Bisecting Lewis X in Hybrid-Type N-Glycans of Human Brain Revealed by Deep Structural Glycomics

Abstract

The importance of protein glycosylation in the biomedical field requires methods that not only quantitate structures by their monosaccharide composition, but also resolve and identify the many isomers expressed by mammalian cells. The art of unambiguous identification of isomeric structures in complex mixtures, however, did not yet catch up with the fast pace of advance of high-throughput glycomics. Here, we present a strategy for deducing structures with the help of a deci-minute accurate retention time library for porous graphitic carbon chromatography with mass spectrometric detection. We implemented the concept for the fundamental N-glycan type consisting of five hexoses, four Nacetylhexosamines and one fucose residue. Nearly all of the 40 biosynthetized isomers occupied unique elution positions. This result demonstrates the unique isomer selectivity of porous graphitic carbon. With the help of a rather tightly spaced grid of isotope-labeled internal N-glycan, standard retention times were transposed to a standard chromatogram. Application of this approach to animal and human brain N-glycans immediately identified the majority of structures as being of the bisected type. Most notably, it exposed hybrid-type glycans with galactosylated and even Lewis X containing bisected N-acetylglucosamine, which have not yet been discovered in a natural source. Thus, the time grid approach implemented herein facilitated discovery of the still missing pieces of the N-glycome in our most noble organ and suggests itselfin conjunction with collision induced dissociationas a starting point for the overdue development of isomer-specific deep structural glycomics.

Towards Mapping of the Human Brain N-Glycome with Standardized Graphitic Carbon Chromatography

“…Guided by reports that brain N-glycans are highly fucosylated and bisected and contain galactose primarily in β1,4-linkage but also, to a smaller extent, in β1,3-linkage [ 9 , 11 ], we bio-synthesized a range of N-glycans containing these structural features in addition to the almost complete set of permutations of glycans with five hexose, four N-acetylhexosamine and one fucose residue (H5N4F1) generated for a recent work [ 28 ]. Some studies also reported α1,2-linked fucose to galactose [ 36 , 37 ] as being present in the brain glycome, so we also biosynthesized structures containing this feature.…”

“…N-glycans from human brain were obtained from SDS extracts as recently described [ 28 ]. Neutral and sialylated N-glycans were separated with a PGC cartridge [ 29 ].…”

“…The purified samples were loaded on a PGC column (100 mm × 0.32 mm, 5 µm particle size, Thermo Scientific, Waltham, MA, USA) with 10 mM ammonium bicarbonate as the aqueous solvent A and 80% acetonitrile in solvent A as solvent B, as described in [ 28 ]. In brief, 5.5 min after sample application at 1% B, a gradient from 8 to 22% solvent B was developed over 52.5 min followed by an increase up to 68% B at a flow rate of 6 µL/min.…”

“…Other studies regarding the brain N-glycome likewise devoted their attention to aspects other than exact structures [ 7 , 27 ]. The recent discovery of novel structures with galactosylated bisecting GlcNAc in the man brain [ 28 ] indicated the need for a more detailed structural analysis.…”

“…Here, we present a comprehensive, isomer-specific analysis of the human brain N-glycome based on standardized porous graphitic carbon (PGC)-LC-MS/MS. To achieve this goal, we extended the range of synthetic reference structures with N-glycans substituted with typically occurring features in the brain N-glycome and acquired their normalized retention times as described in our previous publication [ 28 ]. The neutral and the charged structures were separated by PGC-solid phase extraction and pools were analyzed separately.…”