title: Mass spectrometry-based glycoproteomic approach involving lysine derivatization for structural characterization of recombinant human erythropoietin.
abstract: Lysine-containing peptides comprising glycosylation sites derived from recombinant human erythropoietin (rHuEPO) by trypsin or Lys-C and PNGase F dual digestion were derivatized with 2-methoxy-4,5-dihydro-1H-imidazole and its deuterated analogues. In the same reaction, under reducing conditions (beta-mercaptoethanol), cysteines were converted into methyl-cysteines and lysines into Lys-4,5-dihydro-1H-imidazole. Both modifications on cysteines and lysines simplified the CID-MS/MS spectra, while preserving the structural information by yielding y-series ions and improved the mass spectral signal intensity up to 25 times. Moreover, by this approach, the N-glycan occupation sites were unambiguously determined. O-Glycosylation sites as well as O-glycan structures were determined by a LC-MS/MS experiment carried out on dually digested rHuEPO. N-Glycan mixture purified on a graphitized carbon column using a newly developed method that extracted only sialylated carbohydrates was analyzed first using MALDI-TOF in negative linear ion mode with low mass accuracy but without interferences and metastabile ions and then a reflectron with high mass accuracy. After defining the precursor ions, we performed the nanoESI QTOF MS/MS analysis on N-glycans, mainly targeting the distinction between carbohydrates with sialylated antennae and those lacking sialic acid moieties.
authors.affiliation: Pliva-Research & Development Ltd., Prilaz baruna Filipovića 29, 10000 Zagreb, Croatia. mario.cindric@pliva.hr
authors.forename: Mario
authors.initials: M
authors.lastname: Cindrić
authors.forename: Laura
authors.initials: L
authors.lastname: Bindila
authors.forename: Tina
authors.initials: T
authors.lastname: Cepo
authors.forename: Jasna
authors.initials: J
authors.lastname: Peter-Katalinić
mesh_terms: D000595:Amino Acid Sequence; D003545:Cysteine; D003903:Deuterium; D004921:Erythropoietin / Q000737:chemistry* / Q000302:isolation & purification; D006023:Glycoproteins / Q000737:chemistry* / Q000302:isolation & purification; D006031:Glycosylation; D006801:Humans; D008239:Lysine; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D011134:Polysaccharides / Q000737:chemistry / Q000302:isolation & purification; D040901:Proteomics / Q000379:methods*; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification; D014357:Trypsin
chemical_list: D006023:Glycoproteins; D010446:Peptide Fragments; D011134:Polysaccharides; D011994:Recombinant Proteins; D004921:Erythropoietin; D003903:Deuterium; D014357:Trypsin; D008239:Lysine; D003545:Cysteine