Proteomic Analysis Using 2‐D Liquid Separations of Intact Proteins From Whole‐Cell Lysates

Kan Zhu1, Fang Yan1, Kimberly A. O'Neil1, Rick Hamler1, David M. Lubman1, Linda Lin2, Timothy J. Barder2

1 The University of Michigan, Ann Arbor, Michigan, 2 Eprogen, Inc., Darien, Illinois
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 23.3
DOI:  10.1002/0471140864.ps2303s34
Online Posting Date:  February, 2004
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Abstract

This unit describes procedures for 2‐D liquid separations of proteins from whole‐cell lysates. Protocols for protein isoelectric point (pI) fractionation in the first dimension include the use of liquid isoelectric focusing (IEF) and chromatofocusing. The liquid IEF provides a pI‐based fractionation using a batch‐phase electrophoretic method, while chromatofocusing uses a column‐based chromatographic method to generate the pH gradient. Using either method, a second‐dimension fractionation is provided in the liquid phase using nonporous silica‐based reversed‐phase HPLC (NPS‐RP‐HPLC) to generate a 2‐D liquid map of the protein content of the cell. The eluate of the 2‐D liquid fractionation is directly coupled to a mass spectrometer for on‐line detection of the intact molecular weights of proteins. As a result, a multidimensional map of protein expression is obtained that characterizes cellular proteins by pI, hydrophobicity, and intact molecular weight. Such expression maps are useful for differential proteomic comparison between different cell samples.

Keywords: Rotofor; liquid phase IEF; protein separation; nonporous separations; chromatofocusing

     
 
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Table of Contents

  • Basic Protocol 1: Protein Fractionation by Isoelectric Focusing Using the Rotofor Cell
  • Alternate Protocol 1: Ampholyte‐Free Preparative Isoelectric Focusing over Narrow pH Ranges in the Rotofor Cell
  • Alternate Protocol 2: Isoelectric Focusing Using the Rotofor with Ion‐Exchange Resin as Electrolyte
  • Basic Protocol 2: Protein Fractionation by Chromatofocusing
  • Support Protocol 1: Cell Lysis for Protein Fractionation by Chromatofocusing or Isoelectric Focusing Using the Rotofor
  • Basic Protocol 3: Nonporous Reversed‐Phase Chromatography Interfaced to ESI‐MS
  • Support Protocol 2: Integrating pI, MW, AND Abundance Information into 2‐D Map
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Protein Fractionation by Isoelectric Focusing Using the Rotofor Cell

  Materials
  • Protein sample: see e.g., protocol 5; concentration determined, e.g., by Bradford assay (unit 3.4) or Bio‐Rad RC‐DC Protein Assay Kit
  • Cathode electrolyte: 0.1 M NaOH
  • Anode electrolyte: 0.1 M H 3PO 4
  • Rotofor running buffer (see recipe)
  • Bio‐Lyte 3/10 ampholyte (Bio‐Rad)
  • Rotofor system (Bio‐Rad)
  • Circulating water cooling system
  • Power supply with constant power capability
  • 50‐ml conical centrifuge tube
  • 50‐ml syringe with 19‐G, 1.5‐in. needle
  • Harvesting tubes: 12 × 75‐mm culture tubes
  • Cold trap
  • Vacuum pump
  • pH meter
  • Additional reagents and equipment for protein assay (unit 3.4)

Alternate Protocol 1: Ampholyte‐Free Preparative Isoelectric Focusing over Narrow pH Ranges in the Rotofor Cell

  • Rotolyte buffer‐pair mixtures (Bio‐Rad)
In protocol 1, the pH gradient is maintained by thousands of ampholytes. In this ampholyte‐free method, only one of 13 buffer pairs is used at a time. When voltage is applied, the buffer pair generates a pH gradient between the individual pK a values of the buffers. The simple mixture of the buffer pair cannot provide as wide a linear range as the ampholyte‐based method; the typical pH range used in this method is within 1 to 2 pH units. This is a useful method for concentrating a limited number of proteins over a small pH range, but is not suitable for profiling proteins in a complex biosystem. The method eliminates possible interferences from ampholytes, thereby extending its applicability to protein samples incompatible with ampholytes.The thirteen buffer pairs used in this method are listed in the Bio‐Rad Rotofor manual. This series of buffer pairs cover the pH range from pH 2.9 to 11.0. Prior to the experiment, the pI of the target proteins should be known. For optimum resolution, ratios of the selected buffer pairs can be adjusted so that the pI of the protein to be purified falls near the middle of the established pH gradient.The experimental procedure here is essentially the same as that for the ampholyte‐based method (see protocol 1). However, the running buffer and electrolytes used in the anodic and cathodic cells are different. The running buffer is usually composed of 50% (v/v) water/sample and 50% (v/v) Rotolyte buffer‐pair mixtures. A high concentration of buffer mixtures is important for preventing pH distortion when charged proteins migrate. Anode and cathode electrolytes are categorized in the Bio‐Rad Rotolyte instruction manual. Appropriate electrolytes should be used to minimize the pH distortion at the acidic and basic ends.

