Analyzing In Vivo Metabolite‐Protein Interactions by Large‐Scale Systematic Analyses

Xiyan Li1, Michael Snyder1

1 Department of Genetics, Stanford University, Stanford, California
Publication Name:  Current Protocols in Chemical Biology
Unit Number:   
DOI:  10.1002/9780470559277.ch110193
Online Posting Date:  December, 2011
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Metabolites interact with proteins in vivo in various ways other than enzymatic reactions. Profiling of such interactions may help disclose unknown molecular mechanisms that regulate protein functions, and provide potential targets for disease treatment. Here a procedure is described for systematic analyses of metabolite‐protein interactions in vivo. This procedure couples protein affinity purification and mass spectrometry to identify metabolite‐protein interactions. The primary effort can be completed within 1 day and scaled to process hundreds of samples in a batch. Originally developed in yeast, the same principles and protocol can be adapted to other organisms. Curr. Protoc. Chem. Biol. 3:181‐196 © 2011 by John Wiley & Sons, Inc.

Keywords: metabolite‐protein interaction; liquid chromatography; mass spectrometry; LC‐MS; metabolite; protein affinity purification; yeast

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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Affinity Purification of Yeast Protein and Extraction of Protein‐Interacting Metabolites
  • Support Protocol 1: Conjugation of Immunoglobulin to Dynabeads
  • Support Protocol 2: Yeast Growth and Cell Collection
  • Basic Protocol 2: LC‐MS of Protein‐Bound Metabolites
  • Support Protocol 3: LC‐MS Data Processing Using XCMS
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1: Affinity Purification of Yeast Protein and Extraction of Protein‐Interacting Metabolites

  • Rabbit IgG–conjugated Dynabeads (see protocol 2)
  • Lysis buffer (see recipe) with and without DTT and protease/phosphatase inhibitors
  • Zirconia silica beads (Biospec)
  • Cell pellet (stored at −80°C, see protocol 3)
  • Wash buffer 1 (see recipe)
  • Wash buffer 2 (see recipe)
  • 75% (v/v) ethanol (mass spectrometry grade)
  • 15‐well 4‐12% NuPAGE Bis‐Tris gel (Invitrogen)
  • 2× Laemmli SDS sample buffer (for SDS‐PAGE; see recipe)
  • Page Ruler Plus prestained protein ladder (Fermentas)
  • 20× NuPAGE MOPS SDS running buffer (Invitrogen, cat. no. NP0001‐02)
  • ProtoBlue safe colloidal Coomassie staining solution (National Diagnostics, cat. no. EC‐722)
  • Gel drying solution (see recipe)
  • Refrigerated centrifuge
  • Hula mixer (Invitrogen) or similar product
  • Magnetic stand for 1.5/2.0 ml tubes (Invitrogen)
  • Eppendorf protein LoBind tubes (2.0 ml and 1.5 ml)
  • FastPrep cell lyser with an adapter for 2‐ml tubes (Qbiogene)
  • Heat blocks maintained at 42°C and 95°C
  • Glass mass spectrometry vials with inserts
  • Gel‐drying frame
  • Cellophane membrane for gel drying
  • Gel scanner
  • Additional reagents and equipment for SDS‐PAGE (e.g., Gallagher, )
NOTE: Steps 1 to 8 should be carried out in a cold room at 4°C.NOTE: Non‐filtered pipet tips should be used to avoid introducing polymers that are often found in filters.NOTE: Nitrile gloves are preferred to create a cleaner background on LC‐MS.

Support Protocol 1: Conjugation of Immunoglobulin to Dynabeads

  • 10 mg (11 mg/ml) rabbit IgG (ChromaPure) in 0.01 mM sodium phosphate buffer, pH 7.6/0.25 M NaCl (Jackson ImmunoResearch, cat. no. 011‐000‐003)
  • Bradford protein assay kit and bovine gamma globulin as standard
  • Epoxy Dynabeads (Invitrogen, 300‐mg size, cat. no. 143.02D)
  • Dynabeads antibody coupling kit (Invitrogen, cat. no. 143.11D) containing C1, C2, LB, HB, SB solutions
  • 143 mM (∼50 mg/ml) n‐dodecyl glucoside (nDG, Sigma, cat. no. D8035‐1g) in methanol for downstream mass spectrometry application
  • 0.05% (v/v) Tween‐20
  • Sodium azide
  • BS3 (Bis(sulfosuccinimidyl) suberate (Thermo Scientific, cat. no. PI‐21580, 21585, or 21586; optional)
  • BS3 conjugation buffer: 20 mM HEPES or 20 mM NaPO 4/0.15 M NaCl (pH 7.5)
  • BS3 quenching buffer: 1 M Tris⋅Cl (pH 7.5)
  • Refrigerated microcentrifuge
  • Magnetic stand for 1.5/2.0 ml tubes (Invitrogen)
  • Eppendorf protein LoBind tubes (2.0 ml and 1.5 ml)
  • 15‐ml conical centrifuge tubes (e.g., BD Falcon)
  • Hula mixer (Invitrogen) or similar product

Support Protocol 2: Yeast Growth and Cell Collection

  • Yeast strain
  • SC‐URA solid medium plates with glucose (see recipe)
  • SC‐URA liquid medium with glucose or raffinose (see recipe)
  • 3× YP/Gal (see recipe)
  • 30°C incubator
  • 1‐liter flask
  • Platform shaker capable of accommodating 1‐liter flasks
  • Spectrophotometer
  • 500‐ml centrifuge bottles
  • Centrifuge with JA‐10 or SLA‐3000 rotor
  • 15‐ml conical centrifuge tubes
  • 2‐ml thick‐wall screw‐cap tubes

