Analysis of Protein O‐GlcNAcylation by Mass Spectrometry

Junfeng Ma1, Gerald W. Hart1

1 Department of Biological Chemistry, The Johns Hopkins University, School of Medicine, Baltimore, Maryland
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 24.10
DOI:  10.1002/cpps.24
Online Posting Date:  February, 2017
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

O‐linked β‐DN‐acetyl glucosamine (O‐GlcNAc) addition (O‐GlcNAcylation), a post‐translational modification of serine/threonine residues of proteins, is involved in diverse cellular metabolic and signaling pathways. Aberrant O‐GlcNAcylation underlies the initiation and progression of multiple chronic diseases including diabetes, cancer, and neurodegenerative diseases. Numerous methods have been developed for the analysis of protein O‐GlcNAcylation, but instead of discussing the classical biochemical techniques, this unit covers O‐GlcNAc characterization by combining several enrichment methods and mass spectrometry detection techniques [including collision‐induced dissociation (CID), higher energy collision dissociation (HCD), and electron transfer dissociation (ETD) mass spectrometry]. © 2017 by John Wiley & Sons, Inc.

Keywords: BEMAD; CID; enrichment; ETD; HCD; mass spectrometry; O‐GlcNAc; O‐GlcNAcome; O‐GlcNAcomics; site mapping

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Introduction
  • Basic Protocol 1: Detection of O‐GlcNAcylation by Using CID/HCD Mass Spectrometry
  • Support Protocol 1: High pH Reversed‐Phase HPLC (RPLC)
  • Basic Protocol 2: O‐GlcNAc Site Mapping by Using ETD Mass Spectrometry
  • Basic Protocol 3: O‐GlcNAc Site Mapping by Using β‐Elimination Michael Addition with Dithiothreitol (BEMAD) Followed by CID/HCD Mass Spectrometry
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Detection of O‐GlcNAcylation by Using CID/HCD Mass Spectrometry

  Materials
  • Cells of interest (in culture) and appropriate culture medium
  • Phosphate‐buffered saline (Sigma, cat. no. 3813‐10PAK; dissolve one package into 1 liter of deionized water)
  • Ice
  • Cell lysis buffer (e.g., 50 mM Tris·Cl, pH 7.5, 150 mM NaCl, 1% Triton X‐100, 1 mM NaF, 1 mM beta‐glycerophosphate, 2 μM PUGNAc, and 1× Protease Inhibitor Cocktail), ice cold
  • BCA assay kit or Bradford assay kit and additional reagents and equipment for measuring protein concentration
  • Acetone, prechilled to −20°C
  • Urea
  • 100 mM ammonium bicarbonate (NH 4HCO 3)
  • 0.5 M dithiothreitol (DTT)
  • 0.5 M iodoacetamide (freshly prepared)
  • Trypsin, proteomic grade (Promega)
  • LC‐MS grade Trifluoroacetic acid (TFA)
  • LC‐MS grade formic acid (FA)
  • Magic C18 AQ, 5 µm, 100 Å (Michrom Bioresources)
  • LC‐MS grade acetonitrile (Burdick and Jackson, cat. no. LC‐015)
  • Solvent A (0.1% formic acid in water; Burdick and Jackson, cat. no. LC‐452)
  • Solvent B (0.1% formic acid in acetonitrile; Burdick and Jackson, cat. no. LC‐441)
  • Centrifuge
  • Pipets
  • Probe‐tip sonicator (e.g., Branson)
  • 1.5‐ml microcentrifuge tubes
  • C18 spin column (The Nest Group) or C18 Sep‐Pak column (Waters)
  • Vacuum centrifuge or SpeedVac
  • 0.45‐μm filters
  • 75‐µm × 15‐cm C18 analytical column with a 15‐µm emitter
  • LTQ‐Orbitrap Velos (or other mass spectrometers) coupled with nano HPLC system
  • Proteome Discoverer 1.4 or other software

Support Protocol 1: High pH Reversed‐Phase HPLC (RPLC)

