginosa sulfatase enzyme for anti-doping applications.

The outcome of the project will be an improved sulfatase enzyme for processing drug metabolites that will complement E. coli beta-glucuronidase.
Including the improved enzyme in the methods used to process drug metabolites will increase the sensitivity of analysis and allow doping to be detected for a longer period after an athlete takes a banned drug. We expect
this improved P. aeruginosa sulfatase will join E. coli beta-glucuronidase and
also become an indispensable tool used by anti-doping laboratories in the fight against doping.

Main Findings: 

Previously, we had identified a bacterial sulfatase, Pseudomonas aeruginosa arylsulfatase (PaS), and found it to compare favourably with commercially available sulfatase preparations (Drug Test. Anal. 2015, 7, 903–911). However, the activity towards β-configured anabolic androgenic steroid sulfates (testosterone sulfate, TS, for example) was several orders of magnitude lower than aryl sulfates such as estrone sulfate. Furthermore, the activity towards α-configured steroid sulfates, such as etiocholanolone 3-sulfate (ECS) or epitestosterone 17sulfate (ETS) and androsterone 3-sulfate (AS) was undetectable. Thus at the outset of our project our goals were to achieve:

• A 10-50-fold improvement for testosterone 17-sulfate and significant activity for other targeted steroid sulfates.
• An improved pH profile with maximum activity closer to neutral pH.

These enhancements in activity and scope will deliver new enzyme preparations suitable for anti-doping applications.

We took two approaches to improve PaS in parallel. The first approach made use of the published structure and our modelling to predict how steroid sulfates bind with the enzyme and identify nearby residues for targeted mutagenesis and screening. The second used directed evolution with random mutagenesis and subsequent selection for improved activity. In both cases we selected for variants with greater affinity and catalytic activity for TS hydrolysis (measured as Vmax/Km or initial rates).

In summary the major aims of the project have been met with a 270-fold improvement (Vmax/Km) for TS hydrolysis, and an improvement in substrate scope with PaS mutants derived from the project capable of hydrolysing ECS and ETS at significant rates where the parent PaS enzyme could not. Further mutants developed by the project show significantly increased absolute and relative activity at pH 7.

Code: 13A25JP 

Detection of rEPO and analogues and their differentiation from endogenous EPO has been hampered by two major reasons: similarity and sensitivity.  Regarding similarity, we already evidenced, through glycan analysis, the presence of minor amounts of N-glycolyl-neuraminic acid (Neu5Gc, MW: 325.27 u) in the recombinant preparations, while in the endogenous hormone, N-acetyl-neuraminic (Neu5Ac, MW: 309.27 u). The direct analysis of sialic acids had the drawback of losing the information of what protein were they attached to. The analysis of glycopeptides where the exogenous moiety is present and the aminoacid sequence unequivocally corresponds to EPO will be a perfect solution. However, sensitivity, particularly for glycopeptides, is greatly reduced given their microheterogeneity. O-glycopeptides, being far less heterogeneous, would the obvious choice. New instrumentation (AB Sciex 6500 QTRAP IonDrive) is able to detect as little as sub-attomol amounts of material. This sensitivity is, at last, compatible with the amounts present in urine of the precious non-human EPO glycopeptides. When combined with advances in immunopurification developed these years, the resulting method for detection of the presence of rEPO or NESP in human urine or serum/plasma will be quite straightforward, as any other doping control screening method and will offer unequivocal evidence. 
Thus, the hypothesis of this project is that MS sensitivity has reached a status in which EPO glycopeptides, particularly O-glycopeptides are detectable in urine or blood samples. The MS differences between endogenous and recombinant EPOs (including NESP) will provide specific detection of the recombinant species as proof of doping. 
The objective of the project are: 
To develop an optimized method of immunopurification and enzymatic digestion of rEPOs. 
To optimize separation and MS conditions for each O-glycopeptide and scale-up sensitivity using the latest state-of-the-art MS instrumentation from AB Sciex.

Main Findings: 

The main objective of the project was to develop an MS‐based analytical procedure for the detection of an EPO O‐glycopeptide containing the non‐human monosaccharide N‐glycolylneuraminic acid (Neu5Gc) using latest generation QTrap‐MS instruments as an unambiguous proof of the exogenous origin of the hormone (i.e. rEPO or analogues). The EPO O‐glycopeptide resulting from a tryptic digest (EAISPPDAAS*AAPLR) was chosen as it is unique for EPO and shows the lowest glycan heterogeneity, thus maximizing signal sensitivity while the peptide backbone will make it unique for EPO. 
A method was developed based on pre‐concentration by immunopurification using MAIIA cartridges followed by tryptic hydrolysis and LC‐MS analysis in MRM mode. The peptide glycoform containing two sialic acids was found to be most abundant; the endogenous‐like glycoform containing two N‐acetyl‐neuraminic acids (Neu5Ac) and its non‐human counterpart containing one Neu5Ac and one N‐glycolyl–neuraminic acid (Neu5Gc). The triply charged species ([M+3H]+3) at m/z 805.0 and 810.3 respectively were therefore selected as the precursor ions for target detection. Scale‐up sensitivity was evaluated comparing standard, micro and nano‐LC‐QTrap6500‐MS systems from AB Sciex with a ca. 10 fold increase in sensitivity between each step. The LOD achieved in nano‐LC‐Qtrap‐MS for the rEPO standard was 80 attomol rEP/ul, quivalent to the final concentration of extracted EPO expected from 20 mL of urine sample of ca. 2 IU/L. However, when urine samples were spiked with low concentrations of rEPO and taken through the procedure, the matrix effect prevented the detection of those low amounts.  For that reason different approaches were tested to circumvent the problem. On one hand, a polyclonal antibody was raised against the EPO peptide backbone (EAISPPDAASAAPLRC), modified with a terminal cysteine residue for synthetic reasons. The polyclonal antibody proved to recognize both the peptide and the EPO‐O‐glycopeptide, making it ideal for further purification of the sample before instrumental analysis. In parallel, magnetic beads coated with titanium dioxide were tested, as they are able to selectively bind acidic residues, as the sialic acids present in the O‐glycopeptide. The approach proved to be successful in quantitatively binding the O‐glycopeptides and elute them under conditions compatible with MS analysis. 
A final sample preparation method needs to be developed taking advantage of those tested methodologies making the unambiguous detection of a non‐human rEPO O‐glycopeptide amenable to MS detection.