Maria Lorna A. De Leoz, Xinjian Yan, Xiaoyu Yang, Lisa Kilpatrick, Yuxue Liang, Pedatsur Neta, Stephen E. Stein, and Michael J. Tarlov
The majority of therapeutic proteins are glycosylated, i.e., proteins are modified by the attachment of sugar molecules in complex chains called glycans. Alteration in glycosylation may significantly modify the safety, stability, and efficacy of therapeutic glycoproteins. To aid in the ability to monitor changes in these proteins during manufacturing and storage, we are developing a reference library of all identifiable components derived from the digestion of these molecules. Specifically, we are measuring, evaluating, and storing reference mass spectra for enzymatically-released glycans and derived glycopeptides using high-resolution MS with spectra generated by both ion trap and collision cell fragmentation.
Glycopeptides and glycans were released from therapeutic glycoproteins using trypsin and Peptide-N-glycosidase F, respectively. After desalting and purification by solid phase extraction, the glycoforms were analyzed on a nanospray-Velos/Orbitrap* instrument with identification by fragmentation in the ion trap by resonant collision-induced dissociation (CID) and in the collision cell (HCD) over a range of energies. A clustering algorithm was used to obtain hundreds of quality consensus spectra for each glycoform.
To determine the identity of glycans, consensus spectra of glycans from therapeutic drugs are searched against the consensus spectra of individual glycans using the NIST MS Search program (http://chemdata.nist.gov/mass-spc/ms-search/). Due to the oxonium peaks observed in HCD spectra, it is possible to identify glycan spectra in therapeutic antibodies for de novo glycans using ProMS. These peaks are not readily seen in CID due to the 1/3 cut-off rule.
To identify glycopeptides in MS/MS spectra, ProMS Glyco was developed to search for the presence of oxonium ions and oligosaccharide neutral losses in the HCD MS2 spectra. Several complex and high mannose glycans were observed from the glycan profiles. Glycosylation sites and their glycan heterogeneity were determined from the C18 LC-MS/MS tryptic digest runs.
The attention to producing reproducible and high quality tandem MS spectra at several energies, combined with the high resolution, high mass accuracy, and effective fragmentation of the MS tools used, shows promise in expanding the scope of the NIST mass spectral library to glycoproteomics. This would be valuable in characterizing glycan heterogeneity and sites of glycosylation in therapeutic antibodies.
* Reference to commercial equipment or supplies does not imply endorsement by the University of Maryland or the National Institute of Standards and Technology.