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

Abstract

The brain N-glycome is known to be crucial for many biological functions, including its involvement in neuronal diseases. Although large structural studies of brain N-glycans were recently carried out, a comprehensive isomer-specific structural analysis has still not been achieved, as indicated by the recent discovery of novel structures with galactosylated bisecting GlcNAc. Here, we present a detailed, isomer-specific analysis of the human brain N-glycome based on standardized porous graphitic carbon (PGC)-LC-MS/MS. To achieve this goal, we biosynthesized glycans with substitutions typically occurring in the brain N-glycome and acquired their normalized retention times. Comparison of these values with the standardized retention times of neutral and desialylated N-glycan fractions of the human brain led to unambiguous isomer specific assignment of most major peaks. Profound differences in the glycan structures between naturally neutral and desialylated glycans were found. The neutral and sialylated N-glycans derive from diverging biosynthetic pathways and are biosynthetically finished end products, rather than just partially processed intermediates. The focus on structural glycomics defined the structure of human brain N-glycans, amongst these are HNK-1 containing glycans, a bisecting sialyl-lactose and structures with fucose and N-acetylgalactosamine on the same arm, the so-called LDNF epitope often associated with parasitic worms.

Non-targeted isomer-sensitive N-glycome analysis reveals new layers of organ-specific diversity in mice

“…Brain N-glycosylation is known to be crucial for neurodevelopmental processes and for normal adult brain functions [47][48][49][50] and aberrant glycosylation often induces neurological disfunctions and is involved in other diseases like brain cancer 51 . The importance of glycosylation and the implications of aberrant glycosylation in this organ has been described by others 31,[52][53][54][55][56] and has recently been reviewed by Klaric et al 57 . Given the wealth of glycome information on this organ, we chose the brain to illustrate the advantages of isomer resolved PGC-LC-MS/MS data set and to demonstrate comparability with other studies.…”

“…Importantly, these biosynthetically distant, yet isobaric glycan structures of identical composition would not be discriminated without chromatographic separation of the sample. This highlights that elution rules and relative elution positions for PGC chromatography, which are extensively described elsewhere 14,25,31,34,61,62 , are a valuable tool for the exact identification of isomers and a vital resource for the analysis of this dataset. In addition, the highly reproducible MS/MS data provided here can be used to confirm particular isomers or to identify unknown structures via spectral matching, as already described elswhere 61 .…”

“…7) shows that at least four isomers are present at the MS1 level. Based on relative retention time recently described 14,31 , we could identify two hybrid type glycans (one of them with an elongated bisecting GlcNAc) and a bi-antennary galactosylated structure with a core fucose. Importantly, these biosynthetically distant, yet isobaric glycan structures of identical composition would not be discriminated without chromatographic separation of the sample.…”

“…More recently, owing to its exceptional chromatographic selectivity, porous graphitic carbon (PGC) emerged as a new state of the art in glycome analysis. Coupled to MS, the separation power of PGC, which is able to separate even closely related isomers 25 , became a new gold standard in isomer-specific analysis of glycans, and is nowadays routinely applied by many groups for the analysis of both, Oglycans [26][27][28][29][30] and N-glycans 14,[30][31][32][33][34][35][36][37][38][39] . Complementing existing glycan data-repositories with critical isomer-specific analysis, we here report on and make publicly available a consistent and complete PGC-LC-MS/MS N-glycome dataset of 23 different mouse tissues (n=2), which is, to our knowledge, the currently most comprehensive mouse N-glycome data set.…”

Glycosylation—The Most Diverse Post-Translational Modification

“…Technical innovations have led to improvements in N -glycosylation analyses in recent years. The study of Helm et al [ 2 ] uses today’s facilities to investigate the N -glycome of the human brain. In a technically complex study, typical structures of the brain were first bio-synthesised and then compared with 68 real N -glycan structures, with particular focus on the determination of isomers.…”