Matrix-assisted laser-desorption time-of flight ionisation and high-performance liquid chromatography–electrospray ionisation mass spectral analyses of two glycosylated recombinant epoetins

Stanley, Shawn M.R.; Poljak, Anne

doi:10.1016/s1570-0232(02)00824-3

Cited by 34 publications

(30 citation statements)

References 12 publications

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“…Endogenous human EPO has a mass of ca 30 kDa, contains two intramolecular disulphide bonds, three N-linked sites of glycosylation (at asparagines residues 24, 38 and 83) and one O-linked site of glycosylation (at serine residue 126) [5]. Recombinant forms of this protein contain the same amino acid backbone, but differ in their glycosylation pattern due to their expression in different cells lines.…”

Section: Introductionmentioning

confidence: 99%

UPLC-MS/MS Method for the Identification of Recombinant Human Erythropoietin Analogues in Horse Plasma and Urine

Scarth¹,

Seibert²,

Brown³

et al. 2011

Chromatographia

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Recombinant human erythropoietin (rhEPO) analogues are known to have been used in horse sports for their assumed performance enhancing properties. Recently, several authors have published liquid chromatographictandem mass spectrometric (LC-MS/MS) methods for confirming the presence of rhEPO analogues in horse plasma. In the current study, an improved LC-MS/MS confirmatory procedure for rhEPO, darbepoetin (DPO) and continuous erythropoietin receptor activator (CERA) in horse plasma was developed and validated. The method was also adapted for and applied to urine samples for the first time. Similar to previously published plasma assays, the methods utilise size exclusion and immunoaffinity extraction prior to tryptic cleavage, enzymatic deglycosylation and LC-MS/MS analysis of the resulting signature tryptic peptides (rhEPO/CERA T5, rhEPO/CERA/DPO T6 and DPO T9). However, the novel application of UPLC chromatography significantly improves the run time of the method compared to nano-or micro-LC and its robustness compared to nano-LC. Furthermore, recombinant canine EPO was found to serve as an effective internal standard, thus allowing confidence in interpretation of the success/ failure of every step in the procedure. Limits of detection for confirming the presence of rhEPO, CERA and DPO in plasma were 0.1, 0.25 and 0.05 ng mL -1 , respectively, which were equal to or lower than limits achieved using previously published LC-MS/MS based methods. Limits of detection for confirming the presence of rhEPO, CERA and DPO in urine were 0.05, 0.15 and 0.025 ng mL -1 and the analysis of urine samples collected from horses administered rhEPO (Eprex TM ) or DPO (Aranesp TM ) demonstrated the use of this matrix as a suitable alternative in situations where plasma is not available.

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Section: Introductionmentioning

confidence: 99%

UPLC-MS/MS Method for the Identification of Recombinant Human Erythropoietin Analogues in Horse Plasma and Urine

Scarth¹,

Seibert²,

Brown³

et al. 2011

Chromatographia

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“…Intact EPO has been characterized based on isoelectric focusing [8], 2-D gel electrophoresis [10], capillary zone electrophoresis [11][12][13][14][15][16][17], capillary isoelectric focusing [14], and matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) [13,18]. More detailed methods include the characterization of the peptides by CE [19], LC and CE coupled to ESI-MS [20][21][22], and LC-MALDI-TOF-MS [23] after proteolytic degradation.…”

Section: Introductionmentioning

confidence: 99%

Glycoform characterization of intact erythropoietin by capillary electrophoresis‐electrospray‐time of flight‐mass spectrometry

2005

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Glycosylated proteins often show a large variation in their glycosylation pattern, complicating their structural characterization. In this paper, we present a method for the accurate mass determination of intact isomeric glycoproteins based on capillary electrophoresis-electrospray-time of flight-mass spectrometry. Human recombinant erythropoietin has been chosen as a showcase. The approach enables the on-line removal of nonglycosylated proteins, salts, and neutral and negatively charged species. More important, different glycosylation forms are separated both on the base of differences in the number of negatively charged sialic acid residues and the size of the glycans. Thus, 44 glycoforms and in total about 135 isoforms of recombinant human erythropoietin, taking also acetylation into account, could be distinguished for the reference material from the European Pharmacopeia. Distinct glycosylation differences for samples from different suppliers are clearly observed. Based on the accurate mass an overall composition of each single isoform is proposed, perfectly in agreement with data on glycan and glycopeptide analysis. This method is an ideal complement to the established techniques for glycopeptide and glycan analysis, not differentiating branching or linkage isoforms, but leading to an overall composition of the glycoprotein. The presented strategy is expected to improve significantly the ability to characterize and quantify isomeric glycoforms for a large variety of glycoproteins.

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“…In addition, MALDI mass spectra from high molecular mass biopolymers are simpler and easier to interpret because no series of multiply charged ions are generated. When a large glycoprotein with multiple glycosylation sites is directly analyzed by MALDI‐TOF‐MS, only an average molecular mass can be calculated because the TOF analyzer is not able to resolve the molecular ions of the different glycoforms 5–9. The on‐line coupling of MALDI‐TOF‐MS to high‐performance separation techniques, e.g.…”

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confidence: 99%

Towards a reliable molecular mass determination of intact glycoproteins by matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry

Giménez¹,

Benavente²,

Barbosa³

et al. 2007

Rapid Comm Mass Spectrometry

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Different matrices and sample-matrix preparation procedures have been tested in order to study their influence on the matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectra of intact glycoproteins, which present different degrees of glycosylation (human transferrin; bovine fetuin; bovine alpha(1)-acid-glycoprotein; recombinant human erythropoietin; and the novel erythropoiesis stimulating protein). Using sinapinic acid (SA) and the fast evaporation method, the studied glycoproteins became susceptible to fragmentation at any laser intensity, suggesting that this 'hot' matrix is unsuitable for a reliable molecular mass determination of glycosylated compounds. In contrast, 2,5-dihydroxybenzoic acid (DHB) and 6-aza-2-thiothymine (ATT), with an adequate sample-matrix preparation, provided improved results. Samples containing DHB after crystallization by vacuum drying demonstrated the best performance because the labile functional groups from the glycoforms were apparently fragmented to a lower extent. The average molecular masses obtained using this methodology were in all cases a better estimation than those values reported in the literature. The results were reproducible, and sensitivity was similar to that obtained with SA and the fast evaporation method. These excellent results suggest that this MALDI-TOF-MS methodology could be useful for an improved determination of the average molecular mass values of microheterogeneous compounds such as glycoproteins, glycosylated compounds or, in general, molecular mass values of molecules with similar labile functional groups.

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Matrix-assisted laser-desorption time-of flight ionisation and high-performance liquid chromatography–electrospray ionisation mass spectral analyses of two glycosylated recombinant epoetins

Cited by 34 publications

References 12 publications

UPLC-MS/MS Method for the Identification of Recombinant Human Erythropoietin Analogues in Horse Plasma and Urine

UPLC-MS/MS Method for the Identification of Recombinant Human Erythropoietin Analogues in Horse Plasma and Urine

Glycoform characterization of intact erythropoietin by capillary electrophoresis‐electrospray‐time of flight‐mass spectrometry

Towards a reliable molecular mass determination of intact glycoproteins by matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry

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