Visualization of O‐GlcNAc Glycosylation Stoichiometry and Dynamics Using Resolvable Poly(ethylene glycol) Mass Tags

Peter M. Clark1, Jessica E. Rexach1, Linda C. Hsieh‐Wilson2

1 These authors contributed equally to this work, 2 California Institute of Technology, Pasadena, California
Publication Name:  Current Protocols in Chemical Biology
Unit Number:   
DOI:  10.1002/9780470559277.ch130153
Online Posting Date:  December, 2013
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Abstract

O‐linked N‐acetylglucosamine (O‐GlcNAc) glycosylation is a dynamic protein posttranslational modification with roles in processes such as transcription, cell cycle regulation, and metabolism. Detailed mechanistic studies of O‐GlcNAc have been hindered by a lack of methods for measuring O‐GlcNAc stoichiometries and the interplay of glycosylation with other posttranslational modifications. We recently developed a method for labeling O‐GlcNAc‐modified proteins with resolvable poly(ethylene glycol) mass tags. This mass‐tagging approach enables the direct measurement of glycosylation stoichiometries and the visualization of distinct O‐GlcNAc‐modified subpopulations. Here, we describe procedures for labeling O‐GlcNAc glycoproteins in cell lysates with mass tags. Curr. Protoc. Chem. Biol. 5:281‐302 © 2013 by John Wiley & Sons, Inc.

Keywords: O‐linked N‐acetylglucosamine; glycosylation; posttranslational modifications; chemoenzymatic labeling; poly(ethylene glycol); protein subpopulations

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Labeling O‐GlcNAc Residues on Proteins with a Resolvable Mass Tag Using Oxime Chemistry
  • Alternate Protocol 1: Labeling O‐GlcNAc Residues in Cell Lysates with a Resolvable Mass Tag Using [3+2] Azide‐Alkyne Cycloaddition Chemistry
  • Support Protocol 1: Expression And Isolation of Y289L GalT
  • Support Protocol 2: Data Analysis for Calculating Protein Glycosylation Stoichiometries
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Labeling O‐GlcNAc Residues on Proteins with a Resolvable Mass Tag Using Oxime Chemistry

  Materials
  • Cell or tissue pellet
  • Lysis buffer (see recipe) or nondenaturing buffer supplemented with β‐N‐acetylglucosaminidase inhibitor, e.g., 10 µM O‐(2‐acetamindo‐2‐deoxy‐D‐glucopyranosylidene)amino N‐phenylcarbamate (PUGNAc; Toronto Research Chemicals) and phosphatase inhibitors, e.g., 1 mM NaVO 3, 29 mM, NaF, and 0.5 mM Na 2MoO 4
  • Methanol
  • Chloroform
  • Water: Milli‐Q purified, or equivalent
  • Ketone labeling resuspension buffer (see recipe)
  • 20% (v/v) Triton X‐100
  • 100 mM N‐2‐hydroxyethylpiperazine‐N′‐2‐ethanesulfonic acid (HEPES), pH 7.9
  • 100 mM MnCl 2
  • 5 mM NaCl
  • 50× complete protease inhibitor cocktail: dissolve one Complete Protease Inhibitor Tablet, EDTA‐free (Roche) to 50× concentration in 1 ml water
  • 10 mM uridine 5′‐diphospho‐2‐acetonyl‐2‐deoxy‐α‐D‐galactopyranose diammonium salt (UDP‐ketogalactose; Khidekel et al., )
  • 2 mg/ml mutant Y289L galactosyltransferase (Y289L GalT; protocol 3)
  • Mass tag resuspension buffer (see recipe)
  • 60 mM aminooxy‐functionalized poly(ethylene glycol) (aminooxy‐functionalized PEG; Rexach et al., ), checked for quality by mass spectrometry for the presence of a single collection of peaks centered around 2 kDa or 5 kDa, respectively
  • 1.8 M sodium acetate buffer, pH 3.89
  • Neutralization buffer: 1% (w/v) SDS/100 mM HEPES, pH 7.9
  • 1% (w/v) SDS
  • 4% to 12% Bis‐Tris NuPAGE gradient gel (Invitrogen)
  • Positive control lysate (e.g., adult rat brain or 293T cell lysate)
  • Sonicator (Vibra‐Cell 130 watts; Sonics)
  • Refrigerated centrifuge
  • 1.5‐ml microcentrifuge tubes
  • Test tube inverter (Fisher Scientific)
  • Table‐top test tube rocker
  • Additional reagents and equipment for determining protein concentration (Olson and Markwell, ), carrying out SDS‐PAGE (Gallagher, ), and performing immunoblottimg (Gallagher, , Ursitti et al., )

