Detection and Analysis of Proteins Modified by O‐Linked N‐Acetylglucosamine

Natasha E. Zachara1, Keith Vosseller2, Gerald W. Hart1

1 The Johns Hopkins University School of Medicine, Baltimore, Maryland, 2 Drexel University College of Medicine, Philadelphia, Pennsylvania
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 17.6
DOI:  10.1002/0471142727.mb1706s95
Online Posting Date:  July, 2011
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Abstract

O‐GlcNAc is a common post‐translational modification of nuclear, mitochondrial, and cytoplasmic proteins that is implicated in the etiology of type II diabetes and Alzheimer's disease, as well as cardioprotection. This unit covers simple and comprehensive techniques for identifying proteins modified by O‐GlcNAc, studying the enzymes that add and remove O‐GlcNAc, and mapping O‐GlcNAc modification sites. Curr. Protoc. Mol. Biol. 95:17.6.1‐17.6.33. © 2011 by John Wiley & Sons, Inc.

Keywords: signal transduction; glycosylation; O‐GlcNAc; method(s); detection; analysis; galactosyltransferase; O‐linked; glycosylation

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Increasing the Stoichiometry of O‐GlcNAc on Proteins Before Analysis
  • Basic Protocol 2: Detection of Proteins Modified by O‐GlcNAc Using Antibodies
  • Basic Protocol 3: Detection of Proteins Modified by O‐GlcNAc Using the Lectin sWGA
  • Support Protocol 1: Control for O‐Linked Glycosylation
  • Basic Protocol 4: Detection and Enrichment of Proteins Using WGA‐Agarose
  • Support Protocol 2: Digestion of Proteins with Hexosaminidase
  • Alternate Protocol 1: Detection of Proteins Modified by O‐GlcNAc Using Galactosyltransferase
  • Support Protocol 3: Autogalactosylation of Galactosyltransferase
  • Support Protocol 4: Assay of Galactosyltransferase Activity
  • Basic Protocol 5: Detection of Proteins Modified by O‐GlcNAc Using Metabolic Labeling
  • Basic Protocol 6: Characterization of Labeled Glycans by β‐Elimination and Chromatography
  • Basic Protocol 7: BEMAD
  • Basic Protocol 8: Enrichment of O‐GlcNAc‐Modified Peptides by Lectin Weak‐Affinity Chromatography (LWAC)
  • Basic Protocol 9: Assay for OGT Activity
  • Support Protocol 5: Desalting the O‐GlcNAc Transferase
  • Basic Protocol 10: Assay for O‐GlcNAcase Activity
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Tables
     
 
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Materials

Basic Protocol 1: Increasing the Stoichiometry of O‐GlcNAc on Proteins Before Analysis

  Materials
  • Cells of interest growing in monolayer culture, and appropriate culture medium
  • 1 mM Thiamet‐G (Caymen Chemicals) stock in 1 M HEPES, pH 7.5 (filter sterilize and store in aliquots up to 6 to 12 months at –80°C)
  • 100 mM PUGNAc (Toronto Research Biochemicals) stock in PBS, pH 7.5 (filter sterilize and store in aliquots up to 6 to 12 months at –80°C)
  • 500 mM glucosamine stock in 500 mM HEPES, pH 7.5 (make just prior to use; filter sterilize)
  • Ice
  • 100‐mm tissue culture dishes
  • Humidified water‐jacket CO 2 incubator
  • Additional reagents and equipment for SDS‐PAGE (unit 10.2) and electroblotting (unit 10.8)

Basic Protocol 2: Detection of Proteins Modified by O‐GlcNAc Using Antibodies

  Materials
  • Purified or crude protein (e.g., protocol 1) separated by SDS‐PAGE (unit 10.2) and electroblotted to polyvinylidene difluoride (PVDF; unit 10.8) or nitrocellulose (preferred to PVDF) (duplicate blots are needed)
  • Blocking buffer (3% milk w/v in TBST)
  • Antibody: CTD 110.6 ascites (Covance) diluted 1/2500 in antibody dilution buffer (3% BSA w/v in TBST; see recipe for TBST)
  • N‐acetylglucosamine (GlcNAc; Sigma)
  • TBST (see recipe)
  • HRPO‐conjugated anti–mouse IgM diluted 1/5000 in antibody dilution buffer (3% BSA w/v in TBST)
  • TBS (see recipe)
  • ECL kit (Amersham‐Pharmacia Biotech; GE Healthcare Life Sciences)
  • Additional reagents and equipment for visualization with chromogenic and luminescent substrates (units 10.8& 18.4)
IMPORTANT NOTE: Volumes are given for 9 × 14–cm membranes washed in 10.5 × 15.5–cm boxes. Volumes can be scaled down or up, but membranes should be covered.

