Protein Identification and Characterization by Mass Spectrometry

Scott D. Patterson1

1 Amgen, Inc., Thousand Oaks, California
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 10.22
DOI:  10.1002/0471142727.mb1022s41
Online Posting Date:  May, 2001
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

This overview describes some of the new technologies that can be employed to facilitate rapid identification and characterization of proteins, including the use of correlative approaches for protein identification, rapid posttranslational modification analysis, identification of components in complex mixtures, and direct mass analysis of gel‐separated proteins. The mass spectrometric methods referred to in this overview include matrix‐assisted laser desorption/ionization mass spectrometry (MALDI‐MS) and electrospray ionization mass spectrometry (ESI‐MS).

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

Table of Contents

  • Protein Identification Using MS‐Based Correlative Approaches
  • Direct MS Analysis of Gel‐Separated Proteins and Other Multi‐Dimensional Approaches for Protein Separation and Mass Measurement
  • Rapid Posttranslational Modification Analysis
  • High‐Throughput Protein Identification
  • Conclusion
  • Literature Cited
  • Figures
  • Tables
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

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

Figures

Videos

Literature Cited

Literature Cited
   Amankwa, L.N., Harder, K., Jirik, F., and Aebersold, R. 1995. High‐sensitivity determination of tyrosine‐ phosphorylated peptides by on‐line enzyme reactor and electrospray‐ionization mass‐spectrometry. Protein Sci. 4:113‐125.
   Bean, M.F., Annan, R.S., Hemling, M.E., Mentzer, M., Huddleston, M.J., and Carr, S.A. 1995. LC‐MS methods for selective detection of posttranslational modifications in proteins: Glycosylation, phosphorylation, sulfation, and acylation. In Techniques in Protein Chemistry VI. (J.W. Crabb, ed.) pp. 107‐116. Academic Press, San Diego.
   Betts, J.C., Blackstock, W.P., Ward, M.A., and Anderton, B.H. 1997. Identification of phosphorylation sites on neurofilament proteins by nanoelectrospray mass spectrometry. J. Biol. Chem. 272:12922‐12927.
   Charbonneau, H. and Tonks, N.K. 1992. 1002 protein phosphatases? Annu. Rev. Cell Biol. 8:463‐493.
   Cohen, S.L. 1996. Domain elucidation by mass spectrometry. Structure 4:1013‐1016.
   Cohen, S.L. and Chait, B.T. 1997. Mass spectrometry of whole proteins eluted from sodium dodecyl sulfate–polyacrylamide gel electrophoresis gels. Anal. Biochem. 247:257‐267.
   Coligan, J.E. Dunn, B.M. Ploegh, H.L. Speicher, D.W., and Wingfield, P.T. (eds.). 1997. Current Protocols in Protein Science, John Wiley & Sons New York.
   Cordwell, S.J., Wasinger, V.C., Cerpa‐Poljak, A., Duncan, M.W., and Humphery‐Smith, I. 1997. Conserved motifs as the basis for recognition of homologous proteins across species boundaries using peptide‐mass fingerprinting. J. Mass Spectrom. 32:370‐378.
   Cottrell, J.S. 1994. Protein identification by peptide mass fingerprinting. Pept. Res. 7:115‐124.
   Craig, A.G., Hoeger, C.A., Miller, C.L., Goedken, T., Rivier, J.E., and Fischer, W.H. 1994. Monitoring protein kinase and phosphatase reactions with matrix‐assisted laser desorption/ionization mass spectrometry and capillary zone electrophoresis: Comparison of the detection efficiency of peptide‐phosphopeptide mixtures. Biol. Mass Spectrom. 23:519‐528.
   Craig, A.G., Fischer, W.H., Rivier, J.E., McIntosh, J.M., and Gray, W.R. 1995. MS‐based scanning methodologies applied to Conus venom. In Techniques in Protein Chemistry VI. (J.W. Crabb, ed.) pp. 31‐38. Academic Press, San Diego.