Basic Protocol 2: LC‐MS of Protein‐Bound Metabolites

  • Metabolite samples (ideally freshly prepared within 1 to 2 days; see protocols above)
  • Mobile phase solutions: gradient elution of LC often uses 10% and 90% acetonitrile in water for ESI, and 10% to 100% methanol for APCI; buffer reagents, such as 10 mM ammonium acetate, can be added to improve LC peak shape stability—pH can be adjusted with acetic acid or ammonium hydroxide as needed to help ionization of analytes
  • Strong wash, weak wash, and needle wash solutions: weak and strong wash solutions are the same as the start and end mobile phases; for example, 10% and 90% acetonitrile in water can be used as weak and strong wash solutions for a reversed‐phase gradient—needle wash is similar to weak wash; do not use buffer or salt reagents in wash solutions
  • Reversed‐phase UPLC columns for low polarity to very hydrophobic metabolites: UPLC C18, hexyl/phenyl, or C8 columns from Waters
  • Normal‐phase or HILIC columns for polar hydrophilic metabolites: UPLC Amide, T3, or other columns from Waters
  • Waters Acquity UPLC‐coupled Thermo Exactive Orbitrap mass spectrometer, equipped with an electrospray ionization (ESI) probe or an atmospheric pressure chemical ionization (APCI) probe
NOTE: ESI and APCI complement each other to expand the detection scope. APCI is especially suitable to detect thermostable nonpolar hydrophobic molecules with a molecular weight below 1000 Da. APCI is preferred when it can detect the metabolites of interest. ESI has broader detection coverage in chemical properties and molecular weight than APCI, yet it requires optimization of many parameters for sound performance.
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Literature Cited

Literature Cited
   Gallagher, S. 2006. One‐dimensional SDS gel electrophoresis of proteins. Curr. Protoc. Mol. Biol. 75:10.2A.1‐10.2A.37.
   Gallego, O., Betts, M.J., Gvozdenovic‐Jeremic, J., Maeda, K., Matetzki, C., Aguilar‐Gurrieri, C., Beltran‐Alvarez, P., Bonn, S., Fernandez‐Tornero, C., Jensen, L.J., Kuhn, M., Trott, J., Rybin, V., Müller, C.W., Bork, P., Kaksonen, M., Russell, R.B., and Gavin, A.C. 2010. A systematic screen for protein‐lipid interactions in Saccharomyces cerevisiae. Mol. Syst. Biol. 6:430.
   Gelperin, D.M., White, M.A., Wilkinson, M.L., Kon, Y., Kung, L.A., Wise, K.J., Lopez‐Hoyo, N., Jiang, L., Piccirillo, S., Yu, H., Gerstein, M., Dumont, M.E., Phizicky, E.M., Snyder, M., and Grayhack, E.J. 2005. Biochemical and genetic analysis of the yeast proteome with a movable ORF collection. Genes Dev. 19:2816‐2826.
   Li, X. and Snyder, M. 2011. Metabolites as global regulators: a new view of protein regulation: Systematic investigation of metabolite‐protein interactions may help bridge the gap between genome‐wide association studies and small molecule screening studies. Bioessays 33:485‐489.
   Li, X., Gianoulis, T.A., Yip, K.Y., Gerstein, M., and Snyder,, M. 2010. Extensive in vivo metabolite‐protein interactions revealed by large‐scale systematic analyses. Cell 143:639‐650.
   Lowenadler, B., Jansson, B., Paleus, S., Holmgren, E., Nilsson, B., Moks, T., Palm, G., Josephson, S., Philipson, L., and Uhlen, M. 1987. A gene fusion system for generating antibodies against short peptides. Gene 58:87‐97.
   Morozov, V.N., Morozova, T.Y., Johnson, K.L., and Naylor, S. 2003. Parallel determination of multiple protein metabolite interactions using cell extract, protein microarrays and mass spectrometric detection. Rapid Commun. Mass Sp. 17:2430‐2438.
   Nilsson, B., Moks, T., Jansson, B., Abrahmsen, L., Elmblad, A., Holmgren, E., Henrichson, C., Jones, T.A., and Uhlen, M. 1987. A synthetic IgG‐binding domain based on staphylococcal protein A. Protein Eng. 1:107‐113.
   Smith, C.A., Want, E.J., O'Maille, G., Abagyan, R., and Siuzdak, G. 2006. XCMS: Processing mass spectrometry data for metabolite profiling using nonlinear peak alignment, matching, and identification. Anal. Chem. 78:779‐787.
   Tagore, R., Thomas, H.R., Homan, E.A., Munawar, A., and Saghatelian, A. 2008. A global metabolite profiling approach to identify protein‐metabolite interactions. J. Am. Chem. Soc. 130:14111‐14113.
   Zhu, H., Bilgin, M., Bangham, R., Hall, D., Casamayor, A., Bertone, P., Lan, N., Jansen, R., Bidlingmaier, S., Houfek, T., Mitchell, T., Miller, P., Dean, R.A., Gerstein, M., and Snyder, M. 2001. Global analysis of protein activities using proteome chips. Science 293:2101‐2105.
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