UC San Diego

Polyethylene glycol (PEG)-derivatized proteins are important pharmaceuticals and the characterization of their PEGylation patterns are of great importance to regulatory agencies for their approval. Accurate quantification of the PEGylated sites are technically challenging. The PEG moiety attached to the protein is very bulky and hinders protease digestion, with the removal of PEG a succinyl linker (100.01 Da) is still attached and can be used for identification of PEGylated sites. The dePEGylated protein can also be used in comparison to the Native protein to determine the percent occupancy of PEG on lysine. Here, we present an improved mass spectrometry based method using iTRAQ reagents (AB SCIEX) to quantitate ADI, a PEGylated protein. By using LC coupled with tandem mass spectrometry (QTOF), PEGylated lysines are characterized by measuring the ratio of 114: 115 iTRAQ reagent tags, on Native and dePEGylated protein. 24 of 27 lysines were determined to have a succinyl modification. 14 of 27 lysines were quantified to determine their percent occupancy