   Ding, J., Burkhart, W., and Kassel, D.B. 1994. Identification of phosphorylated peptides from complex mixtures using negative‐ion orifice‐potential stepping and capillary liquid chromatography/electrospray ionization mass spectrometry. Rapid Commun. Mass Spectrom. 8:94‐98.
   Eckerskorn, C., Strupat, K., Schleuder, D., Hochstrasser, D., Sanchez, J.‐C., Lottspeich, F., and Hillenkamp, F. 1997. Analysis of proteins by direct‐scanning infrared‐MALDI mass spectrometry after 2D‐PAGE separation and electroblotting. Anal. Chem. 69:2888‐2892.
   Eng, J.K., 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.
   Fabris, D., Vestling, M.M., Cordero, M.M., Doroshenko, V.M., Cotter, R.J., and Fenselau, C. 1995. Sequencing electroblotted proteins by tandem mass spectrometry. Rapid Commun. Mass Spectrom. 9:1051‐1055.
   Gonzalez, J., Takao, T., Hori, H., Besada, V., Rodriguez, R., Padron, G., and Shimonishi, Y. 1992. A method for determination of N‐glycosylation sites in glycoproteins by collision‐induced dissociation analysis in fast atom bombardment mass spectrometry: Identification of the positions of carbohydrate‐linked asparagine in recombinant alpha‐amylase by treatment with peptide‐N‐glycosidase F in 18O‐labeled water. Anal. Biochem. 205:151‐158.
   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.
   Houthaeve, T., Gausepohl, H., Mann, M., and Ashman, K. 1995. Automation of micro‐preparation and enzymatic cleavage of gel electrophoretically separated proteins. FEBS Lett. 376:91‐94.
   Hsieh, Y.L., Wang, H., Elicone, C., Mark, J., Martin, S.A., and Regnier, F. 1996. Automated analytical system for the examination of protein primary structure. Anal. Chem. 68:455‐462.
   Huang, J.M., Wei, Y.F., Kim, Y.H., Osterberg, L., and Matthews, H.R. 1991. Purification of a protein histidine kinase from the yeast Saccharomyces cerevisiae. The first member of this class of protein kinases. J. Biol. Chem. 266:9023‐9031.
   Huddleston, M.J., Bean, M.F., and Carr, S.A. 1993. Collisional fragmentation of glycopeptides by electrospray ionization LC/MS and LC/MS/MS: Methods for selective detection of glycopeptides in protein digests. Anal. Chem. 65:877‐884.
   Huddleston, M.J., Annan, R.S., Bean, M.F., and Carr, S.A. 1994. Selective detection of Thr‐, Ser‐, and Tyr‐phosphopeptides in complex digests by electrospray LC‐MS. In Techniques in Protein Chemistry V. (J.W. Crabb, ed.) pp. 123‐130. Academic Press, San Diego.
   Hunter, T. 1987. 1001 protein kinases. Cell 50:823‐829.
   James, P. 1997. Of genomes and proteomes. Biochem. Biophys. Res. Commun. 231:1‐6.
   James, P., Quadroni, M., Carafoli, E., and Gonnet, G. 1994. Protein identification in DNA databases by peptide mass fingerprinting. Protein Sci. 3:1347‐1350.
   Jensen, O.N., Podtelejnikov, A., and Mann, M. 1996a. Delayed extraction improves specificity in database searches by matrix ‐assisted laser desorption/ionization peptide maps. Rapid Commun. Mass Spectrom. 10:1371‐1378.
   Jensen, O.N., Vorm, O., and Mann, M. 1996b. Sequence patterns produced by incomplete enzymatic digestion or one ‐step Edman degradation of peptide mixtures as probes for protein database searches. Electrophoresis 17:938‐944.
   Jungblut, P., Thiede, B., Zimnyarndt, U., Muller, E.C., Scheler, C., Wittmann‐Liebold, B., and Otto, A. 1996. Resolution power of 2‐dimensional electrophoresis and identification of proteins from gels. Electrophoresis 17:839‐847.
