Characterization of N‐ and O‐glycopeptides of recombinant human erythropoietins as potential biomarkers for doping analysis by means of microscale sample purification combined with MALDI‐TOF and quadrupole IT/RTOF mass spectrometry

Abstract

The structural characterization of the O- and N-glycan structures of three different commercially available recombinant human erythropoietins (rhEPOs) is represented by means of a microscale sample purification using ZipTip technology and MALDI-TOF and MALDI low-energy CID MS. Glycopeptides were released from rhEPO samples by a differential endoproteolytic digestion to obtain site-specific glycosylation patterns. Mass accuracies in the range of +/- 0.04% obtained by the high-resolution TOF instrument allowed an unambiguous assignment of N-glycan structures via glycan database software. Furthermore, the O-glycan structures were directly analyzed on the glycopeptide level by MS/MS experiments. Principally, site-specific glycosylation was found to be very similar for the three different rhEPOs (EPO-alpha, EPO-beta, and novel erythropoiesis stimulating protein (NESP)) but exhibiting quantitative differences in distinct O- and N-glycan moieties. Significant differences were found in the degree of sialylation and acetylation. Especially, a considerable degree of variation of the O-acetylation of sialic acid residues could be realized on the glycan structures of O- and N-glycopeptides, whereas EPO-alpha and EPO-beta could be clearly differentiated from NESP solely on the O-glycopeptide level.

Comprehensive Glycoproteomic Analysis of Chinese Hamster Ovary Cells

“…There have been many efforts to characterize the glycosylation of EPO in CHO cells, but most of the work focused on glycomics profiling or glycosite identification. 59 With our large-scale IGPs identification method, the N-linked glycans of EPO at specific glycosylation sites were extensively characterized. Owing to the limitation that the first and second glycosites cannot be cleavage by trypsin, only two glycosite-containing peptides from EPO were identified with one peptide containing 2 glycosites (EAE N # IT TGCAEHCSLNE N # IT VPDTK and GQALLV N # S SQPWEPLQLHVDK (Table S5).…”

Inhibition of poly-LacNAc biosynthesis with release of CMP-Neu5Ac feedback inhibition increases the sialylation of recombinant EPO produced in CHO cells

“…For example, we categorized N-glycans having four HexNAc into bi-antennary group because two HexNcA attached to asparagine on a protein, and leaving two HexNAc attached on N-glycan core as two antennae. Although polylactosaminylated bi- or tri-antennary N-glycans are biologically possible, most polylactosaminylation on recombinant EPOs was found in tetra-antennary N-glycans 24 , 26 , 27 . Indeed, polylactosaminylation was observed to have primarily on tetra-antennary rather than bi- or tri-antennary N-glycans by our previous study 22 .…”

Glycoform characterization of erythropoietin combining glycan and intact protein analysis by capillary electrophoresis – electrospray – time‐of‐flight mass spectrometry

“…Structural characterization of rHuEPO has been usually performed by peptide mapping after enzymatic digestion [2,[8][9][10] or glycan analysis after chemical or enzymatic release [11][12][13]. At the intact protein level, rHuEPO has been mainly analysed by CZE [14][15][16][17], IEF [18][19][20], 2-DE [21], CIEF [22] and MALDI-TOF-MS [23].…”