N-Glycan Analysis
Jake S. Yang
Oct 25, 2013
Center for Biomarker Discovery and Translation
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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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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.
Diverse glycosylation
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Technology Innovation
Carbohydrates and Glycobiology, Science, Vol.291, No. 5512, pp. 2263-2502.
glycoproteins
glycans
Glycan isolation, tissue imaging
Glycoprotein Immobilization for Glycan Extraction (GIG)Reversible Hydrazone Solid-Phase Extraction (rHSPE)Sialic Acid ModificationGlycan profiling by GIG-chipLCQuantitative glycomics (iARTs)
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.
Glycan chip imaging
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N-glycan workflowSample
(protein extraction from tissue or cell)
Buffer exchange(amine-free)
GIG (solid-phase)(protein immobilization)
rHSPE(glycan reducing-end capture)
Sialic acid(modification and quantitation)
iARTs(isobaric quantitation)
chipLC(microchip)
On beads(glycan capture)
On slide(glycan imaging)
Detection(MALDI-MS)
Detection(MALDI or ESI - MS)
Solid-phase
detection
separation
quantitation
modification
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o Glycan extraction
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)
Current methods
Enzyme C18/C8 Carbo modify CarboMS
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
O
H
proteinH2N NH
H
protein
reductive amination
Immobilization on solid-phase: Immobilization in pH 10 on N-terminus and lysine
1S. Yang et al., Anal. Chem. 2013, 85(11), 5555-5561.2P. Shah et al., Anal. Chem. 2013, 85 (7), 3606-3613.
3G.J. Rademaker et al., Anal. Biochem. 1998, 257, 149-160.
immobilize modify2
enzyme
-elimination3
MS
wash
MS
Aldehyde beads
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Complex sialic acids
About 50 different sialic acids known [Schauer, 2009]
O
HO
HOHO
OHOH CO2H
OH
2-Keto-3-deoxynononic acid (Kdn)
O
HO
H2NHO
OHOH CO2H
OH
Neuraminic acid (Neu)
O
HO
HN
HO
OHOH CO2H
OH
N-Acetylneuraminic acid (Neu5Ac)
O
O
HO
HN
HO
OHOH CO2H
OH
N-Glycolylneuraminic acid (Neu5Gc)
O
HO
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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 quantitationDemonstration of sialylated N-glycan isotope labeling by mixing 1:1 light to heavy (p-
toluidine)
O OR
OHO
HO
HNAc
OHHO
HO
O OR
HNO
HO
HNAc
OHHO
HOH2N
EDC @pH 4.5 – 5.5Sialic acid
P-toluidine
[P. Shah et al., Anal. Chem. 2013]
amidation
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GIG integration on a microchip
A
B
C
interface
[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
1). Needle insertion 2). Union and capillary installation
3). AminoLink bead packing
4). Graphitized carbon packing
1 2
3 4
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GIG-chipLC operation
Cap needle C Inject proteins from needle B Conjugate proteins to AminoLink beads Release glycans and wash column Cap needle B, go to 2)
1) Protein capture and glycan release
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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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
GIG-chipLC: mouse glycan analysis
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o Abundant oligomannoses are observed on mouse tissueo Sialylated N-glycans are observed in mouse blood serumo Less number of N-glycans are expected without LC
separation ( # of N-glycans < 50)
Identification of glycans without chipLC
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Mouse blood serum, 400 g of serum proteins Mouse blood serum, 200 g of serum proteins
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
GIG-chipLC reproducibility
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Mouse N-glycan profiling
657 31Mouse tissue Mouse serum
[S. Yang et al., manuscript under review, 2013]
o Detected unique unsialylated N-glycans in tissue onlyo Observed mature and sialylated structures from tissue and serumo Demonstrated GIG-chipLC as a simple and robust platform for glycomic
analysis
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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
Glycan quantification
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GIG-iARTs
[S. Yang et al., Anal. Chem., 2013 (accepted)
GIG iARTs
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Improved sensitivity and quantification15 N-glycans identified, 17 others confirmed as glycans
gp120
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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.
Summary
GIG: a robust technique for glycomic analysis
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Acknowledgements
Dr. Don DeVoe
Dr. Shuwei Li
FundingNational Institute of HealthNational Heart, Lung and Blood Institute (NHLBI)Programs of Excellence in Glycoscience (PEG)With Prof. HartNational Cancer InstituteThe Early Detection Research Network (EDRN)Clinical Proteomic Tumor analysis Consortium (CPTAC)
Dr. Jennifer Van EykSarah Parker
Dr. Scott Kuzdzal
Brian Field
Dr. David GrahamDavid Colquhoun
Dr. Kevin Yarema
All members of CBDT
Dr. Hui Zhang
Dr. Daniel Chan
Dr. Lori Sokoll
Dr. Zhen Zhang