   Kassel, D.B., Consler, T.G., Shalaby, M., Sekhri, P., Gordon, N., and Nadler, T. 1995. Direct coupling of an automated 2‐dimensional microcolumn affinity chromatography‐capillary HPLC system with mass spectrometry for biomolecule analysis. In Techniques in Protein Chemistry VI. (J.W. Crabb, ed.) pp. 39‐46. Academic Press, San Diego.
   Klose, J. 1975. Protein mapping by combined isoelectric focusing and electrophoresis of mouse tissues: A novel approach to testing for induced point mutations in mammals. Humangenetik 26:231‐243.
   Krishna, R.G. and Wold, F. 1993. Post‐translational modification of proteins. Adv. Enzymol. Relat. Areas Mol. Biol. 67:265‐298.
   Kuster, B., Wheeler, S.F., Hunter, A.P., Dwek, R.A., and Harvey, D.J. 1997. Sequencing of N ‐ linked oligosaccharides directly from protein gels: In‐gel deglycosylation followed by matrix‐assisted laser desorption/ionization mass spectrometry and normal‐ phase high ‐ performance liquid chromatography. Anal. Biochem. 250:82‐101.
   Lamond, A.I. and Mann, M. 1997. Cell biology and the genome projects—a concerted strategy for characterizing multiprotein complexes by using mass spectrometry. Trends Cell Biol. 7:139‐142.
   Liao, P.C., Leykam, J., Andrews, P.C., Gage, D.A., and Allison, J. 1994. An approach to locate phosphorylation sites in a phosphoprotein: Mass mapping by combining specific enzymatic degradation with matrix ‐ assisted laser desorption/ionization mass spectrometry. Anal. Biochem. 219:9‐20.
   Loo, J.A. 1997. Studying noncovalent protein complexes by electrospray ionization mass spectrometry. Mass Spec. Rev. 16:1‐23.
   Loo, R.R.O., Stevenson, T.I., Mitchell, C., Loo, J.A., and Andrews, P.C. 1996. Mass‐spectrometry of proteins directly from polyacrylamide gels. Anal. Chem. 68:1910‐1917.
   Loo, R.R.O., Mitchell, C., Stevenson, T.I., Martin, S.A., Hines, W., Juhasz, P., Patterson, D., Peltier, J., Loo, J.A., and Andrews, P.C. 1997. Sensitivity and mass accuracy for proteins analyzed directly from polyacrylamide gels: Implications for proteome mapping. Electrophoresis 18:382‐390.
   Mann, M. and Wilm, M. 1994. Error‐tolerant identification of peptides in sequence databases by peptide sequence tags. Anal. Chem. 66:4390‐4399.
   Matsui, N.M., Smith, D.M., Clauser, K.R., Fichman, J., Andrews, L.E., Sullivan, C.M., Burlingame, A.L., and Epstein, L.B. 1997. Immobilized pH gradient two‐dimensional gel electrophoresis and mass spectrometric identification of cytokine‐regulated proteins in ME‐180 cervical carcinoma cells. Electrophoresis 18:409‐417.
   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.
   Mortz, E., O' Connor, P.B., Roepstorff, P., Kelleher, N.L., Wood, T.D., McLafferty, F.W., and Mann, M. 1996. Sequence tag identification of intact proteins by matching tandem mass spectral data against sequence data bases. Proc. Natl. Acad. Sci. U.S.A. 93:8264‐8267.
   Nelson, R.W., Krone, J.R., Bieber, A.L., and Williams, P. 1995. Mass spectrometric immunoassay. Anal. Chem. 67:1153‐1158.
   O'Farrell, P.H. 1975. High‐resolution two‐dimensional gel electrophoresis of proteins. J. Biol. Chem. 250:4007‐4021.
   Opiteck, G.J., Lewis, K.C., Jorgenson, J.W., and Anderegg, R.J. 1997a. Comprehensive on‐line LC/LC/MS of proteins. Anal. Chem. 69:1518‐1524.
   Opiteck, G.J., Jorgenson, J.W., and Anderegg, R.J. 1997b. Two‐dimensional SEC/RPLC coupled to mass spectrometry for the analysis of peptides. Anal. Chem. 69:2283‐2291.
   Pappin, D.J.C., Rahman, D., Hansen, H.F., Bartlet‐Jones, M., Jeffery, W., and Bleasby, A.J. 1995. Chemistry, mass spectrometry and peptide‐mass databases: Evolution of methods for the rapid identification and mapping of cellular proteins. In Mass Spectrometry in the Biological Sciences. (A.L. Burlingame, and S.A. Carr, eds.) pp. 135‐150. Humana Press, Totowa, N.J.
