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


CONCLUSIONS
Oligosaccharide profiling together with top- and middle-down methods enabled: 1) detection of heterogeneous glycosylated protein species and sites in intact IgG1 and fusion proteins with high mass accuracy, 2) estimation of relative abundance levels of protein species in the sample, 3) confirmation of the protein termini structural information, and 4) improved sequence coverage by MALDI-ISD analysis for the internal regions of the proteins without sample pre-fractionation.
Department of Pharmaceutical Sciences, School of Pharmacy, University of Maryland, Baltimore, MD, USA.
Bao Quoc
BQ
Tran
MedImmune, LLC, Gaithersburg, MD, USA.
Christopher
C
Barton
MedImmune, LLC, Gaithersburg, MD, USA.
Jinhua
J
Feng
MedImmune, LLC, Gaithersburg, MD, USA.
Aimee
A
Sandjong
Department of Pharmaceutical Sciences, School of Pharmacy, University of Maryland, Baltimore, MD, USA.
Sung Hwan
SH
Yoon
Department of Pharmaceutical Sciences, School of Pharmacy, University of Maryland, Baltimore, MD, USA.
Shivangi
S
Awasthi
Department of Pharmaceutical Sciences, School of Pharmacy, University of Maryland, Baltimore, MD, USA.
Tao
T
Liang
Department of Pharmaceutical Sciences, School of Pharmacy, University of Maryland, Baltimore, MD, USA.
Mohd M
MM
Khan
Department of Pharmaceutical Sciences, School of Pharmacy, University of Maryland, Baltimore, MD, USA.
David P A
DPA
Kilgour
Department of Pharmaceutical Sciences, School of Pharmacy, University of Maryland, Baltimore, MD, USA. Electronic address: dgoodlett@rx.umaryland.edu.
David R
DR
Goodlett
Department of Pharmaceutical Sciences, School of Pharmacy, University of Maryland, Baltimore, MD, USA. Electronic address: ygoo@rx.umaryland.edu.
Young Ah
YA
Goo
D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000737:chemistry; D002460:Cell Line; D006031:Glycosylation; D006801:Humans; D007074:Immunoglobulin G / Q000096:biosynthesis / Q000737:chemistry; D013058:Mass Spectrometry; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry
D000911:Antibodies, Monoclonal; D007074:Immunoglobulin G; D011993:Recombinant Fusion Proteins
Glycosylation; MALDI-ISD; Middle-down; Oligosaccharide profiling; Protein species; Top-down