Characterization of poly(ethylene glycol) and PEGylated products by LC/MS with postcolumn addition of amines

PEGylation of peptides and proteins presents significant challenges for structural characterization due to the heterogeneity of the poly(ethylene glycol) (PEG), the number of PEG moieties attached, and the site(s) of PEGylation. In this work, a novel and powerful methodology using a postcolumn addition combined with LC/MS was developed and applied to examine high molecular weight (>/=20 kDa) PEG as well as PEGylated peptide and protein products. The PEG and PEGylated compounds were eluted from RP-HPLC, and the HPLC stream was mixed with diethylmethylamine (DEMA) or triethylamine (TEA) through a T mixer coupled to a time-of-flight mass spectrometer. With these conditions, PEG is diethylmethyl- or triethyl-ammoniated instead of protonated while the protein or peptide remains protonated. The charges for PEG and the PEGylated compounds were greatly reduced, and there was no convolution among differently charged ions, even for 40 kDa PEG or tri-20 kDa PEGylated IgG4 heavy chain. Mass accuracies (<0.01%) obtained are similar to large molecular weight proteins. By selecting specific amines, such as DEMA, commercially available software was used to deconvolute the spectra composed of the diethylmethylammoniated PEG or the diethylmethylammoniated and protonated PEGylated peptide or protein to obtain accurate masses. The examples presented in this report demonstrate that the methodology can be used to elucidate different PEG structures and modifications, such as oxidation and maleimide ring opening of PEGylated peptides or proteins.