Characterisation of intact recombinant human erythropoietins applied in doping by means of planar gel electrophoretic techniques and matrix-assisted laser desorption/ionisation linear time-of-flight mass spectrometry


  • Gerald Stübiger,

    1. Institute of Chemical Technologies and Analytics, Vienna University of Technology, Getreidemarkt 9/164, A-1060 Vienna, Austria
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  • Martina Marchetti,

    1. Institute of Chemical Technologies and Analytics, Vienna University of Technology, Getreidemarkt 9/164, A-1060 Vienna, Austria
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  • Marietta Nagano,

    1. Institute of Chemical Technologies and Analytics, Vienna University of Technology, Getreidemarkt 9/164, A-1060 Vienna, Austria
    2. ARC Seibersdorf Research, Doping Control Laboratory, A-2444 Seibersdorf, Austria
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  • Christian Reichel,

    1. ARC Seibersdorf Research, Doping Control Laboratory, A-2444 Seibersdorf, Austria
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  • Günter Gmeiner,

    1. ARC Seibersdorf Research, Doping Control Laboratory, A-2444 Seibersdorf, Austria
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  • Günter Allmaier

    Corresponding author
    1. Institute of Chemical Technologies and Analytics, Vienna University of Technology, Getreidemarkt 9/164, A-1060 Vienna, Austria
    • Institute of Chemical Technologies and Analytics, Vienna University of Technology, Getreidemarkt 9/164, A 1060 Vienna, Austria.
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  • Partly presented at 22nd Manfred Donike Workshop on Doping Analysis, Cologne (Germany), 7–12 March 2004, by G. Stübiger.


Our experiments show that it is possible to detect different types of recombinant human erythropoietins (rhEPOs), EPO-α, EPO-β and novel erythropoesis stimulating protein (NESP), based on exact molecular weight (MW) determination by matrix-assisted laser desorption/ionisation mass spectrometry (MALDI-MS) applying a high-resolution time-of-flight (TOF) mass analyser in the linear mode. Detection limits for the highly purified, intact glycoproteins were achievable in the low fmol range (25–50 fmol) using a sample preparation method applying a hydrophobic sample support (DropStop®) as MALDI target surface. These results are very promising for the development of highly sensitive detection methods for a direct identification of rhEPO after enrichment from human body fluids. During our investigation we were able to differentiate EPO-α, EPO-β and NESP based on distinct molecular substructures at the protein level by specific enzymatic reactions. MW determination of the intact molecules by high resolving one-dimensional sodium dodecyl sulfate /polyacrylamide gel electrophoresis (1D SDS-PAGE) and isoform separation by planar isoelectric focusing (IEF) was compared with MALDI-MS data. Migration differences between the rhEPOs were observed from gel electrophoresis, whereby MWs of 38 kDa in the case of EPO-α/β and 49 kDa for NESP could be estimated. In contrast, an exact MW determination by MALDI-MS based on internal calibration revealed average MWs of 29.8 ± 0.3 kDa for EPO-α/β and 36.8 ± 0.4 kDa for NESP. IEF separation of the intact rhEPOs revealed the presence of four to eight distinct isoforms in EPO-α and EPO-β, while four isoforms, which appeared in the more acidic area of the gels, were detected by immunostaining in NESP. A direct detection of the different N- or O-glycoform pattern from rhEPOs using MALDI-MS was possible by de-sialylation of the glycan structures and after de-N-glycosylation of the intact molecules. Thereby, the main glycoforms of EPO-α, EPO-β and NESP could be characterised based on their N-glycan composition. A microheterogeneity of the molecules based on the degree of sialylation of the O-glycan was observable directly from the de-N-glycosylated protein. Copyright © 2005 John Wiley & Sons, Ltd.

Human erythropoietin (hEPO) is a glycoprotein hormone which is mainly produced by the kidneys of adult humans. The gene encoding for hEPO is located on chromosome 7 of the human genome and encodes a protein of 193 amino acids (AA) including a 27 AA signal peptide.1 The intact protein consists of a 165 (DesArg166) or 166 AA sequence2 after polypeptide processing containing three N-glycosylation (Asn24, 38 and 83) and one O-glycosylation (Ser126) sites3, 4 with a calculated molecular weight (MW) of 18 240 Da (165 AA).5, 6 Post-translational carbohydrate addition leads to a glycoprotein with an average MW of 30.4–34 kDa as was determined by sedimentation equilibrium experiments and slab gel electrophoresis, respectively.7, 8

Since hEPO acts as a stimulator of red blood cell production in the bone marrow, it is mainly involved in the regulation of the oxygen supply of the human body. Endogenous hEPO production itself is mostly regulated by the oxygen demand of the organism. Hypoxia leads to a rapid increase in the transcription of the EPO gene in the EPO-producing cells, resulting in increased hEPO serum levels within less than 2 h. It is presumed that a transmembrane haemoprotein acts as oxygen sensor and that its deoxygenated form activates a transcription factor which then enhances EPO gene transcription in the nucleus.9 Aside from hypoxia, factors like hGH, ACTH, IL-3, angiotensin II or adrenalin are capable of increasing endogenous hEPO production,10, 11 too. The isolation of the EPO gene in 1985 enabled the transfection of mammalian cell lines (CHO, BHK, etc.) for the large-scale production of recombinant human erythropoietin (rhEPO) by biotechnological methods.12

The first rhEPO (EPO-α) was approved in 1987 by the FDA for therapeutic use in the USA. Since rhEPOs and derivatives (e.g. NESP, pegylated EPO, continuous erythropoiesis receptor activator (CERA)) became available partly as therapeutic agents (e.g. Erypo®, ESPO®, Epogin®, Procrit®, NeoRecormon®, Aranesp®), they have been increasingly abused as doping substances to improve endurance performances in sports.13 Due to their oxygen transfer enhancing capabilities, the International Olympic Committee (IOC) banned rhEPOs in 1990. However, misuse of rhEPO in sports was not detectable at that time. The current list of prohibited substances in sports, ‘The 2004 Prohibited List’ issued by the World Anti-Doping Agency (WADA)14 specifies EPO as a prohibited substance (‘peptide hormones’) and as a prohibited method (‘enhancement of oxygen transfer’). The use of EPO is banned both in and out of competition.