  Materials
  • Protein digests (see protocol 1, step 15)
  • Solvent A: add 10 ml TEABC stock (Sigma, cat. no. T‐7408) to 1 liter water
  • Solvent B: add 10 ml TEABC stock (Sigma, cat. no. T‐7408) to 1 liter of 90% ACN
  • Triethylammonium bicarbonate (TEABC stock) buffer 1.0 M, pH 8.4‐8.6 (Sigma, cat. no. T‐7408)
  • 1% formic acid
  • Agilent 1100 or other HPLC systems
  • XBridge C18, 5 μm 4.6 × 20 mm guard column (Waters)
  • XBridge C18, 5 μm 4.6 × 250 mm analytical column (Waters)
  • Vacuum centrifuge

Basic Protocol 2: O‐GlcNAc Site Mapping by Using ETD Mass Spectrometry

  Materials
  • Fractionated peptides (see protocol 2Support Protocol)
  • 10 mM HEPES
  • Click‐iTO‐GlcNAc Enzymatic Labeling System (Life Technologies) containing:
    • MnCl 2
    • UDP‐GalNAz
    • GalT1
  • Peptide:N‐glycosidase F (PNGase F; New England Biolabs)
  • Calf intestinal phosphatase (CIP; New England Biolabs)
  • PC Biotin‐alkyne (Jena Biosciences; protect from light)
  • Sodium ascorbate (Sigma)
  • TBTA {Tris‐[(1‐benzyl‐1H‐1,2,3‐triazol‐4‐yl) methyl]amine, Anaspec}
  • tert‐Butanol (Sigma)
  • Dimethyl sulfoxide (DMSO)
  • 20 mM CuSO 4
  • 100% MeOH
  • 200 mM NaH 2PO 4/300 mM sodium acetate (pH 3.0)
  • 5 mM KH 2PO 4,/25% ACN (pH 3.0)
  • Ammonium hydroxide (NH 4OH)
  • High Capacity Neutravidin Agarose (Thermo Fisher Scientific)
  • Phosphate‐buffered saline (Sigma, cat. no. 3813‐10PAK; dissolve one package into 1 liter of deionized water)
  • Pipets
  • 4°C incubator
  • C18 Spin column (Nest Group)
  • Vortex mixer
  • Centrifuge
  • Aluminum foil
  • SCX spin column (Nest Group)
  • Parafilm
  • 15‐ml conical tubes
  • 200‐μl PCR thin‐walled tubes
  • UV lamp (Blak‐Ray Lamp, Model XX‐15; UVP)
  • 1.5‐ml microcentrifuge tubes
  • SpeedVac
  • LTQ OrbitrapVelos ETD mass spectrometer or other ETD‐enabled mass spectrometers
  • Open Mass Spectrometry Search Algorithm (OMSSA) or other software

Basic Protocol 3: O‐GlcNAc Site Mapping by Using β‐Elimination Michael Addition with Dithiothreitol (BEMAD) Followed by CID/HCD Mass Spectrometry

  Materials
  • Desalted and dried protein digest (e.g., fractionated peptides from the protocol 2Support Protocol)
  • Working BEMAD buffer (see recipe)
  • Synthetic O‐GlcNAc peptides (see Greis et al., )
  • Trifluoroacetic acid (TFA)
  • Thiopropyl‐Sepharose resin (Sigma)
  • Thiol column buffer (i.e., PBS with 1 mM EDTA, freshly prepared!)
  • Thiol elution buffer (i.e., PBS, 1 mM EDTA, and 20 mM DTT, freshly prepared!)
  • Acetonitrile (ACN)
  • C18 spin column (Nest Group)
  • SpeedVac
  • Rotator
  • LTQ‐OrbitrapVelos (or other mass spectrometers) coupled with nanoHPLC system
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

