Quantitative Glycomics of Cultured Cells Using Isotopic Detection of Aminosugars with Glutamine (IDAWG)

Meng Fang1, Jae‐Min Lim1, Lance Wells1

1 Complex Carbohydrate Research Center, University of Georgia, Athens, Georgia
Publication Name:  Current Protocols in Chemical Biology
Unit Number:   
DOI:  10.1002/9780470559277.ch090207
Online Posting Date:  April, 2010
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Abstract

IDAWG (Isotopic Detection of Aminosugars With Glutamine) is a newly reported, in vivo, stable isotopic labeling strategy for quantitative glycomics of cultured cells. Detailed procedures are provided for glycan analysis using IDAWG including labeling, release of both N‐ and O‐linked glycans, permethylation, and mass spectrometry analysis. The methods for data processing and calculations are also introduced here but have not yet been automated. Curr. Protoc. Chem Biol. 2:55‐69. © 2010 by John Wiley & Sons, Inc.

Keywords: IDAWG; stable isotopic labeling; quantitative glycomics; cell culture; glycan analysis

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Cell Culture for IDAWG Labeling
  • Basic Protocol 2: N‐ and O‐linked Glycan Analysis with IDAWG
  • Support Protocol 1: Calculating Relative Ratios of Glycans
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Cell Culture for IDAWG Labeling

  Materials
  • Cells
  • Gln‐free medium appropriate for cells (Invitrogen)
  • Amide‐15N‐Gln (98%) (Cambridge Isotope Laboratories, Inc.)
  • Normal‐abundance 14N‐Gln (L‐glutamine; Invitrogen, cat. no. 21051024)
  • Phosphate‐buffered saline (PBS; see recipe)
  • Appropriate tissue culture equipment
  • Cell scrapers

Basic Protocol 2: N‐ and O‐linked Glycan Analysis with IDAWG

  Materials
  • Cell pellets from cell culture (see protocol 1)
  • Phosphate‐buffered saline (PBS; see recipe)
  • Methanol, HPLC grade, ice cold
  • Chloroform, HPLC grade
  • 18.2 MΩ (Milli‐Q) water
  • Acetone, HPLC grade, ice cold
  • Source of dry N 2
  • 40 mM NH 4HCO 3, pH 8
  • 2 mg/ml trypsin (Sigma‐Aldrich, cat. no. T8003) in 40 mM NH 4HCO 3, pH 8 (store at −20°C)
  • 2 mg/ml chymotrypsin (Sigma‐Aldrich, cat. no. C4129) in 40 mM NH 4HCO 3, pH 8 (store at −20°C)
  • 2 M urea in 40 mM NH 4HCO 3 (prepare fresh)
  • Acetonitrile, HPLC grade
  • 5% and 10% (v/v) acetic acid (HPLC grade)
  • 20% (v/v) isopropanol (HPLC grade) in 5% acetic acid
  • 40% (v/v) isopropanol (HPLC grade) in 5% acetic acid
  • Isopropanol, HPLC grade
  • 100 mM sodium phosphate, pH 7.5
  • 7.5 µg/ml Peptide‐N‐glycosidase F (PNGase F), store at 4°C (ProZyme, http://www.prozyme.com/)
  • 1 M sodium borohydride (prepare fresh)
  • AG 50W–X8 resin stock (see recipe)
  • 1 M HCl
  • 9:1 methanol:glacial acetic acid (HPLC grade)
  • 50% w/w sodium hydroxide solution
  • Anhydrous methanol (99.8%, Sigma)
  • Anhydrous dimethylsulfoxide (DMSO; 99.9%, Sigma)
  • Iodomethane (99.5%, Aldrich)
  • Dichloromethane, HPLC grade
  • 1 mM NaOH in 50% methanol
  • 10‐ml Dounce homogenizer
  • 8‐ml screw‐top glass tubes, precleaned with methanol
  • End‐over‐end rotator
  • VWR Clinical 50 centrifuge or equivalent
  • Pierce Reacti‐Vap Evaporating Unit and Reacti‐Therm Heating/Stirring Module
  • Heating block (e.g., Fisher Isotemp 125D)
  • Bakerbond SPE Octadecyl (C 18) disposable extraction columns (J.T. Baker)
  • Speed‐Vac evaporator
  • Bath sonicator (Branson Ultrasonic Cleaner; Model 1510R‐MT)
  • 500‐µl microsyringe
  • Fused‐silica emitter (360 × 75 × 30 µm, SilicaTip; New Objective, http://www.newobjective.com/)
  • LTQ‐Orbitrap XL mass spectrometer with nano ESI source (ThermoFisher) or equivalent
NOTE: In this protocol, we introduce the method of mixing samples by weighing protein powder (in step 15). Alternatively, cells labeled light and heavy can be combined before step 1, based on accurate equal cell numbers.
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Figures

