Protein Profiling by Microscale Solution Isoelectrofocusing (MicroSol‐IEF)

Xun Zuo1, KiBoem Lee1, Nadeem Ali‐Khan1, David W. Speicher1

1 The Wistar Institute, Philadelphia, Pennsylvania
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 22.6
DOI:  10.1002/0471140864.ps2206s39
Online Posting Date:  March, 2005
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Abstract

Sample prefractionation is essential for more comprehensive coverage and reliable detection of low‐abundance proteins in complex proteomes. An efficient and reproducible new method for sample prefractionation is microscale solution isoelectrofocusing (MicroSol‐IEF), in which samples are separated into chambers defined by membranes of specific pH, yielding well resolved fractions on the basis of isoelectric point (pI). The output seamlessly interfaces with narrow‐pH‐range 2‐D gels, enhancing data obtained from protein profiling studies, including quantitative proteome comparisons. This unit presents the MicroSol‐IEF method using the ZOOM IEF Fractionator with either commercially available or custom‐made pH partition membranes. Alternative configurations are possible for separating samples into different numbers of fractions with various pH ranges and volumes. A detailed method is provided for preparing custom pH membranes. In addition, methods are provided for evaluating the effectiveness of the prefractionation, using 1‐D and 2‐D gel electrophoresis. Approaches for quantitative protein profiling that incorporate MicroSol‐IEF are also discussed.

     
 
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Table of Contents

  • Strategic Planning
  • Basic Protocol 1: Prefractionation of Proteomes into Five Fractions using the ZOOM IEF Fractionator
  • Alternate Protocol 1: Separating Proteomes into Seven Fractions using Custom‐Made Partition Membranes
  • Support Protocol 1: Preparation of an Acrylamide/Immobiline Partition Membrane at a Specific pH
  • Support Protocol 2: Evaluation of the Effectiveness of ZOOM IEF Prefractionation using 1‐D Minigels
  • Support Protocol 3: Evaluation of the Effectiveness of MicroSol‐IEF Prefractionation using 2‐D Gels
  • Basic Protocol 2: Detection of Fractionated Proteins using High‐Resolution, Narrow‐pH‐Range 2‐D Gels
  • Support Protocol 4: Detection of Large Fractionated Proteins using High Resolution, Large‐Pore 1‐D Gels
  • Support Protocol 5: Combination of ZOOM IEF with Two‐Dimensional Differential Gel Electrophoresis (2‐D DIGE) for Quantitative Comparison of Proteins
  • Alternate Protocol 2: Analysis of Fractionated Proteins using LC‐MS/MS Methods
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Prefractionation of Proteomes into Five Fractions using the ZOOM IEF Fractionator

  Materials
  • 1× Microsol‐IEF anode buffer (see recipe)
  • 1× Microsol‐IEF cathode buffer (see recipe)
  • Protein sample (e.g., cell extract)
  • MicroSol‐IEF sample buffer (see recipe)
  • ZOOM IEF Fractionator (Invitrogen; Fig. )
  • pH partition membranes (ZOOM pH Disks; Invitrogen): six different pH membranes (pH 3.0, 4.6, 5.4, 6.2, 7.0, and 10.0)
  • 0.22‐µm Ultrafree‐MC micro‐filter (Millipore)
  • Power supply capable of operating at low currents (0.1 to 1 mA) and at least 1000 V
  • 24‐well tissue culture plates (Becton Dickinson Labware)

Alternate Protocol 1: Separating Proteomes into Seven Fractions using Custom‐Made Partition Membranes

  • Eight custom‐made pH partition membranes (see protocol 3)

Support Protocol 1: Preparation of an Acrylamide/Immobiline Partition Membrane at a Specific pH

