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

nije evidentirano