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-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.
Cindrić
Mario
M
Pliva-Research & Development Ltd., Analytics, Prilaz baruna Filipovića 29, 10000 Zagreb, Croatia. mario.cindric@pliva.hr
Cepo
Tina
T
Galić
Nives
N
Bukvić-Krajacić
Mirjana
M
Tomczyk
Nick
N
Vissers
Johaness P C
JP
Bindila
Laura
L
Peter-Katalinić
Jasna
J
D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid; D004220:Disulfides / Q000737:chemistry; D016179:Granulocyte Colony-Stimulating Factor / Q000737:chemistry* / Q000302:isolation & purification; D007093:Imidazoles / Q000737:chemistry; D007202:Indicators and Reagents; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D011092:Polyethylene Glycols / Q000737:chemistry*; D011994:Recombinant Proteins; D012997:Solvents; D021241:Spectrometry, Mass, Electrospray Ionization; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
C447410:2-methoxy-4,5-dihydro-1H-imidazole; D004220:Disulfides; D007093:Imidazoles; D007202:Indicators and Reagents; D010446:Peptide Fragments; D011994:Recombinant Proteins; D012997:Solvents; D016179:Granulocyte Colony-Stimulating Factor; D011092:Polyethylene Glycols