Sample Preparation and In‐Solution Protease Digestion of Proteins for Chromatography‐Based Proteomic Analysis

Michael P. Washburn1

1 Stowers Institute for Medical Research, Kansas City, Missouri
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 23.6
DOI:  10.1002/0471140864.ps2306s53
Online Posting Date:  August, 2008
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Abstract

The adoption of chromatography‐based proteomics approaches has resulted in the development of new methods for optimal use of these technologies. One such technology, named multidimensional protein identification technology (MudPIT), directly couples liquid chromatography with tandem mass spectrometry. In the MudPIT approach, digested protein samples are directly loaded onto a microcapillary column packed sequentially with reversed‐phase and strong cation exchange resins. Once digested protein samples are loaded onto the column, the column is placed in‐line between a high‐performance liquid chromatography system and an electrospray ionization tandem mass spectrometer (ESI‐MS/MS). The digested protein samples for MudPIT analysis must be directly compatible with ESI‐MS/MS, so the sample preparation and digestion protocols must be optimized for this purpose. The primary objective of all of the protocols in this unit is to yield a final digested protein mixture of less than 300 µl that can then be directly loaded onto a MudPIT column. Curr. Protoc. Protein Sci. 53:23.6.1‐23.6.11. © 2008 by John Wiley & Sons, Inc.

Keywords: multidimensional protein identification technology; MudPIT; proteomics; protein; proteolysis in solution; trypsin digestion

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Preparation of Protein Samples Prior to Enzymatic Digestion and MudPIT Analysis
  • Basic Protocol 2: Enzymatic Digestion of Proteins in Solution for Direct Analysis by MudPIT
  • Basic Protocol 3: Extraction and Digestion of Hydrophobic Proteins with Detergent
  • Commentary
  • Literature Cited
  • Tables
     
 
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Materials

Basic Protocol 1: Preparation of Protein Samples Prior to Enzymatic Digestion and MudPIT Analysis

  Materials
  • Benzonase (Sigma)
  • 10 µg to 500 µg protein sample at 0.1 µg/µl (minimum suggested concentration)
  • 100 mM Tris·Cl, pH 8.5 (see appendix 2E)
  • 6.1 N trichloroacetic acid (TCA, Sigma)
  • Acetone, 4°C
  • 100 mM Tris·Cl, pH 8.5 (see appendix 2E), containing 8 M urea (added just before use)
  • 0.1 M Tris(2‐carboxylethyl)‐phosphine hydrochloride (TCEP): dilute 1 M stock (stored up to 6 months at −20°C) 1/10 with water before use
  • 0.5 M iodoacetamide (IAM)
  • Gel‐loading micropipet tips
  • Refrigerated microcentrifuge (e.g., Beckman Coulter 22R)
  • Speed vacuum system with an SPD SpeedVac, RVT 400 Refrigerated Vapor Trap, VN100 Vapor Net Controller, DVG50 Digital Vacuum Gauge, VPOF110 Oil Filter, and VLP120 ValuPump (ThermoElectron), or equivalent system

Basic Protocol 2: Enzymatic Digestion of Proteins in Solution for Direct Analysis by MudPIT

  Materials
  • 1 µg/µl endoproteinase Lys‐C (sequencing grade, Roche Applied Science) stock solution: prepare an aqueous solution, dispense into aliquots, and store up to 6 months at −20°C
  • Denatured, reduced, and carboxamidomethylated protein samples ( protocol 1, step 9)
  • 100 mM Tris·Cl, pH 7.5 and pH 8.5 (see appendix 2E)
  • 500 mM calcium chloride
  • 1 µg/µl trypsin (modified sequencing grade, Promega): prepare an aqueous solution, dispense into aliquots, and store up to 6 months at −20°C
  • 90% (w/v) formic acid (e.g., Baker)
  • 1 µg/µl elastase (Calbiochem) stock solution in 10 mM Tris·Cl, pH 8.5 (see appendix 2E): dispense into aliquots and store up to 6 months at −20°C
  • Subitlisin A (Calbiochem) stock solution in 10 mM Tris·Cl, pH 8.5 (see appendix 2E): dispense into aliquots and store up to 6 months at −20°C
  • TCA‐precipitated protein ( protocol 1, step 7a)
  • 100 mM sodium carbonate, pH 11.5
  • Urea
  • 0.1 M Tris(2‐carboxylethyl)‐phosphine hydrochloride (TCEP): dilute 1 M stock (stored up to 6 months at −20°C) 1/10 with water before use
  • 0.5 M iodoacetamide (IAM)
  • 0.25 µg/µl proteinase K (recombinant PCR grade, Roche Applied Science): prepare an aqueous solution, dispense into aliquots, and store up to 6 months at −20°C
  • 0.25 µg/µl endoproteinase Asp‐N, sequencing grade (Roche Applied Science) stock solution: prepare an aqueous solution, dispense into aliquots, and store up to 6 months at −20°C
  • 1 µg/µl endoproteinase Glu‐C, sequencing grade (Roche Applied Science) stock solution: prepare an aqueous solution, dispense into aliquots, and store up to 6 months at −20°C
  • 0.1 µg/µl endoproteinase Arg‐C (sequencing grade, Roche Applied Science) stock solution: prepare an aqueous solution, dispense into aliquots, and store up to 6 months at −20°C
  • Activation solution (Roche), reconstituted
  • 1 µg/µl chymotrypsin (sequencing grade, Roche Applied Science) stock solution: prepare an aqueous solution, dispense into aliquots, and store up to 6 months at −20°C
  • EDTA
  • Dithiothreitol (DTT)
  • Hybridizing incubator (e.g., Thermomixer, Eppendorf), recommended

