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N-Glycan Analysis Jake S. Yang Oct 25, 2013 Center for Biomarker Discovery and Translation.

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Presentation on theme: "N-Glycan Analysis Jake S. Yang Oct 25, 2013 Center for Biomarker Discovery and Translation."— Presentation transcript:

1 N-Glycan Analysis Jake S. Yang Oct 25, 2013 Center for Biomarker Discovery and Translation

2 2  Center for Biomarker Discovery and Translation  2 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.

3 3  Center for Biomarker Discovery and Translation  3 Aberrant glycosylation associate with diseases

4 4  Center for Biomarker Discovery and Translation  4 Molecular markers are glycoproteins [D. Sidransky, Nat. Rev. Cancer 2002, 2, ]

5 5  Center for Biomarker Discovery and Translation  5 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

6 6  Center for Biomarker Discovery and Translation  6 Technology Innovation Carbohydrates and Glycobiology, Science, Vol.291, No. 5512, pp glycoproteins glycans Glycan isolation, tissue imaging Glycoprotein Immobilization for Glycan Extraction (GIG) Reversible Hydrazone Solid-Phase Extraction (rHSPE) Sialic Acid Modification Glycan profiling by GIG-chipLC Quantitative glycomics (iARTs) Nat. Biotechnol. 2003, 21, Anal. Chem. 2012, 84 (5), Proteomics Clin. Appl. 2012, 6, Anal. Chem. 2013, 85, Anal. Chem. 2013, 85, Anal. Chem. 2013, 85, Anal. Chem. 2013, 85, DOI: /ac Glycan chip imaging

7 7  Center for Biomarker Discovery and Translation  7 N-glycan workflow Sample (protein extraction from tissue or cell) Sample (protein extraction from tissue or cell) Buffer exchange (amine-free) Buffer exchange (amine-free) GIG (solid-phase) (protein immobilization) GIG (solid-phase) (protein immobilization) rHSPE (glycan reducing-end capture) rHSPE (glycan reducing-end capture) Sialic acid (modification and quantitation) Sialic acid (modification and quantitation) iARTs (isobaric quantitation) iARTs (isobaric quantitation) chipLC (microchip) chipLC (microchip) On beads (glycan capture) On beads (glycan capture) On slide (glycan imaging) On slide (glycan imaging) Detection (MALDI-MS) Detection (MALDI-MS) Detection (MALDI or ESI - MS) Detection (MALDI or ESI - MS) Solid-phase detection separation quantitation modification

8 8  Center for Biomarker Discovery and Translation  8 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 EnzymeC18/C8 Carbo modify Carbo MS S. Yang and H. Zhang, Proteomics Clin. Appl. 2012, 11-12,

9 9  Center for Biomarker Discovery and Translation  9 GIG (chemoselective method) Glycoprotein Immobilization for Glycan Extraction (GIG) 1 Immobilization on solid-phase: Immobilization in pH 10 on N-terminus and lysine 1 S. Yang et al., Anal. Chem. 2013, 85(11), P. Shah et al., Anal. Chem. 2013, 85 (7), G.J. Rademaker et al., Anal. Biochem. 1998, 257, immobilizemodify 2 enzyme  -elimination 3 MS wash MS Aldehyde beads

10 10  Center for Biomarker Discovery and Translation  10 Complex sialic acids About 50 different sialic acids known [Schauer, 2009]

11 11  Center for Biomarker Discovery and Translation  11 On GIG: glycan modification and extraction N-glycan modification on solid-phase O-glycan  -elimination [S. Yang et al., Anal. Chem. 2013, 85(11), ]

12 12  Center for Biomarker Discovery and Translation  12 On GIG: sialic acid isotope quantitation Demonstration of sialylated N-glycan isotope labeling by mixing 1:1 light to heavy (p-toluidine) 4.5 – 5.5 Sialic acid P-toluidine [P. Shah et al., Anal. Chem. 2013] amidation

13 13  Center for Biomarker Discovery and Translation  13 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: /ac ]

14 14  Center for Biomarker Discovery and Translation  14 Microchip implementation 1). Needle insertion 2). Union and capillary installation 3). AminoLink bead packing 4). Graphitized carbon packing 12 34

15 15  Center for Biomarker Discovery and Translation  15 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

16 16  Center for Biomarker Discovery and Translation  16 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

17 17  Center for Biomarker Discovery and Translation  17 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) Identification of glycans without chipLC

18 18  Center for Biomarker Discovery and Translation  18 Mouse blood serum, 400  g of serum proteinsMouse 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

19 19  Center for Biomarker Discovery and Translation  19 Mouse N-glycan profiling Mouse tissueMouse serum [S. Yang et al., manuscript under review, 2013] 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

20 20  Center for Biomarker Discovery and Translation  20 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

21 21  Center for Biomarker Discovery and Translation  21 GIG-iARTs [S. Yang et al., Anal. Chem., 2013 (accepted) GIG iARTs

22 22  Center for Biomarker Discovery and Translation  22 Improved sensitivity and quantification 15 N-glycans identified, 17 others confirmed as glycans gp120

23 23  Center for Biomarker Discovery and Translation  23 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

24 24  Center for Biomarker Discovery and Translation  24 Acknowledgements Dr. Don DeVoe Dr. Shuwei Li 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) Dr. Jennifer Van Eyk Sarah Parker Dr. Scott Kuzdzal Brian Field Dr. David Graham David Colquhoun Dr. Kevin Yarema All members of CBDT Dr. Hui Zhang Dr. Daniel Chan Dr. Lori Sokoll Dr. Zhen Zhang


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