From the analytical point of view, the detection of rhEPO in human body fluids faces several difficulties. Most of all, rhEPO levels in human serum/plasma and urine are very low—usually in the low fmol range. Additionally, the time window for the direct detection of rhEPO isoforms is rather short—about 24–72 h after the last rhEPO administration the application can no longer be detected, especially because no accumulation effects are observable. Enlargement of this time window for rhEPO detection is only possible by implementation of additional blood parameters in combination with the measured absolute serum hEPO levels. Parisotto et al. developed statistical models based e.g. on the haemoglobin concentration, serum transferrin receptor concentration, total serum hEPO concentration and reticulocyte percentage.15, 16 These multivariate models allowed the indirect detection of rhEPO abuse for up to 3 weeks after the last injection. However, due to the variability of ELISA results for serum hEPO and serum transferrin receptor, those parameters are suitable for screening purposes, but not for confirmation testing. Another serious drawback is that rhEPO and endogenous hEPO differ only slightly in the composition of their glycoforms. Recently, a significant correlation between the relative concentrations of serum transferrin receptor and basic hEPO isoforms in urine was demonstrated.17 The situation changed with the development of a direct method based on the separation of rhEPO isoforms from human urine by Lasne et al. using isoelectric focusing (IEF).18, 19

In doping control, analytical methods leading to a direct proof of substance abuse are generally preferred. Several analytical methods, including 2D gel electrophoresis, capillary zone electrophoresis and IEF in the gel, have been evaluated for the direct detection of rhEPO and urinary human EPO (uhEPO)20–25 but, currently, the IOC/WADA-accredited IEF method is the only direct method to confirm rhEPO abuse.

In order to develop a particularly faster and more reliable method for the direct detection of rhEPO and future derivatives, we have focused on various mass spectrometric (MS) strategies. For the development of such an approach, a detailed molecular structural elucidation is necessary to find markers for the direct detection of rhEPO molecules (biomarker discovery). There is evidence that primary structure differences exist between uhEPO, serum human EPO (shEPO) and rhEPOs, and that these variations are attributed mainly to the microheterogeneity of the glycan structures including possible modifications.3, 26–33 In our approach such molecular substructure markers on the glycoprotein and/or on the peptide/carbohydrate level should be sought by means of the most sensitive mass spectrometric techniques. The highly complex molecular structure of rhEPOs makes the application of more sophisticated instrumentation necessary. Matrix-assisted laser desorption/ionisation mass spectrometry (MALDI-MS) was applied in some studies for the analysis of intact EPO molecules.34, 35 This technique, which is now widely used in proteomics and glycomics applications,36–38 seems to be a very promising tool for very sensitive detection of rhEPO in doping analysis, allowing also a high degree of automation. The development of a biomarker test that should be applicable for the detection of rhEPO abuse independently from the source of sample (serum, plasma, urine) is planned.


Recombinant human EPO samples

Erypo® from Janssen-Cilag (injection solution, 2000 IU EPO-α/0.5 mL, product no. 2-00131), NeoRecormon® from Roche (injection solution, 500 IU EPO-β/0.3 mL; product no. 30G1/2) and Aranesp® from Amgen (injection solution, 10 μg novel erythropoesis stimulating protein (NESP)/0.4 mL; product no. 1265-E06) were used for our analyses. It should be stated that 1 IU rhEPO corresponds to an amount of 8.4 ng (∼280 fmol).

Chemicals and materials

All chemicals and solvents used were analytical grade except otherwise stated. Urea, dithiothreitol (DTT), β-mercaptoethanol, iodoacetamide (alkylating reagent), silver nitrate (≥99%), 35 wt.% formaldehyde solution in water for protein staining after gel electrophoresis, glycine, phosphate-buffered saline (PBS), tris-(hydroxymethyl)aminomethane (Trizma® base), N,N,N′,N′-tetramethylethylenediamine (TEMED) and caffeic acid were from Sigma (St. Louis, MO, USA). The denaturing reagent guanidinium hydrochloride (GuHCl), the chemicals for protein staining, sodium thiosulfate pentahydrate (99%), anhydrous sodium carbonate (99%) and Coomassie Brilliant Blue R-250, the MALDI matrices sinapinic acid (puriss. ≥99%) and ferulic acid (purum ≥98%), α-cyano-4-hydroxycinnamic acid (CHCA) (puriss., ∼97%), 2,5-dihydroxybenzoic acid (DHB, ≥98%), nor-harmane (puriss. 99%), harmaline (puriss. 99%), 2-hydroxy-5-methoxybenzoic acid (5-methoxysalicylic acid, ≥98%), 2,6-di- and 2,4,6-trihydroxyacetophenone (DHAP, THAP; puriss. ≥99.5%), 3-hydroxypicolinic acid (3-HPA; puriss. for mass spectroscopy, ≥99%) and dibasic ammonium citrate (puriss. ≥99%) were supplied by Fluka (Buchs, Switzerland). Tris(hydroxymethyl)aminomethane (Tris, research grade) was purchased from Serva (Heidelberg, Germany). Tween 80 (Surfact-Amps 80) and trifluoroacetic acid (TFA, ‘sequanal grade’) were supplied by Pierce (Rockford, IL, USA). Acrylamide/bisacrylamide (97/3, w/w), orthophosphoric acid and the organic solvents, ethanol (EtOH), methanol (MeOH), acetonitrile (ACN) and isopropanol, as well as acetic acid, formic acid (FA) and analytical-grade water (specific conductance ≤1 μS/cm), were obtained from Merck (Darmstadt, Germany). Low-fat dry milk as blocking reagent was from Maresi (Vienna, Austria). The streptavidin-biotinylated horseradish peroxidase complex was purchased from BioSpa (Milan, Italy) and the Covalight chemiluminescence kit was from Covalab (Lyon, France).

PVDF membranes (Immobilon-P) for Western blotting and C18 and C4 ZipTip® pipette tips (spherical silica, particle diameter 15 μm, pore size 200 Å and 300 Å, respectively) for desalting of the proteins were obtained from Millipore (Bedford, MA, USA). MicroSpin™ G-25 columns used for protein purification prior to gel electrophoresis, ammonium peroxodisulfate and GelBond PAG film for fixation of polyacrylamide slab gels were purchased from Amersham Biosciences (Piscataway, NJ, USA). All carrier ampholytes (Servalyts 2-4, 4-6, 6-8) were purchased from Serva (Heidelberg, Germany). The monoclonal mouse IgG2A anti-human EPO antibody (clone #AE7A5) was from R&D Systems (Minneapolis, MN, USA) and the biotin-labelled antibody to mouse IgG (H+L) from P.A.R.I.S. (Compiegne, France). Four times concentrated (4×) lithium dodecyl electrophoresis (LDS) sample buffer, twenty times concentrated (20×) 2-morpholinoethanesulfonic acid (MES) electrophoresis running buffer, pre-cast 10% bis-Tris-polyacrylamide NuPAGE gels and the SeeBlue® Plus2 pre-stained MW protein marker were provided by Invitrogen (Carlsbad, CA, USA). The hydrophobic DropStop® surface layer was obtained from Schur Consumer Products (Vejle, Denmark).