   Patterson, S.D. 1994. From electrophoretically separated protein to identification: Strategies for sequence and mass analysis. Anal. Biochem. 221:1‐15.
   Patterson, S.D. 1995. Matrix‐assisted laser‐desorption/ionization mass spectrometric approaches for the identification of gel‐ separated proteins in the 5‐50 pmol range. Electrophoresis 16:1104‐1114.
   Patterson, S.D. 1997. Identification of low‐ to subpicomolar quantities of electrophoretically separated proteins: Towards protein chemistry in the post ‐ genome era. Biochem. Soc. Trans. 25:255‐262.
   Patterson, S.D. and Aebersold, R. 1995. Mass spectrometric approaches for the identification of gel‐ separated proteins. Electrophoresis 16:1791‐1814.
   Patterson, S.D., Thomas, D., and Bradshaw, R.A. 1996. Application of combined mass spectrometry and partial amino acid sequence to the identification of gel‐ separated proteins. Electrophoresis 17:877‐891.
   Pennington, S.R., Wilkins, M.R., Hochstrasser, D.F., and Dunn, M.J. 1997. Proteome analysis: From protein characterization to biological function. Trends Cell Biol. 7:168‐173.
   Resing, K.A., Johnson, R.S., and Walsh, K.A. 1995. Mass spectrometric analysis of 21 phosphorylation sites in the internal repeat of rat profilaggrin, precursor of an intermediate filament associated protein. Biochemistry 34:9477‐9487.
   Robinson, C.V., Gross, M., Eyles, S.J., Ewbank, J.J., Mayhew, M., Hartl, F.U., Dobson, C.M., and Radford, S.E. 1994. Conformation of GroEL‐bound alpha‐lactalbumin probed by mass spectrometry. Nature 372:646‐651.
   Salmeron, A., Ahmad, T.B., Carlile, G.W., Pappin, D., Narsimhan, R.P., and Ley, S.C. 1996. Activation of MEK‐1 and SEK‐1 by Tpl‐2 proto‐oncoprotein, a novel MAP kinase kinase kinase. EMBO J. 15:817‐826.
   Scheele, G.A. 1975. Two ‐dimensional gel analysis of soluble proteins. Characterization of guinea pig exocrine pancreatic proteins. J. Biol. Chem. 250:5375‐5385.
   Schnolzer, M., Jedrzejewski, P., and Lehmann, W.D. 1996. Protease ‐ catalyzed incorporation of O ‐18 into peptide ‐ fragments and its application for protein sequencing by electrospray and matrix‐assisted laser desorption/ionization mass ‐spectrometry. Electrophoresis 17:945‐953.
   Schreiner, M., Strupat, K., Lottspeich, F., and Eckerskorn, C. 1996. Ultraviolet matrix‐assisted laser‐desorption ionization‐mass spectrometry of electroblotted proteins. Electrophoresis 17:954‐961.
   Schuhmacher, M., Glocker, M.O., Wunderlin, M., and Przybylski, M. 1996. Direct isolation of proteins from sodium dodecyl sulfate‐polyacrylamide gel‐electrophoresis and analysis by electrospray‐ionization mass‐spectrometry. Electrophoresis 17:848‐854.
   Shevchenko, A., Jensen, O.N., Podtelejnikov, A.V., Sagliocco, F., Wilm, M., Vorm, O., Mortensen, P., Shevchenko, A., Boucherie, H., and Mann, M. 1996. Linking genome and proteome by mass spectrometry: Large‐scale identification of yeast proteins from two ‐ dimensional gels. Proc. Natl. Acad. Sci. U.S.A. 93:14440‐14445.
   Shevchenko, A., Chernushevich, I., Ens, W., Standin, K.G., Thomson, B., Wilm, M., and Mann, M. 1997. Rapid “de novo” peptide sequencing by a combination of nanoelectrospray, isotopic labeling and a quadrupole/time ‐ of ‐ flight mass spectrometer. Rapid Commun. Mass Spectrom. 11:1015‐1024.
   Strupat, K., Karas, M., Hillenkamp, F., Eckerskorn, C., and Lottspeich, F. 1994. Matrix ‐ assisted laser desorption ionization mass spectrometry of proteins electroblotted after polyacrylamide gel electrophoresis. Anal. Chem. 66:464‐470.