Alternate Protocol 2: Isoelectric Focusing Using the Rotofor with Ion‐Exchange Resin as Electrolyte

  • AG50W‐X8 cation exchanger (hydrogen form; Bio‐Rad)
  • AG1‐X8 anion exchange resin (hydroxide form; Bio‐Rad)
One of the limitations of the Rotofor is the pH gradient distortion at the acidic and basic ends. During electrophoresis, ampholytes and proteins migrate and focus at their pI points in the chamber; however, in the anode and cathode cells, gas generated during electrolysis pushes acid and base through the ion‐exchange membranes and into the focusing chamber. Using ion‐exchange resins in both electrodes where free hydronium and hydroxide ions are in equilibrium with ions attached to the resin can alleviate this problem. The result is that a limited number of free ions are pushed through the membrane and the pH gradient distortion is minimized.In this alternative procedure, the focusing experiment is conducted in the same manner as described in protocol 1; however, instead of loading 0.1 M phosphoric acid in the anode and 0.1 M sodium hydroxide in the cathode, AG50W‐X8 cation exchanger (hydrogen form) and AG1‐X8 anion exchange resin (hydroxide form) are applied to the anode and cathode, respectively. Prior to the experiment, the resin should be washed with deionized water three times, after which it should be resuspended in deionized water for a volume of ∼20 ml, drawn up into a 50‐ml syringe without a needle, and injected into the electode cell until the cell is ∼80% full. When using resins in the electrode chambers, Rotofor running times can be greatly increased as compared to the standard operating conditions.

Basic Protocol 2: Protein Fractionation by Chromatofocusing

  Materials
  • Protein sample: see, e.g., protocol 5; concentration determined, e.g., by Bradford assay unit 3.4 or Bio‐Rad RC‐DC Protein Assay kit
  • CF start buffer (see recipe for pH ranges 8.5 to 4 or 7 to 4)
  • CF elution buffer (see recipe for pH ranges 8.5 to 4 or 7 to 4)
  • 1 M NaCl
  • Desalting column: PD‐10 (Sephadex G‐25; Amersham Pharmacia Biotech)
  • NPS‐RP‐HPLC column (recommended): 33 × 4.6–mm Micra Platinum column (Eprogen)
  • HPLC/FPLC system running at ambient temperature and at a flow rate of 0.2 ml/min, with fraction collector, UV detector (280 nm), pH electrode, flow cell, and flow meter
  • Chromatofocusing (CF) column: HPCF 1‐D column (Eprogen)
  • Additional reagents and equipment for desalting and buffer exchange by column chromatography (unit 8.3)
NOTE: The CF buffers (see Reagents and Solutions for recipes) bracket the pH range over which the separation is to be performed. CF Buffer solutions are also commercially available through Eprogen as part of the 2‐D Proteosep kit. It is preferable to use the premixed buffers to ensure reproducible results.

Support Protocol 1: Cell Lysis for Protein Fractionation by Chromatofocusing or Isoelectric Focusing Using the Rotofor

  Materials
  • Cells for analysis
  • 50 mM Tris⋅Cl, pH 7.8 to 8.2 ( appendix 2E; any pH between these values can be used) containing 1 mM PMSF (add from 0.5 M stock in DMSO or isopropanol; see note below)
  • General lysis buffer (see recipe)
  • Membrane lysis buffers A and B (see reciperecipes)
  • 0.1 M sodium carbonate (ice cold)
  • 50 mM Tris⋅Cl, pH 7.3 ( appendix 2E) containing 1 mM PMSF (add from 0.5 M stock in DMSO or isopropanol; see note below)
  • Ultrasonicator with microtip probe (e.g., Branson; for bacterial lysis)
  • Bath sonicator (for membrane lysis)
  • Centrifuge (refrigerated centrifuge capable of 20,000 × g for general lysis procedure; ultracentrifuge capable of 150,000 × g for membrane lysis procedure)
  • Dry ice/acetone bath
NOTE: PMSF has a half‐life in aqueous solution of ∼1 hr. Therefore, a stock solution of 0.5 M PMSF should be made in DMSO or isopropanol and should be added to the aqueous lysis solutions (50 mM Tris⋅Cl and membrane lysis buffer A) to a final concentration of 0.5 mM (i.e., half the original concentration of 1 mM) every hour. The DMSO or isopropanol stock can be stored at 4°C for up to 1 month.CAUTION: Be aware that PMSF is highly toxic and solutions containing this compound must be handled with caution at all times.

Basic Protocol 3: Nonporous Reversed‐Phase Chromatography Interfaced to ESI‐MS

  Materials
  • Personal computer (speed >200 MHz)
  • MassLynx and MaxEnt (Micromass)
  • MSCombine (S. Parus, University of Michigan)
  • ProteoVue (Eprogen, Inc.)
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Figures

Videos

Literature Cited

Literature Cited
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