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Journal of Proteomics

Comprehensive glycosylation profiling of IgG and IgG-fusion proteins by top-down MS with multiple fragmentation techniques

Bao Quoc Tran, Christopher Barton, Jinhua Feng, Aimee Sandjong, Sung Hwan Yoon, Shivangi Awasthi, Tao Lianga, Mohd M. Khan, David P.A. Kilgour, David R. Goodlett, Young Ah Goo

Department of Pharmaceutical Sciences, School of Pharmacy, University of Maryland, Baltimore, MD, USA.


We employed top- and middle-down analyses with multiple fragmentation techniques including electron transfer dissociation (ETD), electron capture dissociation (ECD), and matrix-assisted laser desorption ionization in-source decay (MALDI-ISD) for characterization of a reference monoclonal antibody (mAb) IgG1 and a fusion IgG protein. Fourier transform ion cyclotron resonance (FT-ICR) or high performance liquid chromatography electrospray ionization (HPLC-ESI) on an Orbitrap was employed. These experiments provided a comprehensive view on the protein species; especially for different glycosylation level in these two proteins, which showed good agreement with oligosaccharide profiling. Top- and middle-down MS provided additional information regarding glycosylation sites and different combinational protein species that were not available from oligosaccharide mapping or conventional bottom-up analysis. Finally, incorporating a limited enzymatic digestion by immunoglobulin G-degrading enzyme of Streptococcus pyogene (IdeS) with MALDI-ISD analysis enabled extended sequence coverage of the internal region of protein without pre-fractionation.

Graphical abstract

Keywords: Top-down; Middle-down; Glycosylation; Protein species; MALDI-ISD; Oligosaccharide profiling.

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