Proteomic Analysis of Pluripotent Stem Cells

Sean C. Bendall1, Aaron T. Booy1, Gilles Lajoie1

1 University of Western Ontario, London, Ontario
Publication Name:  Current Protocols in Stem Cell Biology
Unit Number:  Unit 1B.1
DOI:  10.1002/9780470151808.sc01b01s2
Online Posting Date:  July, 2007
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Abstract

Mass spectrometry (MS)–based proteomics has become one of the most powerful tools for identifying expressed proteins, providing quick insights into molecular and cellular biology. Traditionally, proteins isolated by either one‐ or two‐dimensional gel electrophoresis are digested with a site specific protease. The resulting peptides are subject to one of two forms of analysis: (1) matrix‐assisted laser desorption/ionization time‐of‐flight (MALDI‐TOF) MS, where a “mass fingerprint” of all the peptides in a sample is generated, or (2) electrospray ionization tandem MS (ESI‐MS/MS), where a mass fragmentation spectra is generated for each peptide in a sample. The resulting mass information is then compared to that of a theoretical database created with available genomic sequence information. This unit provides protocols for this type of assessment in embryonic stem cells (ESCs). Curr. Protoc. Stem Cell Biol. 2:1B.1.1‐1B.1.33. © 2007 by John Wiley & Sons, Inc.

Keywords: human embryonic stem cells; proteomics; mass spectrometry; gel electrophoresis; protein digestion; protein sequencing

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Extraction of Protein from hESCs Under Denaturing Conditions
  • Alternate Protocol 1: Extraction of Protein for Two‐Dimensional Gel Electrophoresis
  • Alternate Protocol 2: Extraction of Protein from Whole Cells for Affinity Purification
  • Alternate Protocol 3: Hypotonic Protein Extraction with Cellular Fractionation
  • Basic Protocol 2: Collection of Extracellular Proteins from hESC Culture
  • Basic Protocol 3: Standard Protein Quantification Assay
  • Alternate Protocol 4: Modified Protein Quantification Assay
  • Support Protocol 1: Precipitation of Protein Extracts
  • Basic Protocol 4: Gel Electrophoresis Separation of Protein Extracts
  • Support Protocol 2: Colloidal Coomassie Staining of Polyacrylamide Protein Gels
  • Basic Protocol 5: In‐Gel Trypsin Digestion of Proteins for Identification by Mass Spectrometry
  • Support Protocol 3: Destaining Silver‐Stained Gels
  • Alternate Protocol 5: In‐Solution Trypsin Digestion of Proteins for Identification by Mass Spectrometry
  • Basic Protocol 6: Desalting and Concentration of Proteins/Peptides by Reverse‐Phase Chromatography
  • Basic Protocol 7: Strong Cation Exchange (SCX) Extraction and Fractionation of Samples
  • Reagents and Solutions
  • Commentary
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Extraction of Protein from hESCs Under Denaturing Conditions

  Materials
  • Adherent hESC growing on plates or fresh or frozen (up to 6 months at −30°C) hESC cell pellets washed with PBS (Invitrogen; see Section C in this book for cell culture and preparation information), 106 or more cells per extraction
  • Phosphate‐buffered saline, pH 7.4 (PBS; Invitrogen), 4°C
  • Denaturing cell lysis buffer (see recipe)
  • 1× Laemmli buffer (see recipe)
  • Phosphatase inhibitor cocktail (see recipe), optional
  • Protease inhibitor cocktail (see recipe), optional
  • 20× nuclease cocktail (see recipe), optional
  • 15‐ml centrifuge tube
  • 1‐ml syringe with 22‐G needle, optional
  • Refrigerated centrifuge

Alternate Protocol 1: Extraction of Protein for Two‐Dimensional Gel Electrophoresis

  • Two‐dimensional (2‐D) gel extraction buffer (see recipe)

Alternate Protocol 2: Extraction of Protein from Whole Cells for Affinity Purification

  • Affinity extraction buffer (see recipe)

Alternate Protocol 3: Hypotonic Protein Extraction with Cellular Fractionation

  Materials
  • 107 to 108 fresh hESC growing on tissue culture plates or as a pellet (see Section C in this book for cell culture and preparation information)
  • Phosphate‐buffered saline, pH 7.4 (PBS; Invitrogen), prewarmed to 37°C
  • Hypotonic cell lysis buffer (see recipe)
  • Phosphatase inhibitor cocktail (see recipe)
  • Protease inhibitor cocktail (see recipe)
  • 40‐ml Dounce homogenizer (glass grinder)
  • 15‐ml plastic, conical centrifuge tubes, precoded
  • Temperature‐controlled centrifuge
  • Additional reagents and equipment for concentrating proteins (see protocol 5 or protocol 8) and extracting proteins from nuclei and membrane fractions (see protocol 1 or protocol 2 or protocol 32)