Basic Protocol 3: Detection of Proteins Modified by O‐GlcNAc Using the Lectin sWGA

  Materials
  • Purified or crude protein separated by SDS‐PAGE (unit 10.2) and electroblotted to polyvinylidene difluoride (PVDF; unit 10.8) or nitrocellulose (duplicate blots are needed)
  • 5% (w/v) BSA in TBST (see recipe for TBST)
  • TBST (see recipe)
  • 0.1 µg/ml HRPO‐conjugated sWGA (EY Labs) in TBST (see recipe for TBST): the lectin can be stored at 1 mg/ml in 0.01 M PBS, pH 7.4 ( appendix 22), at –20°C for at least 1 year
  • N‐acetylglucosamine (GlcNAc; Sigma)
  • High‐salt TBST (HS‐TBST): TBST (see recipe) containing 1 M NaCl
  • Tris‐buffered saline (TBS; see recipe)
  • ECL kit (Amersham‐Pharmacia Biotech)
  • Additional reagents and equipment for visualization with chromogenic and luminescent substrates (units 10.8& 18.4)
IMPORTANT NOTE: Volumes are given for 9 × 14–cm membranes washed in 10.5 × 15.5–cm boxes. Volumes can be scaled down or up, but membranes should be covered.

Support Protocol 1: Control for O‐Linked Glycosylation

  Materials
  • Protein samples and controls blotted to PVDF (triplicate blots are needed; nitrocellulose is not suitable as it dissolves in 55 mM NaOH)
  • Tris‐buffered saline (TBS; see recipe)
  • 55 mM NaOH
  • Milli‐Q water
  • TBST (see recipe)
  • 3% (w/v) BSA in TBST (see recipe for TBST)
  • 40°C water bath
  • Additional reagents and equipment for probing protein blots with protein‐specific antibodies (see protocol 2) or lectins (see protocol 3)
IMPORTANT NOTE: Volumes are given for 9 × 14–cm membranes washed in 10.5 × 15.5–cm boxes. Volumes can be scaled down or up, but membranes should be covered.

Basic Protocol 4: Detection and Enrichment of Proteins Using WGA‐Agarose

  Materials
  • cDNA subcloned into an expression vector with an SP6 or T7 promoter (∼0.5 to 1 µg/µl)
  • RRL ITT system kit (Promega)
  • Label: [35S]Met or [35S]Cys, or [14C]Leu
  • WGA‐agarose (Vector Laboratories) WGA wash buffer: PBS ( appendix 22) containing 0.2% (v/v) NP‐40
  • WGA Gal elution buffer (see recipe)
  • WGA GlcNAc elution buffer (see recipe)
  • TCA or methanol
  • 1‐ml tuberculin syringe with glass wool plug at bottom to support chromatography matrix or Bio‐Rad Bio‐Spin disposable chromatography column
  • Liquid scintillation counter
  • Additional reagents and equipment for digesting proteins with hexosaminidase (see protocol 6), desalting (see protocol 15), SDS‐PAGE (unit 10.2), and autoradiography ( appendix 3A)

Support Protocol 2: Digestion of Proteins with Hexosaminidase

  Materials
  • Protein sample for digestion (include a positive control, e.g., ovalbumin)
  • 2% (w/v) SDS
  • 2× hexosaminidase reaction mixture (see recipe)
  • Additional reagents and equipment for SDS‐PAGE (unit 10.2) and electroblotting (unit 10.8)

Alternate Protocol 1: Detection of Proteins Modified by O‐GlcNAc Using Galactosyltransferase

  Materials
  • Protein sample(s)
  • Dithiothreitol (DTT)
  • Sodium dodecyl sulfate (SDS)
  • Label: 1.0 mCi/ml UDP‐[3H]Gal, (17.6 Ci/mM; GE Healthcare) in 70% (v/v) ethanol
  • Nitrogen source
  • 25 mM 5′‐adenosine monophosphate (5′‐AMP) in Milli‐Q water, pH 7.0
  • Buffer H (see recipe)
  • 10× galactosyltransferase labeling buffer (see recipe)
  • Galactosyltransferase, autogalactosylated (see protocol 8)
  • Calf intestinal alkaline phosphatase
  • Unlabeled UDP‐Gal
  • Stop solution: 10% (w/v) SDS/0.1 M EDTA
  • Speed‐Vac evaporator
  • 37°C incubator
  • 100°C water bath
  • 30 × 1–cm Sephadex G‐50 column equilibrated in 50 mM ammonium formate/0.1% (w/v) SDS
  • Liquid scintillation counter
  • Additional reagents and equipment for acetone precipitation of protein (unit 17.10), PNGase F digestion of proteins (unit 17.13), SDS‐PAGE (unit 10.2), and product analysis (see protocol 11)