Basic Protocol 3: Extraction and Digestion of Hydrophobic Proteins with Detergent

  Materials
  • Urea
  • n‐dodecyl‐β‐D‐maltoside (Sigma)
  • 100 mM Tris⋅Cl, pH 8.5 (see appendix 2E)
  • Hydrophobic protein sample containing peripheral and/or integral membrane proteins (e.g., a pellet containing membrane proteins)
  • BCA protein assay kit (Pierce)
  • Hydridizing incubator (e.g., Thermomixer, Eppendorf), recommended
  • Additional reagents and equipment for determining protein concentration using BCA assay (unit 3.4)
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Figures

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Literature Cited

Literature Cited
   Florens, L. and Washburn, M.P. 2006. Proteomic analysis by multidimensional protein identification technology. Methods Mol. Biol. 328: 159‐175.
   Fournier, M.L., Gilmore, J.M., Martin‐Brown, S.A., and Washburn, M.P. 2007. Multidimensional separations‐based shotgun proteomics. Chem. Rev. 107: 3654‐3686.
   Garcia, B.A., Pesavento, J.J., Mizzen, C.A., and Kelleher, N.L. 2007. Pervasive combinatorial modification of histone H3 in human cells. Nat. Methods 4: 487‐489.
   Klammer, A.A. and MacCoss, M.J. 2006. Effects of modified digestion schemes on the identification of proteins from complex mixtures. J. Proteome Res. 5: 695‐700.
   Link, A.J., Eng, J., Schieltz, D.M., Carmack, E., Mize, G.J., Morris, D.R., Garvik, B.M., and Yates, J.R., 3rd. 1999. Direct analysis of protein complexes using mass spectrometry. Nat. 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., 3rd. 2002. Shotgun identification of protein modifications from protein complexes and lens tissue. Proc. Natl. Acad. Sci. U.S.A. 99: 7900‐7905.
   McDonald, W.H., Ohi, R., Miyamoto, D.T., Mitchison, T.J., and Yates, J.R. 2002. Comparison of three directly coupled HPLC MS/MS strategies for identification of proteins from complex mixtures: Single‐dimension LCMS/MS, 2‐phase MudPIT, and 3‐phase MudPIT. Int. J. Mass Spectrom. 219: 245‐251.
   Norris, J.L., Porter, N.A., and Caprioli, R.M. 2003. Mass spectrometry of intracellular and membrane proteins using cleavable detergents. Anal. Chem. 75: 6642‐6647.
   Washburn, M.P., Wolters, D., and Yates, J.R., 3rd. 2001. Large‐scale analysis of the yeast proteome by multidimensional protein identification technology. Nat. Biotechnol. 19: 242‐247.
   Wolters, D.A., Washburn, M.P., and Yates, J.R., 3rd. 2001. An automated multidimensional protein identification technology for shotgun proteomics. Anal. Chem. 73: 5683‐5690.
   Wu, C.C., MacCoss, M.J., Howell, K.E., and Yates, J.R., 3rd. 2003. A method for the comprehensive proteomic analysis of membrane proteins. Nat. Biotechnol. 21: 532‐538.
   Yu, Y.Q., Gilar, M., Lee, P.J., Bouvier, E.S., and Gebler, J.C. 2003. Enzyme‐friendly, mass spectrometry‐compatible surfactant for in‐solution enzymatic digestion of proteins. Anal. Chem. 75: 6023‐6028.
   Zybailov, B., Coleman, M.K., Florens, L., and Washburn, M.P. 2005. Correlation of relative abundance ratios derived from peptide ion chromatograms and spectrum counting for quantitative proteomic analysis using stable isotope labeling. Anal. Chem. 77: 6218‐6224.
   Zybailov, B., Mosley, A.L., Sardiu, M.E., Coleman, M.K., Florens, L., and Washburn, M.P. 2006. Statistical analysis of membrane proteome expression changes in Saccharomyces cerevisiae. J. Proteome Res. 5: 2339‐2347.
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