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

Abstract

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 (13)C 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 (13)C-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.

Determination of true ratios of different N-glycan structures in electrospray ionization mass spectrometry

“…Two nongalactosylated, neutral glycans (GnGnbi and Gn[GnGn]bi in ProGlycAn nomenclature) and one disialoglycan (Na 6-4 Na 6-4 ) were the subjects of this study. A larger panel of structures was used in a study that introduced the application of stable-isotope-labeled glycans for internal calibration [30]. This work focused on IgG glycans and thus on diantennary structures with zero to two galactoses and fucose and up to two sialic acids.…”

“…However, none of these approaches consider the differing molar response of glycans of different size, different number of sialic acids, different general architecture (oligomannosidic vs complex type), different charge states, and different tendency to form adducts with sodium or ammonium in electrospray ionization (ESI) MS [30]. In addition, chemical modification of glycans is essentially not compatible with the one method that has the greatest ability to separate structural isomers; that is, porous graphitic carbon (PGC) chromatography coupled with ESI-MS [2, 3].…”

“…A solution to the problem is the use of internal standards labeled without alteration of the overall structure, which has recently been presented in the form of 13 C-labeled N-acetylated glycans [30]. A range of glycan structures covering the species relevant for the analysis of monoclonal antibodies was prepared, including a disialylated glycan.…”

MIgGGly (mouse IgG glycosylation analysis) - a high-throughput method for studying Fc-linked IgG N-glycosylation in mice with nanoUPLC-ESI-MS

“…Since a single peak can contain multiple glycan structures 36 , a combination of liquid chromatography (LC) based separation of released glycans and MS-based detection serves as the most informative method for glycoprofiling 37 , 38 . Recent progress in chemoenzymatic synthesis of glycans made available panels of synthesized N-glycans 39 – 42 used as standards for absolute quantifications of N-glycans in complex mixtures 39 . Chemoenzymatically engineered pure preparations of specific IgG glycoforms 40 , 41 , 43 , 44 are used to study their functional properties.…”

Chemoenzymatic synthesis of glycopeptides bearing rare N-glycan sequences with or without bisecting GlcNAc

“…The four oligosaccharides 31–34 were deprotected by first incubating with ethylenediamine in n -butanol at 90 °C, 31 which removed all ester and carbonate groups as well as the Phth to generate free amines ( Scheme 6 ). To facilitate future quantification by mass spectrometry, 55 , 56 the free amines were protected with 13 C labeled acetic anhydride to introduce 13 C labels into the GlcNAcs. Subsequent hydrogenolysis with Pearlman's catalyst under a hydrogen atmosphere 57 led to free N-glycans 35–38 .…”