Structural characterization of PEGylated rHuG-CSF and location of PEG attachment sites (CROSBI ID 145149)
Prilog u časopisu | izvorni znanstveni rad | međunarodna recenzija
Podaci o odgovornosti
Cindrić, Mario ; Čepo, Tina ; Galić, Nives ; Bukvić-Krajačić, Mirjana ; Tomczyk, Nick ; Vissers, Johaness P. C. ; Bindila, Laura ; Peter-Katalinić, Jasna
engleski
Structural characterization of PEGylated rHuG-CSF and location of PEG attachment sites
Mass spectrometry structural characterization is an essential tool in validating the quality of PEG-rHu-proteins. However, in either case top-down or bottom-up fashion, the interference of high intensity PEG signals on MS detection or detrimental influence of PEG on protein structure, leads to incomplete structural characterization. We propose here a method that permits complete and reliable structural characterization of PEGylated recombinant human granulocyte-colony stimulating factor (rHuG-CSF). The approach includes on-column 2-methoxy-4, 5-dihydro-1H-imidazole derivatization of digested PEG rHuG-CSF and subsequent LC/MS investigation. By comparing the LC/MS retention of derivatized and underivatized digested PEG rHuG-CSF, location of the PEG attachment within rHuG-CSF could be deduced. Besides, the protein sequence coverage and position of the disulfide bridges was fully deducible from the MS data interpretation. Additionally, ultra performance liquid chromatography– mass spectrometry-to-the-E (UPLC– MSE) was introduced for analysis of label-free digested PEG rHuG-CSF here to enable high resolution and mass accuracy of MS detection and facilitate deep structural insights of peptides.
Mono-PEGylated rHuG-CSF; LC– MS/MS; MALDI-TOF MS; Lys derivatization; MSE; N-terminal PEG-peptide
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Podaci o izdanju
44 (2)
2007.
388-395
objavljeno
0731-7085
10.1016/j.jpba.2007.02.036
Povezanost rada
Kemija, Temeljne medicinske znanosti, Farmacija