Mass Spectrometry: An Outsourcing Guide

Leroi V. DeSouza1, K.W. Michael Siu1, Ronald E. Pearlman2

1 Centre for Research in Mass Spectrometry and Department of Chemistry, York University, Toronto, Ontario, Canada, 2 Centre for Research in Mass Spectrometry and Department of Biology, York University, Toronto, Ontario, Canada
Publication Name:  Current Protocols Essential Laboratory Techniques
Unit Number:  Unit 12.2
DOI:  10.1002/9780470089941.et1202s02
Online Posting Date:  December, 2009
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Abstract

Proteomics is the study of the proteins expressed from the genome of a cell or organism. Analytical mass spectrometry has recently become a powerful tool to study a cell or organism's proteome and is the focus of this chapter. Mass spectrometry can be used for both qualitative and quantitative analysis of proteomes. This chapter will focus on qualitative analysis, addressing questions of what proteins are present in a proteome and what post‐translational modifications may be associated with these proteins. The instrumentation required for mass spectrometric analysis is generally not available in a standard research laboratory or as part of an undergraduate laboratory, being associated in general with a core facility. We will not describe here details of specific operation of the instruments. Here we focus on the common types of analysis presently in routine use and on preparation of samples for routine biological mass spectrometric analysis that will allow most laboratories, including undergraduate and graduate teaching laboratories, to prepare and analyze samples in experiments designed to obtain proteomic information. Curr. Protoc. Essential Lab. Tech. 2:12.2.1‐12.1.18. © 2009 by John Wiley & Sons, Inc.

Keywords: mass spectrometry; polyacrylamide gel electrophoresis; electrospray ionization; matrix assisted laser desorption ionization; collision induced dissociation; time of flight; tandem mass spectrometry

     
 
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Table of Contents

  • Overview and Principles
  • Mass Spectrometric Analysis
  • Protocols
  • Basic Protocol 1: In‐Gel Trypsin Digestion
  • Basic Protocol 2: In‐Solution Trypsin Digestion
  • Basic Protocol 3: Sample Cleanup (“Desalting”)
  • Troubleshooting
  • Limitations of Mass Spectrometry
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: In‐Gel Trypsin Digestion

  Materials
  • Acrylamide gel (unit 7.3) containing protein(s) of interest
  • Solution A: 50 mM ammonium bicarbonate
  • Solution B: 50% acetonitrile/25 mM ammonium bicarbonate
  • Solution C: 50 mM ammonium bicarbonate/10 mM dithiothreitol (DTT)
  • Solution D: 100 mM iodoacetamide/50 mM ammonium bicarbonate
  • Trypsin (porcine, sequencing grade; e.g., Promega)
  • Solution E: 25 mM ammonium bicarbonate
  • Solution F: 5% formic acid/50% acetonitrile (mix 18 ml Milli‐Q H 2O with 2 ml formic acid, adding acid to H 2O; mix the resulting 20 ml with 20 ml acetonitrile using a glass bottle)
  • Scalpel blades
  • Clean glass plate or petri dish
  • Microcentrifuge (e.g., Picofuge from Stratagene))
  • 1.5‐ml microcentrifuge tubes (e.g., EZ Micro tubes from BioRad, Axygen low‐bind tubes, http://www.axygen.com, or other brands where leaching from the tube in the presence of acetonitrile will not occur)
  • SpeedVac evaporator (Savant) or equivalent
  • 56°C water bath or heating block

Basic Protocol 2: In‐Solution Trypsin Digestion

  Materials
  • Protein solution
  • 20 mM Tris⋅Cl, pH 7.5 (unit 3.3)
  • 100 mM dithiothreitol (DTT): dissolve 0.15 g DTT in 10 ml Milli‐Q H 2O
  • 100 mM iodoacetamide: dissolve 0.185 g iodoacetamide in 10 ml of 50 mM ammonium bicarbonate (store protected from light in amber bottle or wrapped in aluminum foil)
  • Trypsin (porcine, sequencing grade; e.g., Promega)
  • 100 mM ammonium bicarbonate: dissolve 0.792 g ammonium bicarbonate in 100 ml Milli‐Q H 2O (freshly prepared ammonium bicarbonate solution should have a pH of ∼ 8.3 at room temperature)
  • Centrifugal filter: Millipore Microcon, MWCO 3000
  • Heat block
  • Additional reagents and equipment for protein assay (unit 2.2)

Basic Protocol 3: Sample Cleanup (“Desalting”)

  Materials
  • Aqueous solution of 0.3% (v/v) trifluoroacetic acid (TFA)
  • Aqueous solution of 60% (v/v) acetonitrile/0.3% (v/v) TFA
  • Matrix solution: 10 mg/ml α‐cyano‐4‐hydroxycinnamic acid in 60% (v/v) acetonitrile (v/v)/0.3% TFA
  • Aqueous solution of 1% (v/v) formic acid
  • Aqueous solution of 60% (v/v) acetonitrile/1.0% (v/v) formic acid
  • ZipTip pipet tips (Millipore)
  • MALDI target plate or nanospray emitter
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Figures

