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