Methods For Identificaiton Of ConjugationSite On PEGylated Protein

The PEG-linking site is one of the key factors to decide the structure and biologicalactivities of PEGylated proteins. The options of appropriate method are crucial to detect thePEG-linking sites. In this study, lysozyme and rh-G-CSF were modified with methoxy PEGsuccinimidyl carboxymethyl ester (mPEG-SC, mean MW5k) respectively, and the monoPEG modified lysozyme/rhG-CSF (PEG-lysozyme/PEG-rhG-CSF) was detected bybiological mass spectrometry technologies. The key points are summarized as follows:1. The method of tryptic digestion coupled with mass spectrometric analysis was studied todetect the PEG-linking sites. The results showed that the method can get the ratio of differentlinking sites without of complex processing, and the PEGlyated peptide sequence wasT1PLGPASSLPQSFLLK16while the coupling sites wre T1and K16with the ratio of96.4:3.6in the conditions of this paper. The method was suitable for PEGlyated proteins with multi-linking-sites, but the PEG chains on the surface of proteins can impact on the digestion so thatthe tryptic digestion conditions should be optimized. Otherwise, the separation of polypeptidein the HPLC-MS analysis also can affect the ionization efficiency and the quantitative results.2. We studied the method of chymotrypsin digestion coupled with mass spectrometricanalysis. The method was suitable for the identification of PEGlyated proteins with multi-linking-sites, and the identification would not be affected by the digestion degree. It turnedout that the PEGlyated peptide sequence was T1PLGPASSLPQSFLLK16, and the couplingsites wre T1and K16with the ratio of96.4:3.6. But the separation of all the PEGlyatedpolypeptides was needed in the method, and the chymotrypsin has much enzyme digestionsites that a few enzymolysis polypeptides were too short for HPLC-MS analysis. The methodwas limited by the protein sequence.3. Explored the method of PEGlyated peptide sepration of HPLC coupled with amino acidcomponents analysis. The results indicated that the PEGlyated peptide sequence wasT1PLGPASSLPQSFLLK16, and the coupling sites wre T1and K16with the ratio of about95:5.The method was based on the amino acid components analysis, but had special requirementsof the sepration of PEGlyated peptide to get the ratio of different linking sites.4. The method based on Edman degradation collected the PEGlyated peptide and detectedits sequence. The experiment result expressed that the PEGlyated peptide sequence containedPLGPASSLPQSFLLK, and the coupling sites wre T1and K16with the ratio of about95:5. Thepurity of PEGlyated peptide was the key factor of the identification of this method. In actual sample analysis, as the protein sequence has differences, the linking sites ofPEGlyated proteins may be inhomogeneous, so the PEG linking sites and the ratios should beidentified by combination of possible methods.The amino acid components analysis andEdman degradation are rapid for qualitative analysis, while the tryptic digestion andchymotrypsin digestion are suitable of multi-linking-sites for quantitative analysis.