Abstract
The identification of PEGylation sites is essential in the characterization of PEGylated therapeutic proteins. This report describes a simple and novel method of finding poly(ethylene glycol) (PEG) conjugation sites in PEGylated proteins by using a hetero-functional biotin-PEG-N-hydroxyl succinimide derivative. PEGylated lysozyme species having a biotin moiety at each PEG chain end were separated and digested by trypsin. Among the digested lysozyme fragments, biotin-terminated PEGylated peptide fragments were purified by a monomeric avidin immobilized column. Their mass was analyzed by matrix-assisted laser desorption ionization time of flight mass spectrometry, directly indicating that PEG was conjugated to lysine 33, 97, 116 residues. Reversed-phase high-pressure liquid chromatography results for the PEGylated peptide fragments exhibited that PEGylation occurred preferentially at lysine 33> lysine 97> lysine 116.
| Original language | English |
|---|---|
| Pages (from-to) | 97-103 |
| Number of pages | 7 |
| Journal | Journal of Pharmaceutical Sciences |
| Volume | 92 |
| Issue number | 1 |
| DOIs | |
| State | Published - Jan 1 2003 |
| Externally published | Yes |
Funding
This work was supported by grants from the Center for Advanced Functional Polymers, KAIST, and the Ministry of Commerce, Industry, and Energy (A00-961-5411-02-3-3), Republic of Korea. We thank Mr. K. T. Eom for his valuable discussion of this work.
| Funders | Funder number |
|---|---|
| Ministry of Commerce, Industry, and Energy | A00-961-5411-02-3-3 |
| Korea Advanced Institute of Science and Technology |
Keywords
- Avidin-biotin
- Identification of PEGylation sites
- MALDI-TOF MS
- Polyethylene glycol (PEG)