   Szczepanowska, J., Zhang, X., Herring, C.J., Qin, J., Korn, E.D., and Brzeska, H. 1997. Identification by mass spectrometry of the phosphorylated residue responsible for activation of the catalytic domain of myosin I heavy chain kinase, a member of the PAK/STE20 family. Proc. Natl. Acad. Sci. U.S.A. 94:8503‐8508.
   Takach, E.J., Hines, W.M., Patterson, D.H., Juhasz, P., Falick, A.M., Vestal, M.L., and Martin, S.A. 1997. Accurate mass measurements using MALDI ‐ TOF with delayed extraction. J. Prot. Chem. 16:363‐369.
   Tang, Q., Harrata, A.K., and Lee, C.S. 1997. Two ‐ dimensional analysis of recombinant E. coli proteins using capillary isoelectric focusing electrospray ionization mass spectrometry. Anal. Chem. 69:3177‐3182.
   Tempst, P., Erdjument‐Bromage, H., Casteels, P., Geromanos, S., Lui, M., Powell, M., and Nelson, R.W. 1995. MALDI‐ TOF mass spectrometry in the protein biochemistry lab: From characterization of cell ‐ cycle regulators to the quest for novel antibiotics. In Mass Spectrometry in the Biological Sciences. (A.L. Burlingame, and S.A. Carr, eds.) pp. 105‐133. Humana Press, Totowa, N.J.
   Tu, G.‐F., Reid, G.E., Zhang, J.‐G., Moritz, R.L., and Simpson, R.J. 1995. C‐terminal extension of truncated recombinant proteins in Escherichia coli with a 10S RNA decapeptide. J. Biol. Chem. 270:9322‐9326.
   Vestal, M.L., Juhasz, P., and Martin, S.A. 1995. Delayed extraction matrix‐assisted laser desorption time‐ of‐flight mass spectrometry. Rapid Commun. Mass Spectrom. 9:1044‐1050.
   Vestling, M.M. and Fenselau, C. 1994a. Poly(vinylidene difluoride) membranes as the interface between laser desorption mass spectrometry, gel electrophoresis, and in situ proteolysis. Anal. Chem. 66:471‐477.
   Vestling, M.M. and Fenselau, C.C. 1994b. Protease digestions on PVDF membranes for matrix‐assisted laser desorption mass spectrometry. In Techniques in Protein Chemistry V. (J.W. Crabb, ed.) pp. 59‐67. Academic Press, San Diego.
   Vestling, M.M. and Fenselau, C. 1995. Surfaces for interfacing protein gel electrophoresis directly with mass spectrometry. Mass Spectrom. Rev. 14:169‐178.
   Watts, J.D., Affolter, M., Krebs, D.L., Wange, R.L., Samelson, L.E., and Aebersold, R. 1994. Identification by electrospray ionization mass spectrometry of the sites of tyrosine phosphorylation induced in activated Jurkat T cells on the protein tyrosine kinase ZAP‐70. J. Biol. Chem. 269:29520‐29529.
   Watts, J.D., Krebs, D.L., Wange, R.L., Samelson, L.E., and Aebersold, R. 1996. Electrospray ionization mass spectrometric investigation of signal transduction pathways: Determination of sites of inducible protein phosphorylation in activated T‐cells. In Biochemical and Biotechnological Applications of Electrospray Ionization Mass Spectrometry (A.P. Snyder, ed.) pp. 381‐407. ACS Symposium Series, Washington, D.C.
   Wilkins, M.R., Pasquali, C., Appel, R.D., Ou, K., Golaz, O., Sanchez, J.C., Yan, J.X., Gooley, A.A., Hughes, G., Humphery‐Smith, I., Williams, K.L., and Hochstrasser, D.F. 1996. From proteins to proteomes—Large‐scale protein identification by 2‐dimensional electrophoresis and amino acid analysis. Bio/Technology 14:61‐65.
   Wilm, M.S. and Mann, M. 1994. Electrospray and Taylor‐Cone theory, Dole's beam of macromolecules at last. Int. J. Mass Spectrom. Ion Process. 136:167‐180.
   Yates, J.R. III, Eng, J.K., McCormack, A.L., and Schieltz, D. 1995. Method to correlate tandem mass‐spectra of modified peptides to amino‐acid‐sequences in the protein database. Anal. Chem. 67:1426‐1436.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library