Glycoproteomics Using Chemical Immobilization

Hui Zhang1

1 Johns Hopkins University, Baltimore, Maryland
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 24.3
DOI:  10.1002/0471140864.ps2403s48
Online Posting Date:  May, 2007
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Abstract

Protein glycosylation is prevalent in proteins destined for extracellular environments, e.g., transmembrane proteins, cell surface proteins, and secreted proteins in tissues and body fluids. These also are the proteins that are most easily accessible for diagnostic and therapeutic purposes. This unit describes methods for solid‐phase extraction of glycopeptides and subsequent identification of glycopeptides as well as glycosylation sites. The extraction is based on the conjugation of glycopeptides to a solid support, using hydrazide chemistry, and the specific release of formerly glycosylated peptides. The recovered peptides are then identified by tandem mass spectrometry. The methods are applied to the analysis of proteins from cells, body fluids, and tissues.

Keywords: Glycoproteomics; glycosylation; protein post‐translational modification; mass spectrometry; proteomics; extracellular proteins; secreted proteins

     
 
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Table of Contents

  • Basic Protocol 1: Solid‐Phase Extraction of Glycopeptides Using Hydrazide Resin
  • Alternate Protocol 1: Solid‐Phase Extraction of Glycoproteins Using Hydrazide Resin
  • Support Protocol 1: Extraction of Glycoproteins from Cultured Cells
  • Support Protocol 2: Collection and Concentration of Secreted Glycoproteins from Cell Culture Medium
  • Support Protocol 3: Collection and Concentration of Glycoproteins from Plasma and Other Body Fluids
  • Support Protocol 4: Extraction of Glycoproteins from Solid Tissues
  • Support Protocol 5: Determination of Efficiency of Glycoprotein Tryptic Digestion or Glycopeptide/Glycoprotein‐Hydrazide Resin Coupling Using SDS‐PAGE and Silver Staining
  • Support Protocol 6: Desalting and Purifying Peptides Using a Reverse‐Phase Column
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Solid‐Phase Extraction of Glycopeptides Using Hydrazide Resin

  Materials
  • 1 mg protein sample (Support Protocols protocol 31 to protocol 64)
  • Lysis buffer: 50 mM KHPO 4, pH 8 ( appendix 2E)/0.1% RapiGest (Waters): prepare fresh
  • Trifluoroethanol (TFE)
  • 50 mM tris (2‐carboxyethyl) phosphine (TCEP; Pierce): store up to 3 months at −20°C
  • 250 mM iodoacetamide: prepare fresh
  • 50 mM and 100 mM ammonium bicarbonate (NH 4HCO 3) buffer, pH 7.8: prepare fresh
  • 0.5 mg trypsin/ml 50 mM acetic acid: translation grade trypsin (Promega) supplied with 50 mM acetic acid
  • Coupling buffer: 100 mM sodium acetate/150 mM NaCl, pH 5.5; store up to 3 months at room temperature
  • 50 mM sodium periodate: prepare fresh and hold in the dark
  • Hydrazide resin (Bio‐Rad)
  • 80% acetonitrile/0.1% trifluoroacetic acid (TFA)
  • 1.5 M NaCl
  • Peptide‐N‐glycosidase F (PNGase F; NEB)
  • 100 mM NaOH
  • 0.3 M and 0.4% (v/v) acetic acid: store up to 3 months at room temperature
  • HPLC solvent A: 0.4% acetic acid/0.005% heptafluorobutyric acid (HFBA); store up to 3 months at room temperature
  • HPLC solvent B: acetonitrile, 0.4% acetic acid/0.005% HFBA; store up to 3 months at room temperature
  • 60°C water bath
  • Sonicator (e.g., Sonicator 3000; Misonix)
  • Test tube rocker
  • 1‐ml glass vial with polyethylene snap cap (e.g., Waters), three for each sample
  • SpeedVac concentrator
  • Four‐port union (Upchurch Scientific) constructed entirely of polyetheretherketone (PEEK; Yi et al., ) connecting to:
    • Microcapillary HPLC column (10 cm × 75 µm i.d.) packed with MAGIC C18 resin (Michrom BioResources) or equivalent
    • Peptide trap cartridge packed with MAGIC C18 resin (Michrom BioResources)
    • High‐voltage ESI emitter
    • Waste line
  • FAMOS autosampler (Dionex) or equivalent
  • HP1100 solvent delivery system (Hewlett‐Packard) or equivalent
  • LCQ ion trap mass spectrometer (Thermo Finnigan) or equivalent
  • SEQUEST software (Eng et al., )
  • Protein database appropriate to the samples of interest
  • PeptideProphet (Proteome Software, http://www.proteomesoftware.com)
  • Additional reagents and equipment for performing SDS‐PAGE ( protocol 7 and unit 10.1) and desalting and purifying peptides ( protocol 8)