Support Protocol 3: Autogalactosylation of Galactosyltransferase

  Materials
  • 10× galactosyltransferase labeling buffer (see recipe)
  • 10,000 U/ml aprotinin
  • 2‐mercaptoethanol
  • UDP‐Gal
  • Saturated ammonium sulfate: >17.4 g (NH 4) 2SO 4 in 25 ml Milli‐Q water
  • 85% ammonium sulfate: 14 g (NH 4) 2SO 4 in 25 ml Milli‐Q water
  • Galactosyltransferase storage buffer (see recipe)
  • 30‐ to 50‐ml centrifuge tubes
  • Refrigerated centrifuge

Support Protocol 4: Assay of Galactosyltransferase Activity

  Materials
  • 1.0 mCi/ml UDP‐[3H]Gal, (17.6 Ci/mM; GE Healthcare) in 70% (v/v) ethanol
  • Nitrogen source
  • 1× galactosyltransferase dilution buffer: galactosyltransferase storage buffer (see recipe) supplemented with 5 mg/ml BSA
  • 10× galactosyltransferase labeling buffer (see recipe)
  • 25 mM 5′‐adenosine monophosphate (5′‐AMP) in Milli‐Q water, pH 7.0
  • “Pre‐gal” sample aliquot (see protocol 8, step 3) and “auto‐gal” sample aliquot (see protocol 8, step 9)
  • 200 mM GlcNAc
  • Dowex AG1‐X8 resin (PO 4 form) slurry in 20% (v/v) ethanol
  • Speed‐Vac evaporator
  • 37°C incubator
  • Pasteur pipets
  • Glass wool
  • 15‐ml scintillation vials
  • Liquid scintillation counter

Basic Protocol 5: Detection of Proteins Modified by O‐GlcNAc Using Metabolic Labeling

  Materials
  • Cells of interest, growing in culture
  • Biosynthetic labeling medium (see recipe)
  • Phosphate‐buffered saline (PBS; appendix 22)
  • Humidified water‐jacket CO 2 incubator
  • Additional reagents and equipment for PNGase F digestion of proteins (unit 17.13), SDS‐PAGE (unit 10.2), and autoradiography ( appendix 3A)

Basic Protocol 6: Characterization of Labeled Glycans by β‐Elimination and Chromatography

  Materials
  • Labeled proteins
  • β‐elimination reagent: 1 M NaBH 4/0.1 M NaOH (prepare fresh)
  • 4 M acetic acid
  • 1.5‐ml screw‐cap microcentrifuge tubes
  • 37°C incubator
  • Additional reagents and equipment for acetone or methanol precipitation of proteins (unit 17.10), size‐exclusion (gel‐filtration) chromatography (unit 10.9), and Dionex chromatography (Townsend et al., , ; Townsend and Hardy, ).

Basic Protocol 7: BEMAD

  Materials
  • ∼1 to 100 pmol of protein sample in 40 mM ammonium bicarbonate, pH 8.0, in microcentrifuge tubes washed with acetonitrile
  • Performic acid oxidation buffer: 45% (v/v) formic acid, 5% (v/v) hydrogen peroxide, in Milli‐Q water (make fresh)
  • Control peptides (Control phospho and O‐GlcNAc modified peptides can be synthesized or are available from Invitrogen, cat. no. C33373)
  • Ice
  • 40 mM ammonium bicarbonate, pH 8.0
  • Trypsin, sequencing grade modified (Promega)
  • Trifluoroacetic acid (TFA)
  • MgCl 2
  • Alkaline phosphatase (Promega)
  • BEMAD solution: 0.1% (v/v) NaOH, 1% (v/v) triethylamine, and 10 mM DTT (make fresh)
  • Buffer A: 1% (v/v) TFA in MilliQ water
  • Thiopropyl Sepharose 6B (GE Healthcare)
  • Thiol column buffer, degassed: PBS, 1 mM EDTA (prepare fresh)
  • Thiol column elution buffer (prepare fresh), degassed: PBS, 1 mM EDTA, and 20 mM DTT
  • 1% (v/v) acetic acid
  • Buffer B: 75% (v/v) acetonitrile, 1% (v/v) TFA in MilliQ water
  • Microcentrifuge tubes rinsed in 50% (v/v) acetonitrile (do NOT use autoclaved tubes); use these tubes throughout the protocol (this reduces contamination of your sample with the plasticizer)
  • Savant Speed‐Vac Concentrator
  • C 18 reversed‐phase macrospin columns (The Nest Group)
  • 37° and 50°C incubator
  • Nanobore reversed‐phased column (Polymicro Technologies)
  • Mass spectrometer, such as a Iontrap or Orbitrap mass spectrometer with nanospray source

