Capillary electrophoretic characterization of PEGylated human parathyroid hormone with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.
A capillary electrophoretic method (CE) for characterizing PEGylated human parathyroid hormone 1-34 (PTH) with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is described. CE was used to optimize the PEGylation of PTH through control of the reaction pH and the molar ratio of reactants with the advantages of minimal sample consumption and high separation capacity. The mono-PEGylated PTH (mono-PEG-PTH) was isolated and then digested with endoproteinase Lys-C. Resistance to Lys-C digestion on the PEGylation sites in the mono-PEG-PTH resulted in patterns of CE electropherograms different from that of the native PTH, and the PEGylation sites were assigned accordingly. The extent of positional isomers present in the mono-PEG-PTH was also determined by quantifying PEGylated fragments in the same CE electropherogram. In conclusion, the CE analysis of the Lys-C-digested sample allowed for simultaneous analysis of the PEGylation site and the extent of positional isomers in the mono-PEG-PTH. The results were confirmed by MALDI-TOF MS. This method will be applicable for characterizing PEGylation of other therapeutic peptides.
Na
Dong Hee
DH
Drug Targeting Laboratory, College of Pharmacy, SungKyunKwan University, 300 Chonchon-dong, Jangan-ku, Suwon City 440-746, Republic of Korea.
Lee
Kang Choon
KC
D000595:Amino Acid Sequence; D019075:Electrophoresis, Capillary / Q000379:methods*; D006801:Humans; D008969:Molecular Sequence Data; D010281:Parathyroid Hormone / Q000032:analysis* / Q000737:chemistry; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
D010281:Parathyroid Hormone