Videos

Literature Cited

Literature Cited
   Aronica, L., Bednenko, J., Noto, T., DeSouza, L.V., Siu, K.W., Loidl, J., Pearlman, R.E., Gorovsky, M.A., and Mochizuki, K. 2008. Study of an RNA helicase implicates small RNA‐noncoding RNA interactions in programmed DNA elimination in Tetrahymena. Genes Dev. 22:2228‐2241.
   Bodner, W.M., Blackburn, R.K., Krise, J.M. and Moseley, M.A. 2003. Exploiting the complementary nature of LC/MALDI/MS/MS and LC/ESI/MS/MS for increased proteome coverage. J. Am. Soc. Mass Spectrom. 14:971‐979.
   Bowman, G.R., Smith, D.G., Michael Siu, K.W., Pearlman, R.E., and Turkewitz, A.P. 2005. Genomic and proteomic evidence for a second family of dense core granule cargo proteins in Tetrahymena thermophila. J. Eukaryot. Microbiol. 52:291‐297.
   Domon, B. and Aebersold, R. 2006. Mass spectrometry and protein analysis. Science 312:212‐217.
   Gromova, I. and Celis, J.E. 2006. Protein detection in gels by silver staining: A procedure compatible with mass spectrometry. In Cell Biology: A Laboratory Handbook, 3rd ed. (J.E. Celis, N. Carter, T. Hunter, K. Simons, J.V. Small, and D. Shotton, eds.) pp. 527‐532. Academic Press, San Diego.
   Guilhaus, M. 1995. Principles and instrumentation in time‐of‐flight mass spectrometry: Physical and instrumental concepts. J. Mass Spectrom. 30:1519‐1532.
   Jacobs, M.E., DeSouza, L.V., Samaranayake, H., Pearlman, R.E., Siu, K.W., and Klobutcher, L.A. 2006. The Tetrahymena thermophila phagosome proteome. Eukaryot. Cell. 5:1990‐2000.
   Kinter, M. and Sherman, N.E. 2000. Protein Sequencing and Identification Using Tandem Mass Spectrometry. Wiley‐Interscience, New York.
   Rabilloud, T. 1990. Mechanisms of protein silver staining in polyacrylamide gels: A 10‐year synthesis. Electrophoresis 10:785‐794.
   Roepstorff, P. and Fohlman, J. 1984. Proposal for a common nomenclature for sequence ions in mass spectra of peptides. Biomed. Mass Spectrom. 11:601.
   Scopes, R.K. 1994. Protein Purification: Principles and Practice. Springer‐Verlag, New York.
   Shevchenko, A., Wilm, M., Vorm, O., and Mann, M. 1996. Mass spectrometric sequencing of proteins from silver‐stained polyacrylamide gels. Anal. Chem. 68:850‐858.
   Smith, D.G., Gawryluk, R.M., Spencer, D.F., Pearlman, R.E., Siu, K.W. and Gray, M.W. 2007. Exploring the mitochondrial proteome of the ciliate protozoon Tetrahymena thermophila: Direct analysis by tandem mass spectrometry. J. Mol. Biol. 374:837‐863.
   Smith, J.C., Northey, J.G., Garg, J., Pearlman, R.E., and Siu, K.W. 2005. Robust method for proteome analysis by MS/MS using an entire translated genome: Demonstration on the ciliome of Tetrahymena thermophila. J. Proteome Res. 4:909‐919.
   Tirumalai, R.S., Chan, K.C., Prieto, D.A., Issaq, H.J., Conrads, T.P., and Veenstra, T.D. 2003. Characterization of the low molecular weight human serum proteome. Mol. Cell Proteomics. 2:1096‐1103.
   Wang, B.H., Dreisewerd, K., Bahr, U., Karas, M., and Hillenkamp, F. 1993. Gas‐phase cationization and protonation of neutrals generated by matrix‐assisted laser desorption. J. Am. Soc. Mass Spectrom. 4:393‐398.
   Washburn, M.P., Wolters, D., and Yates, J.R. III. 2001. Large‐scale analysis of the yeast proteome by multidimensional protein identification technology. Nat. Biotechnol. 19:242‐247.
   Yan, J.X., Wait, R., Berkelman, T., Harry, R.A., Westbrook, J.A., Wheeler, C.H., and Dunn, M.J. 2000. A modified silver staining protocol for visualization of proteins compatible with matrix‐assisted laser desorption/ionization and electrospray ionization‐mass spectrometry. Electrophoresis 17:3666‐3672.
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