  Materials
  • 0.2 M Immobiline stock solutions of desired pH ranges (Amersham Biosciences)
  • 1 M Tris base
  • 1 M acetic acid
  • 30%T/10%C acrylamide/bisacrylamide stock (see recipe)
  • 87% (v/v) glycerol (Amersham Biosciences)
  • 40% ammonium persulfate (Bio‐Rad), prepare immediately before use
  • TEMED (Bio‐Rad)
  • 12% (v/v) glycerol
  • Partition membrane storage solution (see recipe)
  • 15‐ml conical centrifuge tubes
  • Doctor pH software (Amersham Biosciences)
  • Gel support films (Bio‐Rad Laboratories)
  • ∼ 10.4 × 12.6–cm lid from box of 1‐ml pipet tips (e.g., Fisher)
  • Repel‐Silane (Amersham Biosciences)
  • 1.5‐mm slab gel spacers (Bio‐Rad)
  • Precut oval disks of hydrophilic porous polyethylene, 0.67 mm thick (Invitrogen)
  • Sharp scalpel or razor blade
  • 24‐well tissue culture plates (Becton Dickinson Labware)
  • Platform shaker
  • Sealing tape (Fisher)

Support Protocol 2: Evaluation of the Effectiveness of ZOOM IEF Prefractionation using 1‐D Minigels

  Materials
  • Protein fractions from ZOOM IEF fractionator (see protocol 1, steps and )
  • Protein extracts from pH partition membranes (see protocol 1, step )
  • Unfractionated protein sample (e.g., cell extract) that was the starting material for the fractionation in protocol 1
  • SDS sample buffer (unit 10.1)
  • NuPAGE 10% Bis‐Tris gels (Invitrogen)
  • Molecular weight markers (Invitrogen)
  • 20× NuPAGE MES SDS Running Buffer (Invitrogen)
  • Novex Mini‐Cell electrophoresis device (Invitrogen)
  • Gel‐image analysis software (e.g., Discovery Series Quantity One, version 4.2.0)
  • Additional reagents and equipment for 1‐D SDS‐PAGE (unit 10.1) and staining of gels (unit 10.5)

Support Protocol 3: Evaluation of the Effectiveness of MicroSol‐IEF Prefractionation using 2‐D Gels

  Materials
  • Protein fractions from ZOOM IEF fractionator (see protocol 1, steps and )
  • IPG buffers (Amersham Biosciences): pH ranges of the buffers are chosen in accordance with the IPG strips
  • Reducing buffer (see recipe)
  • Alkylating buffer (see recipe)
  • IPG strips: mid‐pH‐range (3.0 pH units wide where possible), either 7 cm (Invitrogen) or 11 cm (from Bio‐Rad or Amersham Biosciences)
  • Second‐dimension gels (also see unit 10.4): 10% SDS‐polyacrylamide gels, either mini‐size (7 × 7 cm) pre‐cast NuPAGE gels (Invitrogen) or medium‐size (11 × 9 cm, Bio‐Rad Criterion gel format)
  • Gel‐image analysis software (e.g., Melanie 2‐D Gel Analysis, version 4.0)
  • Additional reagents and equipment for 2‐D electrophoresis using immobilized pH gradient gel strips (unit 10.4, protocol 6) and staining of gels (unit 10.5)

Basic Protocol 2: Detection of Fractionated Proteins using High‐Resolution, Narrow‐pH‐Range 2‐D Gels

  Materials
  • Protein fractions from ZOOM IEF fractionator (see protocol 1, steps and )
  • MicroSol‐IEF sample buffer (see recipe)
  • IPG buffers (Amersham Biosciences): pH ranges chosen in accordance with the IPG strips used
  • Reducing buffer (see recipe)
  • Alkylating buffer (see recipe)
  • Commercial 24‐cm IPG strips: pH 3 to 6 and pH 7 and 10 strips (Bio‐Rad) or pH 4.0 to 5.0, 4.5 to 5.5, 5.0 to 6.0, 5.5 to 6.7, and 6 to 9 strips (Amersham Biosciences)
  • Second‐dimension gels (also see unit 10.4): large‐size (18 × 19 cm) 10% SDS‐polyacrylamide gels, custom‐made using the Bio‐Rad Protean II format
  • Gel‐image analysis software (e.g., Melanie 2‐D Gel Analysis, version 4.0)
  • Additional reagents and equipment for 2‐D electrophoresis using immobilized pH gradient gel strips (unit 10.4) and staining of gels (unit 10.5)

