Identification of Proteins in Complex Mixtures Using Liquid Chromatography and Mass Spectrometry

Claire Delahunty1

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Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 5.6
DOI:  10.1002/0471143030.cb0505s17
Online Posting Date:  February, 2003
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Abstract

Liquid chromatography techniques have been successfully coupled with mass spectrometers to provide a robust method for the identification of proteins in mixtures. Chromatography can be performed in‐line with the mass spectrometer and data acquisition can be directly interfaced with search algorithms for identification by correlation with databases.

     
 
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Table of Contents

  • Basic Protocol 1: Sample Preparation for Direct Analysis of Peptides in Mixtures
  • Alternate Protocol 1: Sample Preparation by In‐Gel Digestion of Silver‐ or Coomassie‐Stained Spots Following Page
  • Basic Protocol 2: Loading a Proteins Sample for Microcapillary Column Liquid Chromatography
  • Support Protocol 1: Preparing Microcapillary Columns for Liquid Chromatography
  • Basic Protocol 3: Multidimensional Protein Identification Technology for Analyzing Complex Mixtures
  • Basic Protocol 4: Analysis of Liquid Chromatograpgy –Tandem Mass Spectrometry Data
  • Reagents and Solutions
  • Commentary
  • Figures
     
 
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Materials

Basic Protocol 1: Sample Preparation for Direct Analysis of Peptides in Mixtures

  Materials
  • Total cell lysates, insoluble fractions or membrane proteins, soluble fractions, or unknown sample
  • Cyanogen bromide (e.g., Aldrich), for insoluble fractions or membrane proteins only
  • 96% (v/v) formic acid (e.g., Aldrich), for insoluble fractions or membrane proteins only
  • Ammonium bicarbonate (e.g., J.T. Baker), for insoluble fractions only
  • 100 mM Tris, pH 8.5/8 M urea (e.g., Sigma)
  • 1 M Tris (2‐carboxyethyl)phosphine hydrochloride (TCEP; e.g., Pierce)/100 mM ammonium bicarbonate
  • 500 mM iodoacetamide
  • Endoproteinase Lys‐C (Roche Diagnostics)
  • 100 mM CaCl 2 (e.g., Mallinckrodt Baker; appendix 2A)
  • Porozyme bulk immobilized trypsin beads (Applied Biosystems)
  • 88% (v/v) formic acid (e.g., Aldrich)
  • Additional reagents and equipment for trichloroacetic acid (TCA) precipitation (unit 7.1)
CAUTION: Cyanogen bromide is highly toxic and volatile; carry out all work with this compound in a well‐ventilated fume hood. TCA is extremely caustic. Protect eyes and avoid contact with skin when preparing and handling TCA solutions

Alternate Protocol 1: Sample Preparation by In‐Gel Digestion of Silver‐ or Coomassie‐Stained Spots Following Page

  Materials
  • Coomassie‐ or silver‐stained (unit 6.6) one‐ or two‐dimensional acrylamide gel (units 6.1 & 6.4) containing proteins of interest
  • 30 mM potassium ferricyanide
  • 100 mM sodium thiosulfate
  • Acetonitrile (e.g., Fisher), HPLC grade
  • 25 and 100 mM ammonium bicarbonate (J.T. Baker)
  • 10 mM Tris(2‐carboxyethyl)phosphine (TCEP; e.g., Pierce)/100 mM ammonium bicarbonate
  • Iodoacetamide/ammonium bicarbonate solution (see recipe)
  • Trypsin digestion solution (see recipe), 4°C
  • 50 mM ammonium bicarbonate/5 mM CaCl 2 (e.g., Mallinckrodt Baker)
  • 5% (v/v) formic acid (e.g., Aldrich)
  • Glass plate, cleaned with methanol or ethanol
  • Scalpel or razor blades, cleaned with methanol or ethanol
  • Gel‐loading pipet tips

Basic Protocol 2: Loading a Proteins Sample for Microcapillary Column Liquid Chromatography

  Materials
  • Digested protein sample (see protocol 1 or protocol 2)
  • Pre‐equilibrated microcapillary column (see protocol 4)
  • High‐pressure bomb designed for pneumatically loading microcapillaries (see Dongré et al., ) connected to helium gas tank

Support Protocol 1: Preparing Microcapillary Columns for Liquid Chromatography

  • Methanol (e.g., Fisher), HPLC grade
  • C 18 packing material
  • Partisphere strong cation exchange (SCX; Whatman; for biphasic columns only)
  • Buffer A: 5% (v/v) acetonitrile/0.1% (v/v) formic acid
  • Laser puller (model P‐2000; Sutter Instruments)
  • Ceramic scribe
  • 100 × 365–µm (i.d. × o.d.) fused‐silica capillary (J & W Scientific)
  • Alcohol burner
  • PEEK microcross splitter (Upchurch)
  • Quaternary high‐performance liquid chromatography (HPLC) pump