All proteins used for mass spectrometric calibration were purchased from Sigma (St. Louis, MO, USA): serum albumin (bovine, BSA), trypsin (bovine) and enolase (bakers yeast). The cleavage enzymes, recombinant peptide-N-glycosidase F (PNGase F) from Flavobacterium meningosepticum and neuraminidase from Athrobacter urefaciens (modified sequencing grade, containing 0.25 mg/mL BSA), were obtained as lyophilised preparations from Roche (Mannheim, Germany).

SDS-PAGE and planar IEF equipment

Sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS-PAGE) was carried out on a XCell SureLock™ mini-cell electrophoresis system using 10% bis-Tris-polyacrylamide gels (both Invitrogen). Isoelectric focusing (IEF) on slab gels was performed on a Multiphor II (Amersham Biosciences) with an EPS 3501XL power supply and the Nova Blot kit for semidry electroblotting of the gels (both Amersham Biosciences). An oribital shaker (Belly Dancer) was used for membrane incubations and was purchased from Stovall (Castle Scientific, Emersacker, Germany). For chemiluminescent detection, a CCD camera (epoCAM) with image processing software (GASepo) was used (both ARC Seibersdorf Research, Seibersdorf, Austria).


One-dimensional (1D) SDS-PAGE was carried out according to the Laemmli method.39 For SDS-PAGE of the intact rhEPOs, samples were desalted using MicroSpin™ G-25 columns. After desalting, 4× LDS buffer was added. In the case of EPO-α and EPO-β, aliquots of 420 ng for silver staining and 1.3 μg for Coomassie staining were used, respectively. In case of NESP, 200 and 500 ng were applied. 5% β-mercaptoethanol was added and the samples were heated for 10 min at 70°C before centrifugation. Proteins were separated on a 10% bis-Tris-polyacrylamide gel using MES running buffer applying a constant voltage of 200 V for 45 min. Four μL of a prestained protein MW marker were run simultaneously next to the rhEPO lanes for MW determination. Proteins were detected either by Coomassie R-250 staining or by silver staining.40 Enzymatically treated rhEPOs were lyophilised and redissolved in LDS buffer. Separation was performed under the same conditions. Visualisation of de-sialylated and de-N-glycosylated proteins was carried out by silver staining exclusively.

Planar IEF electrophoresis

IEF of rhEPOs was performed as described by Lasne et al.19, 41 Denaturing (7 M urea) polyacrylamide slab gels (5% total acrylamide, 3% crosslinker; 1 mm thickness) were prepared by the capillary gel casting technique. The carrier ampholytes Servalyt 2-4 and Servalyt 4-6 (mixed 1:1; final concentration: 2% w/v, each) were used to establish the pH gradient. Samples were applied on gels near the cathode by using sample application pieces. Each sample contained 1% v/v Tween 80. Prefocusing of the gels was performed without samples at 250 V, 1 mA/cm, 1 W/cm for 30 min (8°C). The main focusing step was performed at 2000 V, 1 mA/cm, 1 W/cm for 3600 Vh. Electrode solutions were 0.5 M phosphoric acid (anode) and 2% w/v Servalyt 6-8 (cathode), respectively. The focused proteins were blotted onto PVDF membranes using the semidry blotting technique (0.8 mA/cm2) with modified Towbin buffer (25 mM Tris, 192 mM glycine in highly purified water) as transfer buffer. Disulfide bridges were cleaved by incubating the membranes in 5 mM DTT/PBS solution for 1 h at 37°C. The membranes were then rinsed briefly in PBS, blocked with 5% low-fat milk in PBS (1 h) and incubated with the monoclonal mouse IgG2A anti-human EPO antibody. After the membranes had been washed with 0.5% low-fat milk in PBS (33 min), the bound antibodies were blotted onto a second PVDF membrane (0.7% acetic acid; 0.8 mA/cm2), blocked again with 5% low-fat milk (1 h) and incubated overnight (cold room, 4°C) with the biotin-labelled antibody to mouse IgG (H+L). After being washed with 0.5% low-fat milk in PBS (33 min), the membranes were incubated with the streptavidin-biotinylated horseradish peroxidase complex (1 h) and washed again with PBS for 33 min. Bands were visualised via chemiluminescent detection (Covalight kit) and CCD camera exposure (epoCAM). Image processing was performed with GASepo software.

Sample purification for MALDI-MS

ZipTip® sample purification was used for subsequent MALDI-MS analysis following the manufacturer's protocol (Millipore). Injection solutions were used either directly or aliquots of lyophilised rhEPOs were dissolved in acidified aqueous solutions prior to the ZipTip® cleanup procedure. If not otherwise stated, aqueous FA was used instead of TFA for pH adjustment of sample solvents throughout our analyses. C4 or C18 tips were used alternatively. Prior to sample application, the tips were activated with ACN/water (1/1 v/v) and equilibrated with aqueous 0.1% FA solution. Immediately after rhEPO sample loading a washing step (five times with 10 μL of methanol/aqueous 0.1% FA, 5/95 v/v) was performed for removal of salts and excipients. Then the bound proteins were eluted five times with 2–3 μL of ACN/0.1% FA (50/50 v/v). The obtained protein solutions were either directly used for subsequent MALDI-MS analyses or were stored at −25°C.

MALDI matrix preparation

Sample solvents and preparation techniques were adjusted with respect to the ZipTip® cleanup procedure. A passive elution of sample from the ZipTip® was used in any case. The following matrix preparations were used during our analyses: sinapinic acid and ferulic acid (5 mg/mL) were dissolved in ACN/water (50:50 v/v), which was adjusted to pH 2 with FA. DHB matrix was prepared as a 8 mg/mL solution in methanol/0.1% aqueous TFA (90:10 v/v), super-DHB (S-DHB) was prepared as a 10 mg/mL solution of DHB in ACN/0.1% aqueous TFA (77:33 v/v) containing 10% 5-methoxysalicylic acid, and THAP (4 mg/mL) was dissolved in ACN/20 mM dibasic ammonium citrate (1:1 v/v). Caffeic acid (10 mg/mL) was dissolved in TFA/water/ACN (0.1:20:80 v/v/v), 2,6-DHAP (10 mg/mL) was dissolved in ACN/0.1% aqueous TFA (33:77 v/v), 3-HPA (40 mg/mL) was dissolved in water/0.1% aqueous TFA (50:50 v/v) and harmaline was prepared as a 10 mg/mL solution in ACN/0.1% aqueous TFA (40:60 v/v). The CHCA/DHB combination matrix consists of equal parts of 20 mg/mL solutions of CHCA in ACN/5% FA (70:30 v/v) and DHB in ACN/0.1% aqueous TFA (70:30 v/v).

Sample and matrix (0.3 μL each) were applied in directly consecutive steps to the MALDI target without previous mixing (see Table 1). In the case of sensitivity tests the used pipette tips were washed 3–5 times with sample solution prior to sample application on the MALDI target. Standard micropipettes (Gilson, Villiers Le Bel, France), which allowed the application of 1–0.1 μL, were used.

