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 N-acetylhexosamines 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.
Highlights
The importance of protein glycosylation in the biomedical field requires methods that quantitate structures by their monosaccharide composition, and resolve and identify the many isomers expressed by mammalian cells
HPLC with amide-functionalized stationary phases has found wide applications offering the advantage of identical molar response of all N-glycan species in a sample
Hydrophilic interaction (HILIC)-HPLC can just about distinguish between core and outer arm fucosylation.[19]
Summary
Thirty-six isomers of the fundamental composition H5N4F1 that may naturally occur in mammals plus four isomers eventually cropping up in glyco-engineered plants were biosynthetized from scaffold glycans by the use of recombinant glycosyltransferases (Figure S1). Brain peak 541c perfectly coeluted with product 3 and was insensitive to galactosidase, as previously observed for bisecting LacNAc.[50] Likewise, the structure exhibited the informative preferential loss of one terminal GlcNAc in positive mode CID (Figure S11) and the matching E- and F-type ions[49] in CID of negative ions (Figure 4). Making use of the very distinct elution positions of glycans with galactose on the 3-arm versus on bisecting GlcNAc (Figure 3a), we probed brain peak 541a with a fucosidase able to act on the Lewis terminus. Substituents on the bisecting GlcNAc can only be guessed from failure of D and E/ F ions to account for a glycans total mass, whereas their effect on retention is pronounced
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