Support Protocol 4: Detection of Large Fractionated Proteins using High Resolution, Large‐Pore 1‐D Gels

  Materials
  • Protein fractions from ZOOM IEF fractionator (see protocol 1, steps and )
  • Unfractionated protein sample (e.g., cell extract) that was the starting material for the fractionation in protocol 1
  • SDS sample buffer (unit 10.1)
  • NuPAGE 3% to 8% Tris‐Acetate gels (Invitrogen).
  • 20× Novex Tris‐Acetate SDS Running Buffer (Invitrogen)
  • Novex Mini‐Cell electrophoresis device (Invitrogen)
  • Gel‐image analysis software (e.g., Discovery Series Quantity One, version 4.2.0)
  • Additional reagents and equipment for1‐D SDS‐PAGE (unit 10.1) and staining of gels (unit 10.5)

Support Protocol 5: Combination of ZOOM IEF with Two‐Dimensional Differential Gel Electrophoresis (2‐D DIGE) for Quantitative Comparison of Proteins

  Materials
  • Protein samples
  • Additional materials and equipment for labeling proteins as for 2‐D DIGE (unit 22.2, Basic Protocol), protein fractionation using the ZOOM IEF Fractionator (see protocol 1 or protocol 2 in this unit), and detection of fractionated proteins using high‐resolution, narrow‐pH‐range 2‐D gels (see protocol 6 in this unit) or high‐resolution, large‐pore 1‐D gels (see protocol 7 in this unit)

Alternate Protocol 2: Analysis of Fractionated Proteins using LC‐MS/MS Methods

  Materials
  • Protein samples
  • ICAT reagents (Applied Biosystems)
  • 10% SDS polyacrylamide gel (unit 10.1)
  • Additional reagents and equipment for ICAT (unit 23.2, Basic Protocol 1), protein fractionation using the ZOOM IEF Fractionator (see protocol 1 in this unit), SDS‐PAGE (unit 10.1), and LC‐MS/MS (unit 23.1)
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Figures