Table 1. Evaluation of MALDI sample preparation techniques for the rhEPO analysis
MatrixMS modeThreshold energyaResolution (FWHM)S/N ratiobPreparation techniquecApplicabilityReference
  • a

    According to instrument-specific laser power settings (1–180).

  • b

    Signal-to-noise ratio determined for singly charged ions unless otherwise stated for all matrices.

  • c

    Sample preparation after ZipTip® cleanup: (1) 0.3 μL matrix and 0.3 μL sample → air drying, (2) 0.3 μL matrix and 0.3 μL sample → air drying → 0.5 μL formic acid (pH ≤ 2.0) → air drying → 0.5 μL matrix → air drying, (3) 0.3 μL sample and 0.3 μL matrix → air drying.

  • —not useful/(+) bad/+ good/++ excellent; n/a no useful mass spectra; * values for doubly- and triply-charged molecules.

Sinapinic acid (5 mg/mL)Pos80–8513–15>100:1(1)++53
Caffeic acid (10 mg/mL)Pos90n/a2:1(1)(+)57
Ferulic acid (5 mg/mL)Pos75–8012–13>100:1(2)++34
2,4,6-THAP (4 mg/mL)Pos/Neg95n/a2:1(2)(+) +(neg mode)34
2,5-DHB (8 mg/mL)Pos90–1008–10>50:1(1)+55
S-DHB (10 mg/mL)Pos90–958–10>50:1(1)+51
2,6-DHAP (10 mg/ml)Pos>100n/an/a(1)58
CHCA/2,5-DHB (20 mg/mL)Pos5013–14>10:1(1)++56
   10–11*>50:1* (abundant multiply charged molecules) 
Harmaline (10 mg/mL)Pos/Neg>100n/an/a(3)59

Usually, stainless-steel targets with ≤3 mm2 sample wells were used for matrix preparations. For sensitivity experiments the following target surfaces were evaluated: brushed-etched-cleaned stainless steel (KA4-type), brushed-cleaned-etched stainless steel (CS4-type), brushed-etched-cleaned-electropolished stainless steel (KA2-type), a hydrophobic surface (DropStop®), and a gold-plated stainless-steel surface of CS4-type. In all cases, after the application of sample and matrix solution, both were allowed to co-crystallise on the target surface at ambient temperature (‘dried-droplet method’).42

Dilution series for sensitivity tests

Starting from the ZipTip® cleanup procedure, the solutions, containing ∼3 pmol/μL EPO-α, EPO-β and NESP, respectively, were diluted by 1:5 and 1:10 (v/v) steps to yield sample concentrations between 600 fmol/μL and 300 amol/μL. Before each dilution step, analyte solutions were sonicated (3 min) and centrifuged (6000 rpm, 30 s). Pipette tips were washed three times with ACN/1% aqueous FA (1:1) and afterwards the sample solution (1 μL) was transferred into a new tube containing neat solvent (4 or 9 μL). After sonication, a 0.3 μL sample was deposited on the MALDI target.

Protein extraction from SDS gel

rhEPO extraction from gel slices for subsequent MALDI-MS analysis of intact EPO molecules was accomplished according to the literature method.43 A solvent system consisting of FA/water/isopropanol 1:3:2 (FWI), which was described as exhibiting superior solubilisation properties for proteins, was used during the extraction procedure.

The Coomassie-stained bands were cut out from the gels by using a scalpel, transferred to a 0.5 mL sample tube, destained with 40% ACN/60% NH4HCO3 (50 mM) to nearly complete transparency and briefly washed with deionised water. Afterwards the gel slices were crushed with a glass rod and extracted with FWI for 24 h at 2°C. The gel particles were separated from solvent by centrifugation and the supernatants were carefully transferred to new sample tubes. Finally, the supernatants were lyophilised, resuspended in 1% FA and purified by ZipTip® for subsequent analysis by MALDI-MS.

Enzymatic digestion


Aliquots of lyophilised rhEPOs were dissolved directly in ready-made enzyme buffer (pH 8.0) containing 1–3 units (U) PNGaseF/μg protein (sonication for 3 min). Afterwards, samples were incubated for 18 h at 37°C on a thermomixer (Eppendorf, Hamburg, Germany) applying 350 rpm.


Aliquots of lyophilised rhEPOs were dissolved in 50 μL 10 mM NaHCO3 (pH 8.0) containing 0.01 U neuraminidase/μg protein (sonication for 3 min). Afterwards, the samples were incubated for 18 h at 37°C on a thermomixer (350 rpm).

Reduction and alkylation for subsequent de-N-glycosylation of NESP

Aliquots of lyophilised NESP were dissolved in 40 μL of denaturation buffer (0.5 M Tris; 6 M GuHCl; pH 8.6). To prevent incomplete solubilisation the reaction vials were sonicated for 10 min. DTT solution (1 μL, 50 mM in deionised water) was added. The resulting mixture was incubated at 37°C for 1 h on a thermomixer (350 rpm), and alkylated by addition of 3 μL of iodoacetamide (0.2 M in 0.5 M Tris; 6 M GuHCl; pH 8.6) for 1 h at 37°C. 2-Mercaptoethanol (1 μL) was added to stop the alkylation reaction. The reduction (DTT) and alkylation (carbamidomethylation) reactions were exclusively performed in the dark.


All measurements and sensitivity tests were performed on an AXIMA-LNR (Shimadzu Biotech Kratos Analytical, Manchester, UK) MALDI time-of-flight (TOF) mass spectrometer applying a nitrogen laser (λ = 337 nm, 3 ns pulse width, maximum pulse rate 10 Hz). The instrument was operated in positive or negative linear ion mode (flight path length 1.2 m) using the molecular mass optimised delayed-extraction (DE) mode for enhanced mass resolution.44 The instrument contains a video-imaging system, which allows direct monitoring of sample spots. The quality of matrix crystallisation can therefore be easily evaluated, which is an advantage in finding an optimal sample preparation. The ion source pressure during MALDI-MS measurements was typically 2.5 × 10−6 mbar or less. The accelerating voltage was set to 20 kV. For external calibration of the instrument, the singly and doubly charged molecules of standard BSA (MW 66.4 kDa) were used. Internal calibration of the intact molecules was achieved by mixing appropriate amounts of the defined protein calibrants trypsin (MW 23.3 kDa) and enolase (MW 46.7 kDa) together with rhEPO directly on the target. De-N-glycosylated molecules were internally calibrated directly by means of the singly and doubly charged ions of the enzyme PNGaseF (MW 34.6 kDa).45 For exact molecular mass determinations, the peak centroid was determined at a signal height of 70%. Each mass spectrum was the result of 100–500 consecutive single laser pulses directed onto the selected ‘sweet spots’ of sample preparation. All MALDI mass spectra shown were smoothed using the company-supplied Savitsky-Golay peak processing algorithm.46


