Naslov
Mass spectrometry-based glycoproteomic approach involving lysine derivatization for structural characterization of recombinant human erythropoietin

Autori
Cindrić, Mario ; Bindila, Laura ; Čepo, Tina ; Peter-Katalinić, Jasna

Izvornik
Journal of proteome research (1535-3893) 5 (2006), 11; 3066-3076

Vrsta, podvrsta i kategorija rada
Radovi u časopisima, članak, znanstveni

Ključne riječi
rHuEPO glycosylation; Lys-4; 5 dihydro-1H-imidazole; N-glycans; O-glycopeptides; MALDI-TOF; LC-MS/MS; nanoESI MS/MS

Sažetak
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-dihydro1H-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 areflectron with high mass accuracy. After defining the precursor ions, we performed the nanoESI QTOFMS/MS analysis on N-glycans, mainly targeting the distinction between carbohydrates with sialylated antennae and those lacking sialic acid moieties.

Izvorni jezik
Engleski

Znanstvena područja
Biologija, Farmacija, Biotehnologija

POVEZANOST RADA