N-Glycan Analysis
Jake S. Yang
Center for Biomarker Discovery and Translation
Oct 25, 2013
Glycosylation play crucial roles
o
Glycosylation is the most
abundant posttranslational
modification (PTM) and
glycans are most structurally
diverse;
o
More than 50% of all proteins
have been modified by
glycans;
o
Glycoforms are depending
upon many factors which are
related to both gene
expression and cellular
metabolism.
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Aberrant glycosylation associate with diseases
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Molecular markers are glycoproteins
[D. Sidransky, Nat. Rev. Cancer 2002, 2, 210-219]
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Diverse glycosylation
o Individual glycosylation sites on the same protein contain
different glycan structures
Reflect cell type and status
Same protein have different glycan structures in different organs (e.g.,
membrane protein Thy-1 in brain vs. lymphocytes, Rudd and Dwek,
1997)
o Changes in peptide sequence or structure could alter the
types of glycan structures attached
o The robust and high-throughput techniques are needed to
understand the roles of glycans in biological activities.
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Technology Innovation
Glycoprotein Immobilization for Glycan Extraction (GIG)
glycans
Reversible Hydrazone Solid-Phase Extraction (rHSPE)
Sialic Acid Modification
Glycan profiling by GIG-chipLC
Quantitative glycomics (iARTs)
glycoproteins
Carbohydrates and Glycobiology, Science, Vol.291, No. 5512, pp. 2263-2502.
Glycan isolation, tissue imaging
Glycan chip imaging
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Nat. Biotechnol. 2003, 21, 660-666.
Anal. Chem. 2012, 84 (5), 2232-2238.
Proteomics Clin. Appl. 2012, 6, 596-608.
Anal. Chem. 2013, 85, 5555-5561.
Anal. Chem. 2013, 85, 3606-3613.
Anal. Chem. 2013, 85, 8188-8195.
Anal. Chem. 2013, 85, DOI: 10.1021/ac4013013.
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N-glycan workflow
Sample
(protein extraction from tissue or cell)
Buffer exchange
(amine-free)
GIG (solid-phase)
(protein immobilization)
rHSPE
(glycan reducing-end capture)
modification
Sialic acid
(modification and quantitation)
quantitation
iARTs
(isobaric quantitation)
separation
chipLC
(microchip)
Solid-phase
On beads
(glycan capture)
On slide
(glycan imaging)
Detection
(MALDI-MS)
detection
Detection
(MALDI or ESI - MS)
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Current methods
o Glycan extraction
Enzyme
C18/C8
Carbo
modify
Carbo
MS
o Potential issues
Non-specific binding
Sample loss (affinity; multiple purification)
Difficulty to removal of reagents after derivatization (sialic acid
modification: reagents severely interfere glycan ionization)
S. Yang and H. Zhang, Proteomics Clin. Appl. 2012, 11-12, 596-608
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GIG (chemoselective method)
Glycoprotein Immobilization for Glycan Extraction (GIG)1
Aldehyde
beads
MS
immobilize
modify2
enzyme
wash
-elimination3
MS
Immobilization on solid-phase: Immobilization in pH 10 on N-terminus and lysine
1S.
Yang et al., Anal. Chem. 2013, 85(11), 5555-5561.
Shah et al., Anal. Chem. 2013, 85 (7), 3606-3613.
3G.J. Rademaker et al., Anal. Biochem. 1998, 257, 149-160.
2P.
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Complex sialic acids
About 50 different sialic acids known [Schauer, 2009]
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On GIG: glycan modification and extraction
N-glycan modification on solid-phase
O-glycan -elimination
[S. Yang et al., Anal. Chem. 2013, 85(11), 5555-5561.]
