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In-depth Characterization and Spectral Library Building of Glycopeptides in the Tryptic Digest of a Monoclonal Antibody Using 1D and 2D LC/MS-MS



Qian Dong, Xinjian Yan, Yuxue Liang, Stephen E. Stein


This work presents a detailed analysis of glycopeptides produced in the tryptic digestion of an IgG biologic drug reference material. Analysis was done by nanospray ESI LC-MS/MS over a wide range of HCD collision energies with both conventional 1D separation for various digestion conditions and a 20-fraction 2D-LC study of a single digest. An extended version of NIST-developed software for analysis of “shotgun” proteomics served to identify the glycopeptides from their precursor masses and product ions for peptides with up to three missed cleavages. A peptide with a single missed cleavage, TKPREEQYNSTYR, was dominant and led to the determination of almost all glycans reported in this study. The 1D studies found a total of 195 different glycopeptide ions containing 30 glycans having different masses. Identities and relative abundances of these studies agreed with published studies on released glycans of this protein. The 2D studies found a total of 247 glycopeptide ions and 60 glycans of different masses, including all of those found in the 1D studies. This is a significantly larger number of glycans than found in any other study of IgG glycosylation. Systematic variations in retention with glycan size were also noted. Detailed analysis of results including considerations based on charge state and fragment identities show that in-source fragmentation had little effect on the present results as long as the charge 4 state of peptide TKPREEQYNSTYR was used for identification and quantification. Energy dependent changes in HCD fragmentation confirmed glycan structures and led to a peak-annotated mass spectral library for use in the identification of these glycopeptides by mass spectral library search techniques. The effectiveness of this method is demonstrated in an analysis of results of a conventional peptide mapping experiment.
ACS Journal of Proteome Research


Glycopeptides, monoclonal antibody, HCD fragmentation, N-glycosylation, quantitation, 2D-LC MS/MS


Dong, Q. , Yan, X. , Liang, Y. and Stein, S. (2016), In-depth Characterization and Spectral Library Building of Glycopeptides in the Tryptic Digest of a Monoclonal Antibody Using 1D and 2D LC/MS-MS, ACS Journal of Proteome Research, [online], (Accessed May 24, 2024)


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Created May 6, 2016, Updated January 27, 2020