SDS-PAGE and planar IEF electrophoresis of the EPO molecules

As first step, 1D SDS-PAGE was applied to determine the MW of the different rhEPOs and the homogeneity of the commercial rhEPO samples. The pharmaceutical EPO-α, EPO-β and NESP preparations were desalted and purified by G-25 microcolumns and separated afterwards on a highly resolving 10% bis-Tris-polyacrylamide gel. Detection limits of the three intact glycoproteins were determined to be 400 and 80 ng in the case of Coomassie and silver staining, respectively. The MW of the glycoproteins was determined by a calibration curve of log10 (MW of the marker proteins) versus migration length in the PA gel.47 A slight migration difference between EPO-α and EPO-β could be realised, whereas EPO-β exhibited a slightly lower MW (36 kDa) than its homologue EPO-α (37 kDa) (Fig. 1). These values were considerably higher (6–7 kDa) than the MW determination by MALDI-MS (see below). This effect may be explained by the fact that charged sugar moieties cannot completely be covered by the detergent, which leads to an altered electrophoretic mobility of glycoproteins compared with the non-glycosylated standard proteins.48 Such behaviour was also found in the case of the highly glycosylated NESP, whose MW was determined to be 49 kDa from SDS-PAGE (12 kDa too high). In conclusion, it can be stated that the three different EPO samples were quite homogeneous and exhibited well-defined bands for the different rhEPOs.

Figure 1.

Electropherograms of native rhEPOs obtained by SDS-PAGE and IEF. (a) SDS-PAGE/Coomassie staining; lane 1a: EPO-α (Erypo, 1.3 μg), lane 2a: EPO-β (NeoRecormon, 1.3 μg), lane 3a: NESP (Aranesp, 500 ng). (b) SDS-PAGE/silver staining; lane 1b: EPO-α (Erypo, 420 ng), lane 2b: EPO-β (NeoRecormon, 420 ng), lane 3b: NESP (Aranesp, 200 ng). Lane M: pre-stained MW marker. (c) IEF/immunoblotting; lane 1c: EPO-α (Erypo, 0.34 ng), lane 2c: EPO-β (NeoRecormon, 0.34 ng), lane 3c: NESP (Aranesp, 0.25 ng).

Figure 1(c) shows a characteristic band pattern in planar IEF after immnostaining49 using the same batches of EPO-α, EPO-β and NESP as were applied previously for SDS-PAGE. In contrast to EPO-α (lane 1c) and EPO-β (lane 2c), NESP (lane 3c) focused near the anode, thus in the more acidic region of the gel,50 which is related to the higher number of acidic terminal sugars in NESP. EPO-α exhibited 4–5 distinct isoforms, while a broader array of up to 7–8 isoforms could be detected in EPO-β. Two of these EPO-β isoforms were clearly located in the more basic region of the gel compared with EPO-α. In contrast, only four clearly visible bands within a narrow pI range could be differentiated in NESP. The absolute detection limit for the immunostaining procedure of the IEF gels was about 0.02 ng for visualisation of the isoform pattern described above. By increasing the applied amount of EPO, the number of detectable bands also increased to a certain extent. The IEF pattern indicates clearly the molecular structural heterogeneity of all three rhEPO samples.

As can be seen in the SDS-PAGE results (Fig. 2), enzymatic removal of the sialic acids attached to the glycan structures did not yield different MWs for all three rhEPOs (results for EPO-β not shown since they were identical to those from EPO-α). As shown in Fig. 2(a), PNGaseF digestion of native EPO-α resulted in a band with a MW of about 19 kDa, which was interpreted as the O-glycosylated form of the molecules. This band migrated in good agreement with the protein standard as was expected from the theoretical MW. In contrast, digestion of NESP without preceding reduction and alkylation turned out to be incomplete. Hence, two protein bands, one of about 28 kDa and one of 19 kDa, could be detected (Fig. 2(b), lane 3b). The upper band was interpreted as the polypeptide chain of NESP still bearing one N-glycan structure (see below). Lane 4b in Fig. 2(b) exhibits the complete de-N-glycosylated NESP in the case of reduction/alkylation prior to enzymatic cleavage. These experiments presented evidence for the similarity of the rhEPOs in the O-glycosylated protein part.

Figure 2.

Electropherograms of enzymatically treated rhEPOs by SDS-PAGE. (a) EPO-α (Erypo): lane 1a: intact EPO-α, lane 2a: desialylated EPO-α, lane 3a: de-N-glycosylated EPO-α. (b) NESP (Aranesp): lane 1b: intact NESP, lane 2b: desialylated NESP, lane 3b: de-N-glycosylated NESP, lane 4b: de-N-glycosylated NESP after reduction and alkylation. Lane M: pre-stained MW marker. The well-defined protein band at about 38 kDa, which can be detected in lanes 3a, 3b and 4b, represents PNGase F. All protein bands were visualised by silver staining.

MALDI-MS of the intact EPO molecules

Molecular weight determination

A considerable difference between the MWs determined by SDS-PAGE (Fig. 1) and MALDI-MS was observed for the rhEPO samples (Fig. 3). MALDI-MS analyses at the threshold laser energy showed an average MW of 30.2 ± 0.4 kDa for the very similar EPO-α and -β samples and of 37.6 ± 0.9 kDa for NESP These results were obtained by a series of measurements (n = 12–17) determined based on external calibration of [M]+ signals of the intact molecules using sinapinic acid as MALDI matrix. Dependent on the applied laser energy a fluctuation of the determined MWs in the range of ±1.3% (EPO-α/β) up to ±2.4% (NESP) was observed when using external calibration. Such effects have already been described in MW determinations of highly glycosylated proteins by MALDI-MS.51, 52 Therefore, internal calibration with the molecular mass calibrants trypsin and enolase was used for exact MW determination.53 In this case molecular masses of 29.8 ± 0.3 kDa for EPO-α/β and 36.8 ± 0.4 kDa for NESP, respectively, were determined (Figs. 4(a) and 4(b)). In order to confirm the above MWs of EPO-α and NESP (determined directly from injection solution after desalting), the proteins were separated by SDS-PAGE, extracted from gel bands and analysed by MALDI-MS. The results (Figs. 4(c) and 4(d)) were in good agreement with the values obtained before. Despite extensive desalting of all samples and calibrants, remaining alkali-salt adduct formation has to be taken into account in the case of MW determination of the intact glycoproteins, especially since sialylated oligosaccharides are known for abundant sodium and/or potassium adduct formation during the desorption/ionisation process in MALDI-MS.54

Figure 3.