Alternate Protocol 1: Solid‐Phase Extraction of Glycoproteins Using Hydrazide Resin

  • 80% acetonitrile
  • 100% methanol
  • Size‐exclusion column, two for each sample: SEC Macro spin column, P‐6 (>6 kDa) gel filtration (The Nest Group)

Support Protocol 1: Extraction of Glycoproteins from Cultured Cells

  Materials
  • Cells of interest, in culture
  • Cell culture medium containing serum and growth factors (e.g., DMEM with 10% FBS; see appendix 3C)
  • Cell culture medium without serum
  • Lysis buffer: 50 mM KHPO 4, pH 8 ( appendix 2E)/ 0.1% RapiGest (Waters); prepare fresh
  • Humidified, 5% CO 2, 37°C cell incubator
  • Sonicator (e.g., Sonicator 3000; Misonix)
  • Additional reagents and equipment for determining cell number ( appendix 3C)

Support Protocol 2: Collection and Concentration of Secreted Glycoproteins from Cell Culture Medium

  Materials
  • Cells of interest, in culture
  • Cell culture medium containing serum and growth factors (e.g., DMEM with 10% FBS; see appendix 3C)
  • Cell culture medium without serum
  • Lysis buffer: 50 mM KHPO 4, pH 8 ( appendix 2E)/ 0.1% RapiGest (Waters); prepare fresh
  • Humidified, 5% CO 2, 37°C cell incubator
  • Centrifugal filter devices (e.g., CentriprepYM10; Millipore)

Support Protocol 3: Collection and Concentration of Glycoproteins from Plasma and Other Body Fluids

  Materials
  • 100 mg frozen tissue
  • Lysis buffer
  • Razor blade
  • Sonicator (e.g., Sonicator 3000; Misonix)

Support Protocol 4: Extraction of Glycoproteins from Solid Tissues

  Materials
  • 5 µg protein/peptide samples ( protocol 1, steps 6 and 8; protocol 2, steps 7 and 9)
  • 3× sample loading buffer (Cell Signaling Technology)
  • 50% methanol/10% acetic acid solution
  • Silver stain kit (Bio‐Rad) including:
    • Silver stain sensitizing solution
    • Staining solution
    • Developing solution
    • Stopping solution
  • Gel dryer
  • Flatbed scanner
  • Additional reagents and equipment for SDS‐PAGE (unit 10.1)

Support Protocol 5: Determination of Efficiency of Glycoprotein Tryptic Digestion or Glycopeptide/Glycoprotein‐Hydrazide Resin Coupling Using SDS‐PAGE and Silver Staining

  Materials
  • 80% acetonitrile/0.1% trifluoroacetic acid (TFA)
  • 0.1% and 1% TFA
  • Peptide samples ( protocol 1, steps 7, 12, and 21; protocol 2, step 17)
  • 1‐cc reverse‐phase columns (C18, Sep‐Pak; Waters), four columns for each sample
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Figures