Literature Cited
  Alfaro, J.F., Gong, C.X., Monroe, M.E., Aldrich, J.T., Clauss, T.R., Purvine, S.O., Wang, Z., Camp, D.G. 2nd, Shabanowitz, J., Stanley, P., Hart, G.W., Hunt, D.F., Yang, F., and Smith, R.D. 2012. Tandem mass spectrometry identifies many mouse brain O‐GlcNAcylated proteins including EGF domain‐specific O‐GlcNAc transferase targets. Proc. Natl. Acad. Sci. U.S.A. 109:7280‐7285. doi: 10.1073/pnas.1200425109.
  Bond, M.R. and Hanover, J.A. 2015. A little sugar goes a long way: The cell biology of O‐GlcNAc. J. Cell Biol. 208:869‐880. doi: 10.1083/jcb.201501101.
  Boyce, M., Carrico, I.S., Ganguli, A.S., Yu, S.H., Hangauer, M.J., Hubbard, S.C., Kohler, J.J., and Bertozzi, C.R. 2011. Metabolic cross‐talk allows labeling of O‐linked β‐N‐acetyl glucosamine‐modified proteins via the N‐acetylgalactosamine salvage pathway. Proc. Natl. Acad. Sci. U.S.A. 108:3141‐3146. doi: 10.1073/pnas.1010045108.
  Chalkley, R.J., Wells, L., and Vosseller, K. 2009a. O‐GlcNAc proteomics: Mass spectrometric analysis of O‐GlcNAc modifications on proteins. Compr. Anal. Chem. 52:353‐374. doi: 10.1016/S0166‐526X(08)00215‐8.
  Chalkley, R.J., Thalhammer, A., Schoepfer R, and Burligame, A.L. 2009b. Identification of protein O‐GlcNAcylation sites using electron transfer dissociation mass spectrometry on native peptides. Proc. Natl. Acad. Sci. U.S.A. 106:8894‐8899. doi: 10.1073/pnas.0900288106.
  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. doi: 10.1006/abio.1996.0047.
  Hahne, H., Sobotzki, N., Tamara, N., Helm, D., Borodkin, V.S., van Aalten, D.M., Agnew, B., and Kuster, B. 2013. Proteome wide purification and identification of O‐GlcNAc‐modified proteins using click chemistry and mass spectrometry. J. Proteome Res. 12:927‐936. doi: 10.1021/pr300967y.
  Hart, G.W. 2014. Three decades of research on O‐GlcNAcylation—a major nutrient sensor that regulates signaling, transcription and cellular metabolism. Front. Endocrinol. 5:183 doi: 10.3389/fendo.2014.00183.
  Holt, G.D. and Hart, G.W. 1986. The subcellular distribution of terminal N‐acetyl glucosamine moieties. Localization of a novel protein‐saccharide linkage, O‐linked GlcNAc. J. Biol. Chem. 261:8049‐8057.
  Khidekel, N., Ficarro, S.B., Peters, E.C., and Hsieh‐Wilson, L.C. 2004. Exploring the O‐GlcNAc proteome: Direct identification of O‐GlcNAc‐modified proteins from the brain. Proc. Natl. Acad. Sci. U.S.A. 101:13132‐13137. doi: 10.1073/pnas.0403471101.
  Khidekel, N., Ficarro, S.B., Clark, M.C., Bryan, M.C., Swaney, D.L., Rexach, J.E., Sun, Y.E., Coon, J.J., Peters, E.C., and Hsieh‐Wilson, L.C. 2007. Probing the dynamics of O‐GlcNAc glycosylation in the brain using quantitative proteomics. Nat. Chem. Biol. 3:339‐348. doi: 10.1038/nchembio881.
  Ma, J. and Hart, G.W. 2013. Protein O‐GlcNAcylation in diabetes and diabetic complications. Exp. Rev. Proteomics 10:365‐380. doi: 10.1586/14789450.2013.820536.
  Ma, J. and Hart, G.W. 2014. O‐GlcNAc profiling: From proteins to proteomes. Clin. Proteomics 11:8. doi: 10.1186/1559‐0275‐11‐8.