Basic Protocol 3: Multidimensional Protein Identification Technology for Analyzing Complex Mixtures

  Materials
  • Microcapillary column loaded with protein sample (see protocol 3)
  • Buffer A: 5% (v/v) acetonitrile/0.1% (v/v) formic acid
  • Buffer B: 80% (v/v) acetonitrile/0.1% (v/v) formic acid
  • Buffer C: 500 mM ammonium acetate (e.g., Sigma)/5% (v/v) acetonitrile/0.1% (v/v) formic acid
  • PEEK microcross splitter (Upchurch)
  • LCQ ion trap mass spectrometer (ThermoFinnigan) including custom platform for nanoelectrospray, or equivalent tandem mass spectrometer
  • XYZ translational positioner (Newport)
  • HPLC system, including quaternary HPLC pump (e.g., Agilent, ThermoFinnigan, Waters; for two‐dimensional LC only) and nitrogen gas source
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Figures

Videos

Literature Cited

Literature Cited
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   Dongr, A.R., Eng, J.K., and Yates, J.R. III. 1997. Emerging tandem‐mass‐spectrometry techniques for the rapid identification of proteins. Trends Biotechnol. 15:418‐425.
   Eng, J., McCormack, A.L., and Yates, J. R. III 1994. An approach to correlate tandem mass spectral data of peptides with amino acid sequences in a protein database. J. Am. Soc. Mass Spectrom. 5:976‐989.
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   Gatlin, C.L., Kleeman, G.R., Hays, L.G., Link, A.J., and Yates, J.R. III 1998. Protein identification at the low femtomole level from silver‐stained gels using a new fritless electrospray interface for liquid chromatography‐microspray and nanospray mass spectrometry. Anal. Biochem. 263:93‐101.
   Gygi, S., Corthals, G.L., Zhang, Y., Rochon, Y., and Aebersold, R. 2000. Evaluation of two‐dimensional gel electrophoresis‐based proteome analysis technology. Proc. Natl. Acad. Sci.U.S.A. 97:9390‐9395.
   Hunt, D.F., Yates, J.R. III, Shabanowitz, J., Winston, S., and Hauer, C.R. 1986. Protein sequencing by mass spectrometry. Proc.Natl. Acad. Sci. U.S.A. 83:6233‐6237.
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   Jensen, O.N., Wilm, M., Shevchenko, A., and Mann, M. 1999b. Sample preparation methods for mass spectrometric peptide mapping directly from 2‐DE gels. In 2‐D Proteome Analysis Protocols (A.J. Link ed.) pp.513‐530. Humana Press Totowa, N.J
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   Link, A.J., Eng, J., Schieltz, D.M., Carmack, E., Mize, G.J., Morris, D.R., Garvik, B.M., and Yates, J.R. III 1999. Direct analysis of protein complexes using mass spectrometry. Nature Biotechnol. 17:676‐682.
   MacCoss, M.J., McDonald, W.H., Saraf, A., Sadygov, R., Clark, J.M., Tasto, J.J., Gould, K.L., Wolters, D., Washburn, M., Weiss, A., Clark, J.I., and Yates, J.R. III 2002. Shotgun identification of protein modifications from protein complexes and lens tissue. Proc.Natl. Acad. Sci. U.S.A. 99:7900‐7905.
   Matsudaira, P. 1987. Sequence from picomole quantities of proteins electroblotted onto polyvinylidene difluoride membranes. J. Biol.Chem. 262:10035‐10038.
   McCormack, A.L., Schieltz, D.M., Goode, B., Yang, S., Barnes, G., Drubin, D., and Yates, J.R. III 1997. Direct analysis and identification of proteins in mixtures by LC/MS/MS and database searching at the low‐femtomole level. Anal. Chem. 69:767‐776.
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   Santoni, V., Malloy, M., and Rabilloud, T. 2000. Membrane proteins and proteomics: Un amour impossible?. Electrophoresis 21:1054‐1076.
   Washburn, M.P. and Yates, J.R. III 2000. New methods of proteome analysis: Multidimensional chromatography and mass spectrometry. In Proteomics: A Trends Guide (W. Blackstock and M. Mann eds.) pp. 27‐31 Elsevier Science London, U.K.
   Washburn, M.P., Wolters, D., and Yates, J.R. III 2001. Large‐scale analysis of the yeast proteome by multidimensional protein identification technology. Nature Biotechnol. 19:242‐247.
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Internet Resources
   http://ncbi.nlm.nih.gov/Ftp/index.html
  National Center for Biotechnology Information's FTP site, from which genome databases can be downloaded.
   http://genome-www.stanford.edu
  Web site for Stanford Genomic Resources, including genome databases.
   http://www.tigr.org
  Web site for The Institute for Genome Research, including genome databases.
   http://prospector.ucsf.edu
  Sites for Web‐based search tools for analyzing MS/MS data.
   http://www.matrixscience.com
   http://prowl.rockefeller.edu/PROWL/pepfragch.html
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