Alternate Protocol 1: Labeling O‐GlcNAc Residues in Cell Lysates with a Resolvable Mass Tag Using [3+2] Azide‐Alkyne Cycloaddition Chemistry

  Additional Materials (also see protocol 1)
  • Azide labeling resuspension buffer (see recipe)
  • 10 mM N‐2‐hydroxyethylpiperazine‐N′‐2‐ethanesulfonic acid (HEPES), pH 7.9
  • Click‐iT O‐GlcNAc Enzymatic Labeling System (Life Technologies), including
    • UDP‐GalNAz (component A)
    • Y289L GalT, labeled as Gal‐T1 (Y289L) (component B)
    • Click‐iT labeling buffer (component C)
  • Click‐iT Protein Reaction Buffer Kit (Life Technologies), including
    • Click‐iT reaction buffer (component A)
    • Click‐iT reaction component B (CuSO 4)
    • Click‐iT reaction buffer additive 1 (component C)
    • Click‐iT reaction buffer additive 2 (component D)
  • 50× complete protease inhibitor cocktail (EDTA‐free): dissolve one Complete Protease Inhibitor Tablet, EDTA‐free (Roche) to 50× concentration in 1 ml water
  • Alkynyl labeling resuspension buffer (see recipe)
  • 10 mM alkynyl‐functionalized poly(ethylene glycol) in DMSO: (alkynyl‐functionalized PEG; mPEG‐alkyne, 2‐kDa or 5‐kDa; Creative PEGWorks), checked for quality by mass spectrometry for presence of a single collection of peaks centered around 2 kDa or 5 kDa, respectively
  • Test tube rocker

Support Protocol 1: Expression And Isolation of Y289L GalT

  Materials
  • BL21(DE3) cells (Lucigen)
  • 289L GalT cDNA in a pET23a plasmid backbone (Ramakrishnan and Qasba, )
  • Luria‐Bentani (LB) agar plates containing 100 µg/ml ampicillin (e.g., see Stevenson, )
  • LB broth containing 100 µg/ml of ampicillin
  • Isopropylthio‐β‐galactoside (IPTG, Sigma‐Aldrich)
  • Phosphate‐buffered saline (PBS, Sigma‐Aldrich)
  • 25% (w/v) sucrose in PBS
  • Y289L resuspension buffer (5 M guanidine hydrochloride/0.3 M sodium sulfite): store up to 1 day at 4°C
  • 2‐nitro‐5‐(sulfothio)‐benzoate (NSTB), made fresh (Thannhauser et al., )
  • 5 M guanidine hydrochloride
  • Refolding solution (see recipe)
  • Dialysis solutions (see recipe)
  • 10‐kDa nominal molecular weight limit (NMWL) dialysis tubing (Spectrum Labs)
  • Coomassie blue stain (see recipe)
  • 5 mM MnCl 2
  • 10 mM 4‐(2‐hydroxyethyl)piperazine‐1‐ethanesulfonic acid (HEPES), pH 7.9
  • 1.5 µl of 10 mM UDP‐ketogalactose (Khidekel et al., )
  • 100 pmol/µl Click‐iT O‐GlcNAc Peptide LC/MS Standard (Life Technologies)
  • Dihydroxybenzoic acid (DHB) matrix (see recipe)
  • 37°C incubator with shaker
  • Spectrophotometer
  • Refrigerated centrifuge
  • Sonicator (Vibra‐Cell 130 watts; Sonics)
  • 10‐kDa NMWL Centricon centrifugal filter unit (Millipore)
  • Additional reagents and equipment for carrying out electroporation (Seidman et al., ), protein quantification (Olson and Markwell, ), SDS‐PAGE (Gallagher, ), and mass spectrometry (Dave et al., )
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Figures

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Literature Cited

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