  Ma, J. and Hart, G.W. 2016. Mass spectrometry‐based quantitative O‐GlcNAcomic analysis. Methods Mol. Biol. 1410:91‐103. doi: 10.1007/978‐1‐4939‐3524‐6_6.
  Ma, J., Liu, T., Wei, A.C., Banerjee, P., O'Rourke B., and Hart G.W. 2015. O‐GlcNAcomic profiling identifies widespread O‐linked β‐N‐acetyl glucosamine modification (O‐GlcNAcylation) in oxidative phosphorylation system regulating cardiac mitochondrial function. J. Biol. Chem. 290:29141‐29153. doi: 10.1074/jbc.M115.691741.
  Nagel, A.K., Schilling, M., Comte‐Walters, S., Berkaw, M.N., and Ball, L.E. 2013. Identification of O‐linked N‐acetyl glucosamine (O‐GlcNAc)‐modified osteoblast proteins by electron transfer dissociation tandem mass spectrometry reveals proteins critical for bone formation. Mol. Cell Proteomics 12:945‐955. doi: 10.1074/mcp.M112.026633.
  Nandi, A., Sprung, R., Barma, D.K., Zhao, Y., Kim, S.C., Falck, J.R., and Zhao, Y. 2006. Global identification of O‐GlcNAc‐modified proteins. Anal. Chem. 78:452‐458. doi: 10.1021/ac051207j.
  Parker, B.L., Gupta, P., Cordwell, S.J., Larsen, M.R., and Palmisano, G. 2011. Purification and identification of O‐GlcNAc‐modified peptides using phosphate‐based alkyne CLICK chemistry in combination with titanium dioxide chromatography and mass spectrometry. J. Proteome Res. 10:1449‐1458. doi: 10.1021/pr100565j.
  Ramirez‐Correa, G.A., Ma, J., Slawson, C., Zeidan, Q., Lugo‐Fagundo, N.S., Xu, M., Shen, X., Gao, W.D., Caceres, V., Chakir, K., DeVine, L., Cole, R.N., Marchionni, L., Paolocci, N., Hart, G.W., and Murphy, A.M. 2015. Removal of abnormal myofilament O‐GlcNAcylation restores Ca2+ sensitivity in diabetic cardiac muscle. Diabetes 64:3573‐3587. doi: 10.2337/db14‐1107.
  Syka, J.E., Coon, J.J., Schroeder, M.J., Shabanowitz, J., and Hunt, D.F. 2004. Peptide and protein sequence analysis by electron transfer dissociation mass spectrometry. Proc. Natl. Acad. Sci. U.S.A. 101:9528‐9533. doi: 10.1073/pnas.0402700101.
  Teo, C.F., Ingale, S., Wolfert, M.A., Elsayed, G.A., Nöt, L.G., Chatham, J.C., Wells, L., and Boons, G.J. 2010. Glycopeptide‐specific monoclonal antibodies suggest new roles for O‐GlcNAc. Nat. Chem. Biol. 6:338‐343. doi: 10.1038/nchembio.338.
  Torres, C‐R. and Hart, G.W. 1984. Topography and polypeptide distribution of terminal N‐acetyl glucosamine residues on the surfaces of intact lymphocytes. J. Biol. Chem. 259:3308‐3317.
  Trinidad, J.C., Barkan, D.T., Gulledge, B.F., Thalhammer, A., Sali, A., Schoepfer, R., and Burlingame, A.L. 2012. Global identification and characterization of both O‐GlcNAcylation and phosphorylation at the murine synapse. Mol. Cell Proteomics 11:215‐229. doi: 10.1074/mcp.O112.018366.
  Vocadlo, D.J., Hang, H.C., Kim, E.J., Hanover, J.A., and Bertozzi, C.R. 2003. A chemical approach for identifying O‐GlcNAc‐modified proteins in cells. Proc. Natl. Acad. Sci. U.S.A. 100:9116‐9121. doi: 10.1073/pnas.1632821100.
