Etanercept is a recombinant Fc fusion protein therapeutic that has a complex distribution of post-translation modifications (PTM), such as N and O-linked glycans. Current CID-based MS/MS can struggle to characterize such PTMs, since side-chain species are typically fragmented via a mechanism that does not produce diagnostic ions related to the PTM localization. Other forms of fragmentation, such as ETD, can maintain side chain information but suffer from low sensitivity and scan rate, as well as inadequate coverage of low charge state peptides. In this study, a new form of fragmentation (electron activated dissociation, or EAD) was employed to comprehensively characterize the glycosylation of etanercept at the peptide level. A comparative analysis between intact/subunit and peptide mapping results was performed as a validation step for the novel EAD workflow. Three N linked glycosylations were detected, as well as nine sites of O-linked glycosylation, with high confidences, via a single, generic, data-dependent LC-MS/MS analysis. Even in challenging cases, exact positioning information of the glycosylation on the peptide could be obtained. EAD also allows for confirmation of amino acid isomers (Leu/IsoLeu and Asp/IsoAsp) via MS/MS analysis, in the same experiment.

 

 

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