Videos

Literature Cited

   Alvarez‐Manilla, G., Warren, N.L., Abney, T., Atwood, J. 3rd, Azadi, P., York, W.S., Pierce, M., and Orlando, R. 2007. Tools for glycomics: Relative quantitation of glycans by isotopic permethylation using 13CH3I. Glycobiology 17:677‐687.
   Aoki‐Kinoshita, K.F. 2008. An introduction to bioinformatics for glycomics research. PLoS Comput. Biol. 4:e1000075.
   Aoki, K., Perlman, M., Lim, J.M., Cantu, R., Wells, L., and Tiemeyer, M. 2007. Dynamic developmental elaboration of N‐linked glycan complexity in the Drosophila melanogaster embryo. J. Biol. Chem. 282:9127‐9142.
   Atwood, J.A. 3rd, Cheng, L., Alvarez‐Manilla, G., Warren, N.L., York, W.S., and Orlando, R. 2008. Quantitation by isobaric labeling: Applications to glycomics. J. Proteome Res. 7:367‐374.
   Ceroni, A., Maass, K., Geyer, H., Geyer, R., Dell, A., and Haslam, S.M. 2008. GlycoWorkbench: A tool for the computer‐assisted annotation of mass spectra of glycans. J. Proteome Res. 7:1650‐1659.
   Dube, D.H. and Bertozzi, C.R. 2005. Glycans in cancer and inflammation: Potential for therapeutics and diagnostics. Nat. Rev. Drug Discov. 4:477‐488.
   Gygi, S.P., Rist, B., Gerber, S.A., Turecek, F., Gelb, M.H., and Aebersold, R. 1999. Quantitative analysis of complex protein mixtures using isotope‐coded affinity tags. Nat. Biotechnol. 17:994‐999.
   Kang, P., Mechref, Y., Kyselova, Z., Goetz, J.A., and Novotny, M.V. 2007. Comparative glycomic mapping through quantitative permethylation and stable‐isotope labeling. Anal. Chem. 79:6064‐6073.
   Liu, H., Sadygov, R.G., and Yates, J.R. 3rd. 2004. A model for random sampling and estimation of relative protein abundance in shotgun proteomics. Anal. Chem. 76:4193‐4201.
   Lowe, J.B. and Marth, J.D. 2003. A genetic approach to mammalian glycan function. Annu. Rev. Biochem. 72:643‐691.
   Morelle, W., Faid, V., Chirat, F., and Michalski, J.C. 2009. Analysis of N‐ and O‐linked glycans from glycoproteins using MALDI‐TOF mass spectrometry. Methods Mol. Biol. 534:5‐21.
   Ong, S.E., Blagoev, B., Kratchmarova, I., Kristensen, D.B., Steen, H., Pandey, A., and Mann, M. 2002. Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics. Mol. Cell. Proteomics 1:376‐386.
   Orlando, R., Lim, J.M., Atwood, J.A. 3rd, Aangel, P.M., Fang, M., Aoki, K., Alvarez‐Manilla, G., Moremen, K.W., York, W.S., Tiemeyer, M., Pierce, M., Dalton, S., and Wells, L. 2009. IDAWG: Metabolic incorporation of stable isotope labels for quantitative glycomics of cultured cells. J. Proteome Res. 8:3816‐3823.
   Radulovic, D., Jelveh, S., Ryu, S., Hamilton, T.G., Foss, E., Mao, Y., and Emili, A. 2004. Informatics platform for global proteomic profiling and biomarker discovery using liquid chromatography‐tandem mass spectrometry. Mol. Cell. Proteomics 3:984‐997.
   Silva, J.C., Denny, R., Dorschel, C.A., Gorenstein, M., Kass, I.J., Li, G.Z., McKenna, T., Nold, M.J., Richardson, K., Young, P., and Geromanos, S. 2005. Quantitative proteomic analysis by accurate mass retention time pairs. Anal. Chem. 77:2187‐2200.
   Wang, W., Zhou, H., Lin, H., Roy, S., Shaler, T.A., Hill, L.R., Norton, S., Kumar, P., Anderle, M., and Becker, C.H. 2003. Quantification of proteins and metabolites by mass spectrometry without isotopic labeling or spiked standards. Anal. Chem. 75:4818‐4826.
   Yue, T., Goldstein, I.J., Hollingsworth, M.A., Kaul, K., Brand, R.E., and Haab, B.B. 2009. The prevalence and nature of glycan alterations on specific proteins in pancreatic cancer patients revealed using antibody‐lectin sandwich arrays. Mol. Cell. Proteomics 8:1697‐1707.
Key Reference
   Orlando et al., 2009. See above.
  Initial description of the IDAWG labeling strategy and showed proof‐of‐principle utility using murine embryonic stem cells.
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