Basic Protocol 2: Collection of Extracellular Proteins from hESC Culture

  Materials
  • Day 4 or 5 hESC on matrigel (see Xu et al., ; Hoffman and Carpenter, ; Wang et al., ), ∼60% to 80% confluent
  • Sterile tissue‐culture phosphate‐buffered saline, pH 7.4 (PBS; Invitrogen), prewarmed to 37°C
  • Protein‐free hESC medium (see recipe), prewarmed to 37°C
  • Denaturing cell lysis buffer (see recipe)
  • 0.22‐µm sterile membrane filter
  • 15‐ml centrifugal filters, 5000 MWCO (e.g., Amicon filters; Millipore)
  • Temperature‐controlled centrifuge
  • Lyophilizer or vacuum concentrator, optional
  • Additional reagents and equipment for culture of hESC (see Section C in this book)

Basic Protocol 3: Standard Protein Quantification Assay

  Materials
  • Protein sample (Basic Protocols protocol 11 and protocol 52; Alternate Protocols protocol 21, protocol 32, and protocol 43)
  • Sample diluent buffer (compatible with sample and assay reagents; e.g., see )
  • 2 mg/ml (or greater) standard protein solution (BSA or IgG)
  • Protein assay reagent (Bio‐Rad)
  • 96‐well polystyrene flat‐bottom microtiter plate
  • 96‐well microtiter plate reader with a 595‐nm filter
  • Graph paper or graphing program

Alternate Protocol 4: Modified Protein Quantification Assay

  • Protein assay resolubilization buffer: 0.1% (w/v) SDS/8 M urea; prepared fresh or stored up to 1 year at −80°C
  • Heating block set to 90°C

Support Protocol 1: Precipitation of Protein Extracts

  • 2% (w/v) deoxycholate (DOC) solution
  • 100% (w/v) trichloroacetic acid (TCA) solution
  • Acetone, cooled to −20°C

Basic Protocol 4: Gel Electrophoresis Separation of Protein Extracts

  Materials
  • IPG strips (24‐cm IPG Immobiline Drystrip pH 3 to 10 NL; GE Healthcare)
  • Mineral oil
  • Ampholytes, pH 3 to 10 (GE Healthcare)
  • 200 µg protein extract ( protocol 2)
  • Two‐dimensional (2‐D) gel rehydration buffer (see recipe) containing protease inhibitors (see recipe)
  • SDS equilibration buffer (see recipe)
  • 1% (w/v) dithiothreitol (DTT) in SDS equilibration buffer (see recipe), freshly prepared
  • 2.5% (w/v) iodoacetamide solution in SDS equilibration buffer (see recipe), freshly prepared
  • Polyacrylamide slab gel (e.g., 25.5 cm × 20.5 cm × 0.15 cm; 10% or 12% (w/v) polyacrylamide recommended for whole‐cell lysate)
  • Protein molecular weight standards for electrophoresis, prestained, broad range (e.g., Bio‐Rad) and recommended loading buffer, optional
  • Agarose sealing solution (see recipe)
  • SDS running buffer (see recipe)
  • Trough for IPG strips
  • Isoelectric focusing apparatus (e.g., Ettan IPGphor manifold and Ettan IPGphor; GE Healthcare)
  • 25‐ml serological pipets with ends cut off, optional
  • Filter paper (absorbant electrophoresis type), optional
  • Polyacrylamide gel electrophoresis apparatus, large format (minimum 25‐cm wide), temperature control recommended (e.g., Bio‐Rad)
  • Microwave or hot water bath

Support Protocol 2: Colloidal Coomassie Staining of Polyacrylamide Protein Gels

  Materials
  • Two‐dimensional gel (2‐D) gel with proteins ( protocol 9)
  • Protein gel fixing solution: 50% (v/v) ethanol 3% (v/v) phosphoric acid
  • Modified Neuhoff's solution (see recipe)
  • Colloidal Coomassie staining solution: modified Neuhoff's solution with 1.2 g/liter of Coomassie G250
  • Container(s) to hold gels
  • Rotary shaker

Basic Protocol 5: In‐Gel Trypsin Digestion of Proteins for Identification by Mass Spectrometry

  Materials
  • Protein sample embedded in polyacrylamide gel ( protocol 9)
  • Acetonitrile/ammonium bicarbonate destaining solution: 1 M ammonium bicarbonate/20% (v/v) acetonitrile
  • Methanol/acetic acid washing solution: 50% (v/v) methanol/5% (v/v) acetic acid
  • Acetonitrile, HPLC grade
  • 10 mM dithiothreitol (DTT), freshly prepared from 1 M stock solution (see recipe)
  • Iodoacetamide
  • 100 mM ammonium bicarbonate
  • 50 mM ammonium bicarbonate, ice‐cold
  • Porcine modified trypsin, sequencing grade (Promega)
  • 10% (v/v) formic acid in deionized (d)H 2O
  • Scalpel
  • 1.5‐ and 0.5‐ml plastic microcentrifuge tubes
  • Centrifuge
  • 37°C water bath or incubator
  • Vacuum concentrator (e.g., SpeedVac)
NOTE: Anytime this protocol mentions the removal of a solution it will be necessary to centrifuge 30 sec at 5000 × g, room temperature, and aspirate the supernatant with a pipet.