  Vosseller, K., Hansen, K.C., Chalkley, R.J., Trinidad, J.C., Wells, L., Hart, G.W., and Burlingame, A.L. 2005. Quantitative analysis of both protein expression and serine/threonine post‐translational modifications through stable isotope labeling with dithiothreitol. Proteomics 5:388‐398. doi: 10.1002/pmic.200401066.
  Vosseller, K., Trinidad, J.C., Chalkley, R.J., Specht, C.G., Thalhammer, A., Lynn, A.J., Snedecor, J.O., Guan, S., Medzihradszky, K.F., Maltby, D.A., Schoepfer, R., and Burlingame, A.L. 2006. O‐Linked N‐acetyl glucosamine proteomics of postsynaptic density preparations using lectin weak affinity chromatography and mass spectrometry. Mol. Cell Proteomics 5:923‐934. doi: 10.1074/mcp.T500040‐MCP200.
  Wang, Z. and Hart, G.W. 2008. Glycomic approaches to study GlcNAcylation: Protein identification, site‐mapping, and site‐specific O‐GlcNAc quantitation. Clin. Proteomics 4:5. doi: 10.1007/s12014‐008‐9008‐x.
  Wang, Z., Pandey, A., and Hart, G.W. 2007. Dynamic interplay between O‐linked N‐acetylglucosaminylation and glycogen synthase kinase‐3‐dependent phosphorylation. Mol. Cell Proteomics 6:1365‐1379. doi: 10.1074/mcp.M600453‐MCP200.
  Wang, Z., Park, K., Comer, F., Hsieh‐Wilson, L.C., Saudek, C.D., and Hart, G.W. 2009. Site‐specific GlcNAcylation of human erythrocyte proteins: Potential biomarker(s) for diabetes. Diabetes 58:309‐317. doi: 10.2337/db08‐0994.
  Wang, Z., Udeshi, N.D., O'Malley, M., Shabanowitz, J., Hunt, D.F., and Hart, G.W. 2010a. Enrichment and site mapping of O‐linked N‐acetyl glucosamine by a combination of chemical/enzymatic tagging, photochemical cleavage, and electron transfer dissociation mass spectrometry. Mol. Cell Proteomics 9:153‐160. doi: 10.1074/mcp.M900268‐MCP200.
  Wang, Z., Udeshi, N.D., Slawson, C., Compton, P.D., Sakabe, K., Cheung, W.D., Shabanowitz, J., Hunt, D.F., and Hart, G.W. 2010b. Extensive crosstalk between O‐GlcNAcylation and phosphorylation regulates cytokinesis. Sci. Signal 3:ra2. doi: 10.1126/scisignal.2000526.
  Wells, L., Vosseller, K., Cole, R.N., Cronshaw, J.M., Matunis, M.J., and Hart, G.W. 2002. Mapping sites of O‐GlcNAc modification using affinity tags for serine and threonine post‐translational modifications. Mol. Cell Proteomics 1:791‐804. doi: 10.1074/mcp.M200048‐MCP200.
  Zachara, N.E., Vosseller, K., and Hart, G.W. 2011a. Detection and analysis of proteins modified by O‐linked N‐acetylglucosamine. Curr. Protoc. Protein Sci. 66:12.8.1‐12.8.33.
  Zachara, N.E., Molina, H., Wong, K.Y., Pandey, A., and Hart, G.W. 2011b. The dynamic stress‐induced “O‐GlcNAc‐ome” highlights functions for O‐GlcNAc in regulating DNA damage/repair and other cellular pathways. Amino Acids 40:793‐808. doi: 10.1007/s00726‐010‐0695‐z.
  Zhao, P., Viner, R., Teo, C.F., Boons, G.J., Horn, D., and Wells, L. 2011. Combining high‐energy C‐trap dissociation and electron transfer dissociation for protein O‐GlcNAc modification site assignment. J. Proteome Res. 10:4088‐4104. doi: 10.1021/pr2002726.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library