MALDI mass spectra of the intact rhEPO molecules on different matrix systems: (a) NESP, (b) EPO-β, and (c) EPO-α on sinapinic acid; (d) NESP, (e) EPO-β, and (f) EPO-α on CHCA/DHB combination matrix; singly, doubly and triply charged molecules are indicated (quadruply charged ions were observed only on CHCA/DHB); approximately 1 pmol each was applied on target; all mass spectra were calibrated externally.

Figure 4.

Molecular weight determination of rhEPOs by MALDI-MS. MW determination of (a) NESP and (b) EPO-α by internal calibration purified out of injection solution (trypsin and enolase were used as internal calibrants) and of (c) NESP and (d) EPO-α after SDS-PAGE by external calibration (1.2 μg EPO-α and 2 μg NESP were separated on SDS gel and subjected to MALDI-MS after re-extraction from gel slices and ZipTip® cleanup); sinapinic acid was used for all measurements.

As clearly indicated by our results, the highly glycosylated NESP could be, even in mixtures, already easily distinguished from the other rhEPOs at the level of the intact protein by means of external calibration. A considerable mass difference of ∼6.7 kDa was observed from EPO-α and -β, which can be unambiguously attributed to the additional N-glycan structures. Systematic molecular mass determinations by internal calibration revealed a broader signal distribution for EPO-β and slight shifts of the average MWs in the range of 300–400 Da to higher masses in the case of EPO-α. These observations may be considered as being the result of subtle molecular structural differences causing different isoform profiles of these molecules, as was shown by IEF separation (Fig. 1(c)).

MALDI matrix evaluation

To determine the highest obtainable sensitivity for MALDI-MS detection of the intact molecules, different MALDI sample preparation strategies were compared (Table 1). In our study the threshold laser energy required for signal generation, the achievable mass spectrometric resolution, and particularly the signal-to-noise (S/N) ratio, were used as evaluation criteria. Another important parameter was the time consumption for the generation of a useful MALDI mass spectrum. A satisfactory performance was recognised only if not more than 200 arbitrary distributed single-laser shots were necessary to find a ‘sweet spot’ of sample preparation. Throughout our analyses, the cinnamic acid derivatives, sinapinic acid and ferulic acid, followed by the benzoic acid derivatives, DHB55 and super-DHB (S-DHB)34 in acidified matrix solutions (pH 1.5–2), were found to be the most useful matrices for the detection of rhEPOs in the positive linear mode. Sinapinic and ferulic acid showed a characteristic crystallisation behaviour. In the case of sinapinic acid, disperse distributed rhombic crystals were formed, while ferulic acid showed needle-shaped crystals (up to 0.5–1 mm in length) from the rim of the target spot. Long lasting signals could be generated from such macro-crystals over a high number of single laser shots (200–500 profiles). A very similar effect was observed in the case of DHB and S-DHB crystal structures. All of these matrices showed almost equal results in terms of S/N ratio and peak resolution (Table 1). An increased tendency to metastable fragmentation could be noticed by an asymmetric peak-tailing effect51 when raising the laser energy, especially on the latter matrices. Matrix concentrations were found to give best results in the range of 3–5 mg/mL. Employing higher matrix concentrations above 10 mg /mL considerably impaired S/N ratios, especially when dealing with more diluted samples (fmol range). On the other hand, reducing the concentration of matrices to ≤1 mg/mL34 had a more detrimental effect on crystal formation. A recently described new matrix combination of DHB and CHCA56 in acidified solvent (containing 5% FA and 0.1% TFA) turned out to be an alternative to ferulic and sinapinic acid. This matrix showed a mixed crystallisation type with fine short-rhombic homogenously distributed crystals and DHB-like needles at the rim, oriented towards the center of the preparation. A significantly lower threshold energy was required for signal generation (∼30%) of the different rhEPOs, but more abundant multiply charged molecules (up to 5+) could be observed. Average MWs of the rhEPOs were found to be approximately 28.4 ± 0.5 kDa for EPO-α or EPO-β and 34.5 ± 0.9 kDa for NESP, respectively, which was slightly lower than for the other evaluated matrices (Fig. 3). These observations may be explained by the ‘hot’ matrix character of CHCA37 that causes both an increase in the protonation efficiency during ionisation and a higher kinetic energy of the molecules after the desorption process. Loss of labile groups should also be taken into account in this matrix system. Interestingly, average MWs of the different rhEPOs, recalculated using the masses of the multiply charged molecules, were about 0.5–1 kDa higher than the masses of the singly charged ions observed directly from the MALDI mass spectra. Under conditions of linearity of calibration, these values seemed to be more accurate than those of the singly charged ions because of better statistics.

The matrices caffeic acid,57 2,6-DHAP,58 3-HPA34 and the β-carbolines (e.g. harmaline and nor-harmane)59 failed the criteria for a positive evaluation throughout our analyses. The only useful matrix for measurements in the negative ion mode was THAP with addition of ammonium citrate, which was very successfully employed for the analyses of sialylated glycans and glycopeptides.60 Although THAP is reported38 to be a useful matrix for measurements in the positive ion mode, it turned out to give worse results in terms of S/N ratio and sensitivity than sinapinic acid, ferulic acid and S-DHB (Table 1).

Sensitivity and different MALDI target surfaces

Different target surfaces and sample supports were tested for sensitivity in order to detect intact rhEPO molecules. In all cases samples were purified by ZipTip® technology prior to sample preparation.61 The absolute binding capacity of the C18 tips was determined by relative quantification on SDS-PAGE to be ≤1 μg rhEPO. Very similar capacities were obtained also in case of C4 tips. Stainless-steel targets with different surface treatment were evaluated (Table 2). The CS4- and KA4-types gave best results, whereas the KA2-type and gold-plated target showed only a limited applicability between the pmol and upper fmol range. A recently introduced hydrophobic surface layer (DropStop®)62 provided a significant improvement in sensitivity for detection down to the lower fmol range (100–50 fmol). Aided by the hydrophobic surface properties, sample droplets (∼0.6 μL) concentrated to very small surface areas (∼100–300 μm2). From 25–50 fmol the quality of sample preparation and crystallisation became significantly more important, showing a considerably reduced reproducibility.

Table 2. Sensitivity assessment on different MALDI target surfaces
EPO-amount*Stainless steelDropStopdGolde
  • ++ high/+ good/(+) preparation dependent/—no signal/n.a. not further analysed, * absolute sample amount deposited on target.

  • a


  • b


  • c


  • d

    Hydrophobic surface (‘one-way’ multilayer-foil DropStop®).

  • e

    Gold-plated stainless steel surface of CS4-type.

  • FA: ferulic acid, SA: sinapinic acid, S-DHB: super-DHB.