Support Protocol 3: Destaining Silver‐Stained Gels

  Materials
  • Silver‐stained sectioned gel pieces ( protocol 11, steps 1 and 2)
  • 30 mM potassium ferricyanide
  • 100 mM sodium thiosulfate
  • Acetonitrile/ammonium bicarbonate destaining solution: 1 M ammonium bicarbonate/20% w/v acetronitrile

Alternate Protocol 5: In‐Solution Trypsin Digestion of Proteins for Identification by Mass Spectrometry

  Materials
  • Protein sample (from protocol 1 or protocol 52 or protocol 2, protocol 32, or protocol 43, extracted using protocol 8)
  • Denaturing cell lysis buffer (see recipe)
  • 50 mM ammonium bicarbonate in dH 2O
  • Dithiothreitol (DTT), solid or 1 M stock solution (see recipe)
  • 100 mM ammonium bicarbonate in deionized (d)H 2O
  • Iodoacetamide, solid
  • Porcine modified trypsin, sequencing grade (Promega)
  • Acetonitrile, HPLC grade
  • Heating block, set at 65°C (optional)
  • 37°C water bath or incubator

Basic Protocol 6: Desalting and Concentration of Proteins/Peptides by Reverse‐Phase Chromatography

  Materials
  • Buffer B: 0.1% (v/v) formic acid in acetonitrile
  • Buffer A: 0.1% (v/v) formic acid in deionized H 2O
  • Sample ( protocol 13)
  • 10% (v/v) formic acid
  • Buffer C: mix Buffer A and Buffer B 1:1
  • Disposable C 18 solid phase extraction (SPE) column, 1 ml capacity ∼100 mg solid phase
  • Vacuum apparatus/manifold
  • 1.5‐ and 0.5‐ml microcentrifuge tube
  • Vacuum centrifuge (e.g., SpeedVac)

Basic Protocol 7: Strong Cation Exchange (SCX) Extraction and Fractionation of Samples

  Materials
  • Mobile phase: 0.1% (v/v) formic acid, 20% (v/v) acetonitrile in H 2O
  • 50 to 500 µg protein sample: total salt concentration (i.e., Na+, K+, NH 4+) <10 mM; diluted with mobile phase or desalted as in protocol 14.
  • KCl stock: 500 mM potassium chloride (KCl)/20% (v/v) acetonitrile/0.1% (v/v) formic acid
  • 0.8 × 50–mm cation exchange column (Bio SCX Series II; Agilent)
  • Analytical HPLC equipment: flow rate of 1 ml/min, minimum 20‐µl sample loop
  • Vacuum centrifuge (e.g., SpeedVac)
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Figures

Videos

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Key References
   Aebersold, R. and Mann, M. 2003. See above.
  General overviews of proteomics. They contain brief descriptions of current applications, popular types of instrumentation, and information on peptide sequencing and identification.
   Domon, B. and Aebersold, R. 2006. See above.
  Basic review of mass spectrometry theory, instrumentation, and application.
   Steen, H. and Mann, M. 2004. See above.
   Glish, G.L. and Vachet, R.W. 2003. See above.
Internet Resources
   http://www.bio‐rad.com/LifeScience/pdf/Bulletin_9004.pdf
  Bio‐Rad Protein assay application manual and compatibility information
   http://www1.amershambiosciences.com/aptrix/upp01077.nsf/Content/2‐D_electrophoresis∼2‐Delectrophoresis_handbook
  GE Healthcare two‐dimensional gel electrophoresis application and troubleshooting guide.
   http://www.bioinfor.com:8080/peaksonline
  Free online search tools for proteomic data.
   http://www.thegpm.org
  Repository of proteomic software and information.
   http://www.matrixscience.com/search_form_select.html
  Tool for parsing and annotating large protein datasets (i.e., convert protein accession numbers to gene accession number, or add gene ontology information to a dataset).
   http://www.proteomecommons.org/
  General proteomic and mass spectrometry information.
   http://www.expasy.org/tools/
   http://tools.proteomecenter.org/software.php
   http://www.biomart.org/
   http://www.ionsource.com/
   http://www.spectroscopynow.com/
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