Videos

Literature Cited

   Bause, E. 1983. Structural requirements of N‐glycosylation of proteins. Studies with proline peptides as conformational probes. Biochem. J. 209:331‐336.
   Desiere, F., Deutsch, E.W., Nesvizhskii, A.I., Mallick, P., King, N.L., Eng, J.K., Aderem, A., Boyle, R., Brunner, E., Donohoe, S., Fausto, N., Hafen, E., Hood, L., Katze, M.G., Kennedy, K.A., Kregenow, F., Lee, H., Lin, B., Martin, D., Ranish, J.A., Rawlings, D.J., Samelson, L.E., Shiio, Y., Watts, J.D., Wollscheid, B., Wright, M.E., Yan, W., Yang, L., Yi, E.C., Zhang, H., and Aebersold, R. 2005. Integration with the human genome of peptide sequences obtained by high‐throughput mass spectrometry. Genome Biol. 6:R9
   Deutsch, E.W., Eng, J.K., Zhang, H., King, N.L., Nesvizhskii, A.I., Lin, B., Lee, H., Yi, E.C., Ossola, R., and Aebersold, R. 2005. Human Plasma Peptide Atlas. Proteomics 5:3497‐3500.
   Eng, J., McCormack, A.L., and Yates, J.R. 3rd. 1994. An approach to correlate tandem mass spectral data of peptides with amino acid sequences in a protein database. J. Am. Soc. Mass Spectrom. 5:976‐989.
   Greis, K.D., Hayes, B.K., Comer, F.I., Kirk, M., Barnes, S., Lowary, T.L., and Hart, G.W. 1996. Selective detection and site‐analysis of O‐GlcNAc‐modified glycopeptides by beta‐elimination and tandem electrospray mass spectrometry. Anal. Biochem. 234:38‐49.
   Kaji, H., Saito, H., Yamauchi, Y., Shinkawa, T., Taoka, M., Hirabayashi, J., Kasai, K., Takahashi, N., and Isobe, T. 2003. Lectin affinity capture, isotope‐coded tagging and mass spectrometry to identify N‐linked glycoproteins. Nat. Biotechnol. 21:667‐672.
   Keller, A., Nesvizhskii, A.I., Kolker, E., and Aebersold, R. 2002. Empirical statistical model to estimate the accuracy of peptide identifications made by MS/MS and database search. Anal. Chem. 74:5383‐5392.
   Li, X.J., Yi, E.C., Kemp, C.J., Zhang, H., and Aebersold, R. 2005. A software suite for the generation and comparison of peptide arrays from sets of data collected by liquid chromatography‐mass spectrometry. Mol. Cell Proteomics 4:1328‐1340.
   Pan, S., Zhang, H., Rush, J., Eng, J., Zhang, N., Patterson, D., Comb, M.J., and Aebersold, R. 2005. High throughput proteome screening for biomarker detection. Mol. Cell Proteomics 4:182‐190.
   Roth, J. 2002. Protein N‐glycosylation along the secretory pathway: Relationship to organelle topography and function, protein quality control, and cell interactions. Chem. Rev. 102:285‐303.
   Yi, E.C., Lee, H., Aebersold, R., and Goodlett, D.R. 2003. A microcapillary trap cartridge‐microcapillary high‐performance liquid chromatography electrospray ionization emitter device capable of peptide tandem mass spectrometry at the attomole level on an ion trap mass spectrometer with automated routine operation. Rapid Commun. Mass Spectrom. 17:2093‐2098.
   Zhang, H., Li, X.J., Martin, D.B., and Aebersold, R. 2003. Identification and quantification of N‐linked glycoproteins using hydrazide chemistry, stable isotope labeling and mass spectrometry. Nat. Biotechnol. 21:660‐666.
   Zhang, H., Yi, E.C., Li, X.J., Mallick, P., Kelly‐Spratt, K.S., Masselon, C.D., Camp, D.G. 2nd, Smith, R.D., Kemp, C.J., and Aebersold, R. 2005. High throughput quantitative analysis of serum proteins using glycopeptide capture and liquid chromatography mass spectrometry. Mol. Cell Proteomics 4:144‐155.
   Zhang, H., Liu, A.Y., Loriaux, P., Wollscheid, B., Zhou, Y., Watts, J., and Aebersold, R. 2006a. Mass spectrometric detection of tissue‐derived proteins in blood. Mol. Cell. Proteomics 6:64‐71.
   Zhang, H., Loriaux, P., Eng, J., Campbell, D., Keller, A., Moss, P., Bonneau, R., Zhang, N., Zhou, Y., Wollscheid, B., Cooke, K., Yi, E.C., Lee, H., Peskind, E.R., Zhang, J., Smith, R.D., and Aebersold, R. 2006b. UniPep, a database for human N‐linked glycosites: A resource for biomarker discovery. Genome Biol. 7:R73.
Key References
   Zhang et al., 2003. See above
  Describes a method to identify and quantify N‐linked glycoproteins. It also applies the method to enrich cell surface proteins and efficiently profile serum proteins by removing albumin from serum samples.
   Kaji et al., 2003. See above
  Describes mass spectrometry analysis of protein glycosylations using combined techniques of lectin affinity purification and isotope‐coded tagging.
Internet Resources
   http://www.unipep.org
  UniPep, a database for human N‐linked glycosites: a resource for biomarker discovery.
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