Videos

Literature Cited

   Giaffreda, E., Tonani, C., and Righetti, P.G. 1993. A pH gradient simulator for electrophoretic techniques in a Windows environment. J. Chromatogr. 630:313‐327.
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   Gygi, S.P., Rist, B., Griffin, T.J., Eng, J., and Aebersold, R. 2002. Proteome analysis of low‐abundance proteins using multidimensional chromatography and isotope‐coded affinity tags. J. Proteome Res. 1:47‐54.
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   Pharmacia Biotech. 1997. Isoelectric membrane formulas for IsoPrime Purification of proteins. IsoPrime Protocol Guide #1. Pharmacia Biotech, Uppsala, Sweden.
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   Rabilloud, T. 1999. Solubilization of proteins in 2‐D electrophoresis: An outline. Methods Mol. Biol. 112:9‐19.
   Rabilloud, T., Adessi, C., Giraudel, A., and Lunardi, J. 1997. Improvement of the solubilization of proteins in two‐dimensional electrophoresis with immobilized pH gradients. Electrophoresis 18:307‐316.
   Ranish, J.A., Yi, E.C., Leslie, D.M., Purvine, S.O., Goodlett, D.R., Eng, J., and Aebersold, R. 2003. The study of macromolecular complexes by quantitative proteomics. Nat. Genet. 33:349‐355.
   Righetti, P.G. 1983. Isoelectric focusing: Theory, Methodology and Application. Elsevier, Amsterdam.
   Righetti, P.G., Wenisch, E., and Faupel, M. 1989. Preparative protein purification in a multi‐compartment electrolyser with immobiline membranes. J. Chromatogr. 475:293‐309.
   Righetti, P.G., Wenisch, E., Jungbauer, A., Katinger, H., and Faupel, M. 1990. Preparative purification of human monoclonal antibody isoforms in a multi‐compartment electrolyser with immobiline membranes. J. Chromatogr. 500:681‐696.
   Righetti, P.G., Castagna, A., and Herbert, B. 2001. Prefractionation techniques in proteome analysis: A new approach identifies more low‐abundance proteins. Anal. Chem. 73:320‐326.
   Righetti, P.G., Castagna, A.C., Herbert, B.H., Reymond, F.R., and Rossier, J.S. 2003. Prefractionation techniques in proteome analysis. Proteomics 3:1397‐1407.
   Speicher, D.W., Lee, K., Tang, H.Y., Echan, L., Ali‐Khan, N., Zuo, X., and Hembach, P. 2004. Current challenges in proteomics: Mining low abundance proteins and expanding protein profiling capacities. In Proceedings of the 16th International Mass Spectrometry Conference (A.E. Ashcroft, A.G. Brenton, and J.J. Monaghan, eds). 16:37‐57.
   Unlu, M., Morgan, M.E., and Minden, J.S. 1997. Difference gel electrophoresis: A single gel method for detecting changes in protein extracts. Electrophoresis 18:2071‐2077.
   Wall, D.B., Kachman, M.T., Gong, S., Hinderer, R., Parus, S., Misek, D.E., Hanash, S.M., and Lubman, D.M. 2000. Isoelectric focusing nonporous RP HPLC: A two‐dimensional liquid‐phase separation method for mapping of cellular proteins with identification using MALDI‐TOF mass spectrometry. Anal. Chem. 72:1099‐1111.
   Weber, G. and Bocek, P. 1998. Recent developments in preparative free flow isoelectric focusing. Electrophoresis 19:1649‐1653.
   Zuo, X. and Speicher, D.W. 2000. A method for global analysis of complex proteomes using sample prefractionation by solution isofocusing prior to two‐dimensional electrophoresis. Anal. Biochem. 284:266‐278.
   Zuo, X. and Speicher, D.W. 2002. Comprehensive analysis of complex proteomes using microscale solution isoelectrofocusing prior to narrow pH range two‐dimensional electrophoresis. Proteomics 2:58‐68.
   Zuo, X., Echan, L., Hembach, P., Tang, H.Y., Speicher, K.D., Santoli, D., and Speicher, D.W. 2001. Towards global analysis of mammalian proteomes using sample prefractionation prior to narrow pH range two‐dimensional gels and using one‐dimensional gels for insoluble and large proteins. Electrophoresis 22:1603‐1615.
   Zuo, X., Hembach, P., Echan, L., and Speicher, D.W. 2002. Enhanced analysis of human breast cancer proteomes using micro‐scale solution isoelectrofocusing combined with high resolution 1‐D and 2‐D gels. J. Chromatogr. B 782:253‐265.
   Zuo, X., Lee, K., and Speicher, D.W. 2004. Electrophoretic prefractionation of proteomes for comprehensive analysis of proteomes. In Proteome Analysis: Interpreting the Genome (D.W. Speicher, ed.), pp. 93‐118. Elsevier Science Inc., New York.
Key References
   Zuo and Speicher, 2000. See above.
  The first publication for development of proteome prefractionation using microscale solution IEF.
   Zuo and Speicher, 2002. See above.
  Demonstrated the enhanced protein profiling of mouse serum by combining narrow pH range 2‐D gels and prefractionation by microscale solution IEF.
   Zuo et al., 2002. See above.
  The first publication to demonstrate the combination of microscale solution IEF and downstream high resolution 1‐D and 2‐D gels for improvement of breast cancer proteome analyses.
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