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On GIG: sialic acid isotope quantitation
Demonstration of sialylated N-glycan isotope labeling by mixing 1:1 light to heavy (p-toluidine)
P-toluidine
amidation
Sialic acid
EDC @pH 4.5 – 5.5
[P. Shah et al., Anal. Chem. 2013]
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GIG integration on a microchip
A
interface
C
B
[S. Yang, S. Toghi Eshighi, H. Chiu, D.L. DeVoe, and H. Zhang, Anal. Chem. 2013, DOI: 10.1021/ac4013013]
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Microchip implementation
2). Union and capillary
installation
1). Needle insertion
1
2
3
4
3). AminoLink bead
packing
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4). Graphitized carbon
packing
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GIG-chipLC operation
1) Protein capture and
glycan release
Cap needle C
Inject proteins from needle B
Conjugate proteins to AminoLink beads
Release glycans and wash column
Cap needle B, go to 2)
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2) Glycan
separation
Cap needle of B and up-cap C
Wash column through needle A
Elute glycans to needle C
Analyze elution by MS
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GIG-chipLC: mouse glycan analysis
o Experimental procedure
Isolation of glycans using GIG
Modification of sialic acids on beads
Separation of N-glycans using porous graphitized carbon
Profiling of N-glycans by Shimadzu Resonance MALDI-MS
o Analyze glycans of mouse heart tissue and blood
serum
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Identification of glycans without chipLC
o Abundant oligomannoses are observed on mouse tissue
o Sialylated N-glycans are observed in mouse blood serum
o Less number of N-glycans are expected without LC
separation ( # of N-glycans < 50)
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GIG-chipLC reproducibility
o
The majority of N-glycans are eluted in respective same fraction.
o
Isomers of N-glycans are observed by porous graphitized carbon.
o
Has advantages using microfluidics
High-throughput, low sample and reagent consumption, fast analysis, and
flexible interfacing
Mouse blood serum, 400 g of serum proteins
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Mouse blood serum, 200 g of serum proteins
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Mouse N-glycan profiling
Mouse tissue
7
65
31
Mouse serum
o
Detected unique unsialylated N-glycans in tissue only
o
Observed mature and sialylated structures from tissue and serum
o
Demonstrated GIG-chipLC as a simple and robust platform for glycomic
analysis
[S. Yang et al., manuscript under review, 2013]
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Glycan quantification
o Glycan quantification is essential for determination for both
fundamental studies of biological activities and biomarker
identification [J. Zaia, Chem. Biol. Rev. 2008]
o A current challenge in the field of glycomics is to determine
how to quantify changes in glycan expression between
different cells, tissues, or biological fluids [J.A. Atwood III, R.
Orlando et. al, J. Proteome Res. 2007]
o MS-based quantification methods include isotope and isobaric
labeling
Isotope: pair-wise measurement, increasing MS complexity
Isobaric: concurrent measurement, improving throughput and sensitivity
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GIG-iARTs
GIG
iARTs
[S. Yang et al., Anal. Chem., 2013 (accepted)
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Improved sensitivity and quantification
15 N-glycans identified, 17 others confirmed as glycans
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gp120
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Summary
o A novel method (GIG) is developed for solid-phase glycan
isolation and modification.
o GIG improves specificity and facilitates glycan modification
with minimizing sample loss using covalent immobilization.
o Glycan structure can be enzymatically analyzed on GIG.
o GIG-chipLC is the high-throughput platform for glycomic
analysis from complex biological samples.
o Isobaric labeling could quantify glycans for clinical application.
GIG: a robust technique for glycomic analysis
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Acknowledgements
Dr. Don DeVoe
All members of CBDT
Dr. Shuwei Li
Dr. Hui Zhang
Dr. Daniel Chan
Dr. Lori Sokoll
Dr. Scott Kuzdzal
Dr. Zhen Zhang
Brian Field
Dr. Jennifer Van Eyk
Sarah Parker
Dr. David Graham
David Colquhoun
Dr. Kevin Yarema
Funding
National Institute of Health
National Heart, Lung and Blood Institute (NHLBI)
Programs of Excellence in Glycoscience (PEG)
With Prof. Hart
National Cancer Institute
The Early Detection Research Network (EDRN)
Clinical Proteomic Tumor analysis Consortium (CPTAC)
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