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  • Title: Structural characterization of PEGylated rHuG-CSF and location of PEG attachment sites.
    Author: Cindrić M, Cepo T, Galić N, Bukvić-Krajacić M, Tomczyk N, Vissers JP, Bindila L, Peter-Katalinić J.
    Journal: J Pharm Biomed Anal; 2007 Jun 28; 44(2):388-95. PubMed ID: 17448619.
    Abstract:
    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-MS(E)) 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.
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