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

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Abstract
Table of Contents
Materials
Figures
Literature Cited

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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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 CaCl₂ (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 CaCl₂ (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
C₁₈ 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

Figure 5.6.1 Integrated two‐dimensional liquid chromatography. Peptides are separated in the first dimension by strong cation exchange (SCX) followed by reversed‐phase (RP) separation and elution into the mass spectrometer. Triangles, circles, and squares represent different peptides.

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Figure 5.6.2 All mass spectrometers are composed of three basic parts: an ionization source, a mass analyzer, and an ion detector. The mass analyzer depicted in this figure is from a tandem mass spectrometer. MS‐1 and MS‐2 indicate the tandem mass spectrometers.

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Figure 5.6.3 In the liquid chromatography–tandem mass spectrometry LC‐MS/MS technique, peptides are first separated in a liquid chromatographic step. Fractions from the chromatography are subjected to MS and selected ions from the MS experiment are further fragmented in an MS/MS experiment.

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Figure 5.6.4 Search algorithms employed for LC‐MS/MS data interpretation search uninterpreted MS/MS spectra against protein and DNA databases. Results are scored on the cross‐correlation fit between a theoretical (or model) spectrum and the tandem mass spectra obtained from the experiment (real spectrum).

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Videos

Literature Cited

Literature Cited
	Aebersold, R.H., Pipes, G., Hood, L.E., and Kent, S.B. 1986. Electroblotting onto activated glass.High efficiency preparation of proteins from analytical sodium dodecyl sulfate‐polyacrylamide gels for direct sequence analysis. J. Biol. Chem. 261:4229‐4238.
	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.
	Fenn, J.B., Mann, M., Meng, C.K., Wong, S.F., and Whitehouse, C.M. 1989. Electrospray ionization for mass spectrometry of large biomolecules Science 246:64‐71.
	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.
	Jensen, O.N., Wilm, M., Shevchenko, A., and Mann, M. 1999a. Peptide sequencing of 2‐DE gel‐isolated proteins by nanospray tandem mass spectrometry. In 2‐D Proteome Analysis Protocols (A.J. Link ed.) pp. 571‐578. Humana Press Totowa, N.J
	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
	Kennedy, R.T. and Jorgenson, J.W. 1989. Quantitative analysis of individual neurons by open tubular liquid chromatography with voltammetric detection. Anal. Chem. 61:1128‐1135.
	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.
	Peng, J. and Gygi, S.P. 2001. Proteomics: The move to mixtures. J. Mass Spectrom. 36:1083‐1091.
	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.
	Wolters, D.A., Washburn, M.P., and Yates, J.R. III 2001. An automated multidimensional protein identification technology for shotgun proteomics. Anal. Chem. 73:5683‐5690.
	Yates, J.R. III 1998a. Mass spectrometry and the age of the proteome. J. Mass Spectrom. 33:1‐19.
	Yates, J.R. III 1998b. Database searching using mass spectrometry data. Electrophoresis 19:893‐900.
	Yates, J.R. III 2000. Mass spectrometry from genomics to proteomics. Trends Genet. 16:5‐8.
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

GO TO THE FULL PROTOCOL:

PDF or HTML at Wiley Online Library

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

Abstract

Table of Contents

Materials

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

Figures

Videos

Literature Cited