79
Views
3
CrossRef citations to date
0
Altmetric
MASS SPECTROMETRY

Characterization of Minor N-linked Glycans on Antibodies Using Endo H Release and MALDI–Mass Spectrometry

, , &
Pages 1711-1724 | Received 25 Feb 2009, Accepted 21 Apr 2009, Published online: 26 Jun 2009
 

Abstract

A MALDI mass spectrometry method using Bruker Daltonic's LIFT technology for MS/MS analysis has been developed for profiling and characterizing low abundant N-glycans from recombinant immunoglobulin G (IgG) antibodies. In this method, Endoglycosidase H (Endo H) released N-glycans are derivatized at their reducing end with 2-aminobenzamide (2-AB) and separated by normal phase chromatography. Endo H hydrolyses the bond between the two GlcNAc residues of the trimannosyl core of high mannose and hybrid N-linked glycans, leaving the core GlcNAc attached to the protein. High mannose and hybrid type N-glycans are released from the glycoprotein whereas the more abundant, complex biantennary type oligosaccharide structures are unaffected. Analysis of Endo H treated glycan moieties by MALDI mass spectrometry identified several minor species of high mannose and hybrid type glycans. Subsequent MALDI TOF MS/MS analysis of the resulting products yielded information about structural features of the high mannose and hybrid type glycans. This study involving Endo H treatment followed by MALDI mass spectrometry coupled with LIFT technology for MS/MS analysis offers a specific and sensitive technique for visualizing, and characterizing minor glycan species.

Acknowledgments

We would like to thank John Valliere-Douglass, Gerd Kleemann, and Michael Treuheit for useful discussions and critical review of the manuscript.

Notes

Note. Sialic acid (N-acetylneuraminic acid), ♦; Galactose (Gal), •; N-acetyl-glucosamine (GlcNac), ▪; and Mannose (Man), ○.

Reprints and Corporate Permissions

Please note: Selecting permissions does not provide access to the full text of the article, please see our help page How do I view content?

To request a reprint or corporate permissions for this article, please click on the relevant link below:

Academic Permissions

Please note: Selecting permissions does not provide access to the full text of the article, please see our help page How do I view content?

Obtain permissions instantly via Rightslink by clicking on the button below:

If you are unable to obtain permissions via Rightslink, please complete and submit this Permissions form. For more information, please visit our Permissions help page.