Page 95 - ebook

Digging into N-glycosylation

in Human Brain tissue and Cerebrospinal Fluid

1,2
1,2
1,2
1,2
Sol Kim , Dea Sik Cho , Heejin Jeong , Myung Jin Oh , and Hyun Joo An 1,2*

1 Asia-Pacific Glycomics Reference Site, Chungnam National University, Daejeon, Korea

2 Graduate School of Analytical Science and Technology, Chungnam National University, Daejeon, Korea

Overview Result

Glycosylation is highly implicated with brain development and functions. Changes

Profiling of N-glycans and distribution

in glycosylation have recently been reported in Alzheimer's disease and

intractable epilepsy. However, the study of human brain glycome are relatively Tissue ECCs

poorly understood compared to other mammalian due to the limited availability

of samples and the lack of analytical tools.

Background

CSF circulation

in CNS Discovey of

Time Intensive Glycan biomarker

Not easily obtained

Localized sampling of tissue CSF ECCs

Very invasive and risky procedure

Brain Biopsy

Spinal Cord

Quick
Cerebrospinal

fluid (CSF)
Easily obtained

Comprehensive tissue profile

Liquid Biopsy
Less invasive and risky procedure

Glycoprotein

*ECC : Extracted Compound Chromatogram ■ High Mannose ■ Complex type ■ Complex/Hybride type ■ Hybride type

Cerebrospinal fluid (CSF), a body fluid that interact directly with the extracellular

fluid of the central nervous system (CNS), serves as a source for the neurological Diversity of N-glycans between tissue and CSF

study. However, a systematic analysis for correlation of glycosylation in the CSF

Top 10 N-glycan list in tissue and CSF Glycan features in tissue and CSF
and brain tissue has not yet been performed. Here, we explored the association

Tissue CSF
of their glycosylation using LC/MS-based glycomics.

Rank Structure NAPI CV Structure NAPI CV
%
%
%
%
Materials & Method
1 12.63 6.07 10.57 3.01

Workflow 2 11.11 9.40 9.40 2.12

Tissue sonication Membrane extraction N-glycan release Amount of those glycans ≥ NAPI 40 %

3 6.50 3.64 7.90 3.66
Differences of glycan in tissue and CSF

4 4.82 13.79 4.96 0.93 Tissue CSF

7_6_4_2 5_4_0_1
X3
4 2.5

5 3.91 9.93 4.61 10.79 3 2.0
NAPI (%) 2 NAPI (%) 1.5

1.0
1
0.5
Brain tissue P-value : 1.84 X 10 -3 0 P-value : 1.32 X 10 -11 0.0
6 3.77 1.90 3.97 7.36 Tissue CSF Tissue CSF
CSF 5_4_0_2
X2 6_6_3_1
Biomarker development LC/MS & MS/MS analysis Solid Phase Extraction (PGC) 3 4
3
2
7 3.27 0.44 2.89 8.67 NAPI (%) NAPI (%) 2

1
1

P-value : 2.62 X 10 -4 0 P-value : 2.37 X 10 -2 0 Tissue CSF
8 3.13 0.93 2.88 16.51 Tissue CSF

5_6_2_1 5_4_1_2

4.5 1.0
4.0 0.8
9 2.95 10.86 2.58 0.55 3.5 0.6
NAPI (%) 3.0 NAPI (%) 0.4

2.5 0.2
10 2.86 8.59 2.51 0.50 P-value : 1.14 X 10 -5 2.0 P-value : 2.67 X 10 -2 0.0
Tissue CSF Tissue CSF

We profiled the N-glycans obtained from human brain and CSF. Membranes

A total of 87 and 68 glycans were identified in human brain and CSF, respectively.
containing various glycoproteins were selectively extracted from brain tissues

Neutral-glycans were dominantly expressed over 70% in both in brain and CSF.
using ultracentrifugation. Protein-bound N-glycans from brain tissue and CSF

Fucosylated-glycan(Hex HexNAc Fuc ) and high-mannose glycan(Man5), known
were subsequently enzymatically released by treating with PNGase F and 3 5 1

as the most abundant N-glycan in brain tissue, were identified as major
enriched by PGC solid-phase extraction. Chromatographic separation and

components in CSF. Interestingly, bi-antennary complex type glycans terminal
structure-specific profiling of brain N-glycans was accomplished by MS analysis

sialic acid epitope were overexpressed in CSF than in tissues.
performed on Q-TOF mass spectrometer coupled to an LC system.

Acknowledgement Summary

• This work was supported by the National Research Foundation of Korea Grant We performed N-glycan profiling to study the diversity of N-glycosylation

funded by the Korean Government (MSIP)(2020, R&D Equipment Engineer between human brain tissue and CSF. The associations were confirmed in the

Education Program, 2014R1A6A9064166). major N-glycans of each sample, and N-glycans with difference were specifically

• We thank Prof. Jeong Ho Lee at KAIST for tremendous help. identified. We will conduct further studies digging into the glycosylation diversity

• We thank LABMS members (Laboratory for Advanced Bio-analytical Mass between CSF and brain tissue to explore the use of CSF as a tissue alternative in

Spectrometry) for their tremendous help. brain disease diagnosis and biomarker discovery.

Homepage : www.agrs.kr / E-mail: sugar@cnu.ac.kr