1–2 pmol++FA +SA++FA, SA, S-DHB+FA-SA++FA, SA, S-DHB+FA, SA
0.2 pmol (200 fmol)(+) FA, SA(+) FA, SA, S-DHB++FA, SA, S-DHB-FA, SA
100 fmol++SA, S-DHB+FA
50 fmol+FA, S-DHB (+) SA
25 fmoln.a.n.a.n.a.(+)FA, S-DHB-SAn.a.
10–20 fmoln.a.n.a.n.a.n.a.
5 fmoln.a.n.a.n.a.n.a.

Sinapinic and ferulic acid gave comparable results on any of the tested surfaces, with the latter showing a slightly better performance on stainless steel and the former on DropStop®. The ‘dried-droplet’ technique produced bigger crystals, which provided superior results to the ‘thin-layer’ method.63 The latter revealed detection limits in the range of 100–200 fmol on DropStop®, only. Also DHB and S-DHB exhibited good sensitivities down to the lower fmol range.

Homogeneity of sample preparation has been considered a prerequisite for MALDI mass spectra quality and sensitivity.42 In contrast, investigations on intact proteins by MALDI-MS revealed that the formation of bigger crystal structures with a higher local concentration of the analytes improves sensitivity.64, 65 Additionally, in our case, the formation of macro-crystal structures seemed to be crucial in reaching the critical local analyte concentration for detection. A slower evaporation of solvent from the droplets on the DropStop® foil at ambient temperatures supported the crystallisation process. The disadvantage of a more inhomogeneous crystal formation was therefore compensated for an apparently higher sensitivity. Moreover, slow crystallisation is known to improve the quality of MALDI mass spectra by salt-exclusion effects.66 Employing the mentioned sample preparation strategy, a detection limit of about 25 fmol (S/N ratio 3:1)—without taking losses during sample manipulations into account—could be determined for the analysis of the intact, fully glycosylated rhEPOs.

MALDI-MS of enzymatically treated EPO molecules


In contrast to the rather broad signal distribution of the intact rhEPO molecules (RFWHM ∼15), which remained unresolved by MALDI-MS in the linear mode, a pattern of distinct peaks could be observed after de-sialylation of the molecules (Fig. 5). The mass shift of approximately 3–6 kDa of the peak maxima to a lower mass range indicated a loss of 10–20 sialic acid residues (MW 291 Da). This observation is in good accordance with the complete removal from the glycan structures by the enzymatic treatment. Removal of sialic acids, and in fact most of the attached alkali salts, had a tremendous effect in terms of resolution enhancement of MALDI mass spectra (RFWHM ∼190). The results clearly indicated that most of the microheterogeneity of rhEPOs detectable by MALDI-MS could be attributed directly to the sialic acid residues, which mainly contributed to the quite unresolvable signal distribution of the intact molecules. After de-sialylation, at least 8–10 individual well-separated peaks were found. These peaks could be clearly attributed to the sum of the glycan structures occupying the one O-glycosylation site and each of the three N-glycosylation sites of EPO-α and EPO-β molecules, respectively.67, 68 Mass differences of ∼370 Da between the main peaks indicated different quantities of N-acetyllactosamine moieties (365 Da) of these glycan structures. A good fit between the observed molecular mass values and the theoretical MWs calculated for the individual isoforms (protein backbone including all possible glycan structures without sialic acids) could be obtained within the range of mass uncertainty. Mass accuracies in the range of ±0.2–0.6% of the average MW could be obtained by external calibration, which was sufficient for an unequivocal assignment of the peaks. Thereby, it turned out that the most intense peaks of EPO-α and EPO-β corresponded well with glycoforms containing 1–2 tri- and 2–3 tetraantennary N-glycans, respectively (Table 3(a)). As mentioned above, alkali adduct formation could not be ruled out.

Figure 5.

MALDI mass spectra of the rhEPOs after de-sialylation: (a) NESP, (b) EPO-β, (c), and (d) EPO-α (the N-glycan composition of the detected glycoforms is indicated, Tri: triantennary N-glycan, Tetra: teraantennary N-glycan, lac: N-acetyllactosamine repeat); approximately 1 pmol each was applied on target; all measurements were performed with sinapinic acid.

Table 3. Main glycoforms of rhEPOs identified after differential enzymatic treatment by MALDI-MS (a) after de-sialylation and (b) after de-N-glycosylation of the molecules
N-Glycan structurem/z theoretaDe-sialo-EPOαm/z observedbDe-sialo-EPOβm/z observedDe-sialo-NESP m/z observed% Dac
3× Tri25 011.125 142.525 167.60.53/0.63
2× Tri+Tetra25 376.525 503.225 573.60.50/0.78
Tri+2× Tetra25 741.725 865.125 936.50.48/0.76
3× Tetra26 106.926 228.026 285.50.46/0.68
2× Tetra+Tetra-lac126 472.126 591.526 622.00.45/0.57
Tetra+2× Tetra-lac126 837.326 956.526 983.60.44/0.55
2× Tri+3× Tetra30 376.730 498.30.41
Tri+4× Tetra30 741.930 839.30.30
5× Tetra31 107.131 191.10.25
4× Tetra+Tetra-lac131 472.331 546.40.23
3× Tetra+2×Tetra-lac131 837.531 913.10.23
2× Tetra+3× Tetra-lac132 202.732 269.10.20
O-Glycan Structurem/z theoretade-N-EPOαm/z observedbde-N-EPOβm/z observedde-N-NESP m/z observed% Dac
  • a

    Theoretical average MW of the glycoforms in Da (O-glycosylated polypeptide chain consisting of 165 AA including the indicated N-glycan structures).

  • b

    Observed m/z values.

  • c

    Mass accuracy as % Da of theoretical MW.

  • —not observed.

no O-glycan18 243.018 246.918 217.10.02/0.14
 18 183.818 191.60.04
O-glycan18 608.118 621.718 617.50.07/0.05
 18 548.918 564.30.08
O-glycan+1 SA18 899.218 906.018 904.30.04/0.03
 18 841.018 853.40.07
O-glycan+2 SA19 190.319 199.519 194.20.05/0.02
 19 131.119 135.60.02

A second series of peaks showing mass increments of ∼150 Da to the main peaks could also be observed in the fine structure of the signals (Fig. 5(d)). These peaks may be attributed to isoforms containing non-fucosylated N-glycans (fucose residue 146 Da), which are known to be expressed to a lesser degree in CHO cells.4 Although EPO-α and EPO-β showed a quite similar isoform distribution, glycoforms containing three tetraantennary N-glycans were more apparent in EPO-α and those with one tri- and two tetraantennary N-glycans in EPO-β, respectively. This observation may now explain the previously noticed mass shift of peak maxima even of the fully glycosylated molecules (see above). Furthermore, de-sialylation of NESP also revealed positive ion MALDI mass spectra with well-resolved glycoforms. The still higher MW of NESP (mass shift of about +5 kDa) compared with EPO-α and EPO-β could be clearly attributed to the two additional N-glycan structures present in NESP. In contrast to EPO-α and EPO-β the number of isoforms detectable was lower and three main glycoforms could be observed. They consist of 3–5 tetraantennary N-glycans bearing 1–2 additional N-acetyllactosamine repeating units (Table 3(a)).