Basic Protocol 8: Enrichment of O‐GlcNAc‐Modified Peptides by Lectin Weak‐Affinity Chromatography (LWAC)

  Materials
  • 100 mM ammonium bicarbonate pH 8.0 (adjust pH with 1 N NaOH)
  • 500 mM DTT stock solution in 100 mM ammonium bicarbonate buffer
  • 500 mM Iodoacetamide solution in 100 mM ammonium bicarbonate buffer (prepare fresh and store in the dark)
  • Sequencing‐grade modified trypsin (Promega, cat. no. V511A)
  • Formic acid
  • 100% acetonitrile
  • Washing buffer: 0.1% (v/v) formic acid in Milli Q water
  • Elution buffer: 80% (v/v) acetonitrile, 25 mM formic acid, in Milli Q water
  • WGA buffer (see recipe)
  • WGA coupled to agarose, 10 ml (Agarose Wheat Germ Agglutinin; Vector Laboratories, cat. no. AL‐1023)
  • 70% (v/v/) ethanol
  • Reagent A: 0.1% (v/v) formic acid in Milli Q water
  • Reagent B: 70% (v/v) acetonitrile, 0.1% (v/v) formic acid in MilliQ water
  • WGA storage buffer (see recipe)
  • WGA column regeneration buffer: 1 M NaCl, pH 3 (adjust pH with acetic acid)
  • Vortex
  • Microcentrifuge
  • 60°C incubator
  • C 18 reversed‐phase macrospin columns (capacity 300 µg; The Nest Group, part no. SMM SS18V)
  • 2‐ml microcentrifuge tubes
  • Kimwipes
  • Savant Speed‐Vac Concentrator
  • 40‐ml empty glass column designed for gravity flow to wash WGA‐agarose
  • 20‐ml empty glass column for chromatography with the frit removed, this should be appropriate for use on an FPLC/HPLC
  • An opaque 12‐meter (∼39 feet) length of Teflon tubing (o.d. 1.59 mm; i.d. 1 mm; volume ∼10 ml) (Upchurch Scientific)
  • AKTA Purifier (Amersham Biosciences) HPLC system, or equivalent HPLC
  • Polyetheretherketone (PEEK) unions containing 0.5‐µm frits (Upchurch Scientific)
  • 100‐µl injection loop
  • 2× 96‐well collection plates, 2‐ml deep (e.g., MASTERBOCK, Greiner)
  • ZipTip (C 18; P10; Millipore, cat. no. ZTC18S096)
  • Mass spectrometer
  • Fit‐flow stoppers (Upchurch Scientific)

Basic Protocol 9: Assay for OGT Activity

  Materials
  • 0.1 mCi/ml UDP‐[3H]GlcNAc (20 to 45 Ci/mmol; NEN Life Science Products) in 70% (v/v)ethanol
  • 25 mM 5′‐adenosine monophosphate (5′‐AMP), in Milli‐Q water, pH 7.0
  • Nitrogen source
  • Crude or purified OGT sample, desalted (see protocol 15)
  • 10× OGT assay buffer (see recipe)
  • CKII peptide substrate (+H 2N‐PGGSTPVSSANMM‐COO): dissolve in H 2O to 10 mM and adjust to pH 7, if necessary
  • 50 mM formic acid
  • Methanol (HPLC‐grade)
  • Scintillation fluid
  • Speed‐Vac evaporator
  • Waters Sep‐Pak C 18 cartridges
  • Scintillation counter

Support Protocol 5: Desalting the O‐GlcNAc Transferase

  Materials
  • Sephadex G‐50 slurry (GE Healthcare)
  • OGT desalting buffer (see recipe)
  • Protein sample for OGT assay in volume ≤200 µl
  • Ice
  • 1‐ml tuberculin syringe
  • 1.5‐ml tubes, prechilled

Basic Protocol 10: Assay for O‐GlcNAcase Activity

  Materials
  • Partially purified O‐GlcNAcase (0.2 to 1 µg) or cell extract sample (20 to 50 µg, precipitated with 30% to 50% ammonium sulfate and desalted).
  • 10× O‐GlcNAcase assay buffer (see recipe)
  • 100 mM (50×) p‐nitrophenol N‐acetylglucosaminide (pNP‐GlcNAc) in DMSO
  • 500 mM Na 2CO 3
  • 96‐well flat‐bottom plates or 1.5‐ml microcentrifuge tubes
  • 37°C incubator
  • Plate reader or spectrophotometer
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Literature Cited

Literature Cited
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