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Chemo-Enzymatic Synthesis of 13C Labeled Complex N‑Glycans As Internal Standards for the Absolute Glycan Quantification by Mass Spectrometry

Methods for the absolute quantification of glycans are needed in glycoproteomics, during development and production of biopharmaceuticals and for the clinical analysis of glycan disease markers. Here we present a strategy for the chemo-enzymatic synthesis of 13C labeled N-glycan libraries and provid...

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Bibliographic Details
Published in:Analytical chemistry (Washington) 2015-11, Vol.87 (22), p.11460-11467
Main Authors: Echeverria, Begoña, Etxebarria, Juan, Ruiz, Nerea, Hernandez, Álvaro, Calvo, Javier, Haberger, Markus, Reusch, Dietmar, Reichardt, Niels-Christian
Format: Article
Language:eng ; jpn
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Summary:Methods for the absolute quantification of glycans are needed in glycoproteomics, during development and production of biopharmaceuticals and for the clinical analysis of glycan disease markers. Here we present a strategy for the chemo-enzymatic synthesis of 13C labeled N-glycan libraries and provide an example for their use as internal standards in the profiling and absolute quantification of mAb glycans by matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) mass spectrometry. A synthetic biantennary glycan precursor was 13C-labeled on all four amino sugar residues and enzymatically derivatized to produce a library of 15 glycan isotopologues with a mass increment of 8 Da over the natural products. Asymmetrically elongated glycans were accessible by performing enzymatic reactions on partially protected UV-absorbing intermediates, subsequent fractionation by preparative HPLC, and final hydrogenation. Using a preformulated mixture of eight internal standards, we quantified the glycans in a monoclonal therapeutic antibody with excellent precision and speed.
ISSN:0003-2700
1520-6882
DOI:10.1021/acs.analchem.5b03135