Enzymatic de-N-glycosylation

The complete removal of all N-glycans from the rhEPOs led to molecules containing only one O-glycosylation. Nevertheless, a microheterogeneity of the remaining O-glycosylated molecules was observed. A combined enzymatic treatment by PNGaseF and neuraminidase revealed that this heterogeneity was mainly caused by a variable degree of sialylation of the O-glycan structures. To a lesser degree, non-O-glycosylated molecules could also be detected, which indicated that some of rhEPO molecules also occurred in non-O-glycosylated forms as already described69 (Figs. 6(a) and 6(b)). A clear identification of the main O-glycoforms by mass spectrometric MW determinations of the de-N-glycosylated molecules was possible by means of internal calibration. Determination of the average MWs of the O-glycoforms could be achieved with a mass accuracy in the range of ±0.02–0.08% (Table 3(b)).

Figure 6.

Comparison of the O-glycoforms of rhEPOs detected by MALDI-MS (a) PNGaseF and sialidase treatment and (b) only PNGAseF treatment of EPO-α; (c) PNGaseF treatment after reduction and alkylation of NESP and (d) PNGaseF treatment of native NESP; SA: sialic acid; all measurements performed on sinapinic acid.

A kinetic study of the overall de-N-glycosylation process using low enzyme concentrations showed that one N-glycan is cleaved very easily from the intact glycoproteins, while the other remaining glycan structures were removed successively to yield the fully de-N-glycosylated protein. Whereas a complete removal of all N-glycans was possible for EPO-α and EPO-β, in the case of NESP a molecule with MW ∼22 kDa turned out to be enzymatically persistent even after an extended incubation time (twice, 24 h). A mass difference of about 3.2 kDa compared with the fully de-N-glycosylated protein indicated that one sialylated N-glycan structure (most likely tetraantennary) was still attached to the polypeptide chain (Fig. 6(d)). This observation confirmed our previous results with 1D gel electrophoresis (Fig. 2(b)). A steric hindrance for PNGaseF is supposed to be responsible for this phenomenon in native NESP. Completion of the reaction was achieved by reduction and alkylation of the protein prior to PNGaseF digestion (Fig. 6(c)). This fact supports the hypothesis that disulfide bridges are also involved in the altered cleavage behaviour of PNGaseF. In addition to other molecular structural features,70 the increased serum half-life of NESP might result from an inaccessibility of cleavage sites for degrading enzymes during catabolism.71

Comparison of the mass spectra of all three rhEPOs revealed that their O-glycans mainly consisted of mono- and disialylated isoforms, which could be clearly distinguished (Figs. 7(a)–7(c)). A mass difference of about 55 Da between signals from de-N-EPO-α/β compared with those of de-N-NESP could be observed, which was in good accordance with the calculated mass difference of the protein sequences (59 Da). Additionally, small variations in the fine structure of the peaks between the O-glycoforms of the rhEPOs were observed in MALDI mass spectra. We suppose post-translational modifications to be responsible for these signals, which could actually not be conclusively resolved by MALDI-MS on the intact protein level.

Figure 7.

Comparison of rhEPOs after de-N-glycosylation: (a) NESP, (b) EPO-β, and (c) EPO-α (measured in positive ion mode using sinapinic acid); (d) EPO-β and (e) EPO-α (measured in negative ion mode using THAP/20 mM ammonium citrate).

For evaluation of the results obtained in the positive ion mode, the de-N-glycosylated rhEPOs were measured in the negative ion mode using THAP as matrix. In this manner, mass spectra of the de-N-glycosylated molecules could be generated, which confirmed the previous results in the positive mode. A very similar signal distribution was obtained with some variance in terms of the relative signal intensities. Dominating peaks were observed from the singly and doubly sialylated O-glycan structures on the protein backbone. In contrast to the positive ion mode, non-glycosylated and non-sialylated structures, respectively, were more suppressed in the corresponding mass spectra of rhEPOs. Doubly sialylated O-glycans seemed to be preferably expressed in EPO-β compared with EPO-α (Figs. 7(d) and 7(e)).


The highly complex glycosylation pattern of rhEPOs makes them very challenging molecules for detailed structural characterisation by mass spectrometric techniques. The detection of rhEPOs in human body fluids became an increasingly important issue in doping control, which makes the application of more sophisticated instrumentation necessary. During our investigations, MALDI-MS turned out to be a very promising tool for the detection and structural characterisation of rhEPOs. Employing a careful matrix preparation strategy, a highly sensitive detection of the intact sialo-glycoproteins was possible. The concentration of the analytes in macro-crystals during the crystallisation process turned out to be a crucial point of the MALDI-MS analysis. The degree of hydrophobicity of the target surface influences the crystallisation process. The highest sensitivity was obtained when the very hydrophobic DropStop® foil was employed as sample support. The limit of detection for all three intact rhEPOs was found to be in the low fmol range (25–50 fmol).

A comparison of rhEPO isoform patterns obtained by IEF separation and our MALDI-MS data revealed a deeper insight into the rhEPO glycoforms. Exact molecular mass determination by means of internal calibration allowed an unambiguous identification of distinct N- and O-glycoforms after different enzymatic treatments. A direct detection of distinct glycoforms of rhEPOs using MALDI-MS in the linear mode was possible after removal of sialic acids from the glycan structures and de-N-glycosylation of the intact molecules, alternatively. In the course of our study, differences between the main isoforms of EPO-α, EPO-β and NESP based on their N-glycan composition could be observed in the MALDI mass spectra. As was clearly observed, the sialic acid residues mostly contribute to the microheterogeneity of the intact molecules. Removal of sialic acid residues by enzymatic treatment led to distinct isoforms, which could be correlated with the N-glycan structures (mainly tri- and tetraantennary). Also from the de-N-glycosylated molecules a microheterogeneity based on the degree of sialylation and additional post-translational modifications of the O-glycan was observable, but could not be ultimately resolved on the intact molecular level. Therefore, an extension of our analyses at the peptide and oligosaccharide level aiming to further investigate the individual glycoforms of rhEPOs by MALDI-MS is in progress thus perhaps allowing their differentiation from u/shEPO in the future.


The authors thank the Bundesministerium für Verkehr, Innovation und Technologie (BMVIT) for their financial support. At the start of the investigations Erypo®, Aranesp® and NeoRecormon® samples were kindly supplied by Janssen-Cilag, Amgen and Roche, respectively. Furthermore, we thank R. Grimm (Agilent Technologies, Waldbronn, Germany) for helpful discussions.