Preparation of Proteins and Peptides for Mass Spectrometry Analysis in a Bottom‐Up Proteomics Workflow

Rebekah L. Gundry1, Melanie Y. White1, Christopher I. Murray1, Lesley A. Kane1, Qin Fu1, Brian A. Stanley1, Jennifer E. Van Eyk1

1 The Johns Hopkins University School of Medicine, Baltimore, Maryland
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 10.25
DOI:  10.1002/0471142727.mb1025s88
Online Posting Date:  October, 2009
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Abstract

This unit outlines the steps required to prepare a sample for MS analysis following protein separation or enrichment by gel electrophoresis, liquid chromatography, and affinity capture within the context of a bottom-up proteomics workflow in which the protein is first broken up into peptides, either by chemical or enzymatic digestion, prior to MS analysis. Also included are protocols for enrichment at the peptide level, including phosphopeptide enrichment and reversed-phase chromatography for sample purification immediately prior to MS analysis. Finally, there is a discussion regarding the types of MS technologies commonly used to analyze proteomics samples, as well as important parameters that should be considered when analyzing the MS data to ensure stringent and robust protein identifications and characterization. Curr. Protoc. Mol. Biol. 88:10.25.1-10.25.23. © 2009 by John Wiley & Sons, Inc.

Keywords: in-solution digestion; in-gel digestion; peptide desalting proteomics; mass spectrometry

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Preparation of Protein from a Polyacrylamide Gel for Mass Spectrometric Analysis
  • Basic Protocol 2: Preparation of Protein from a Liquid Chromatography Fraction for Mass Spectrometric Analysis
  • Basic Protocol 3: Preparation of Protein from Affinity Capture for Mass Spectrometric Analysis
  • Basic Protocol 4: Preparation of Peptide Samples for Mass Spectrometric Analysis Using Phosphopeptide Enrichment Methods
  • Basic Protocol 5: Peptide Sample Cleanup Prior to MS Analysis, MS Instrument Selection, and Database Searching
  • Selecting the Appropriate MS Instrumentation
  • Concepts to Consider when Performing the MS Database Search
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Preparation of Protein from a Polyacrylamide Gel for Mass Spectrometric Analysis

 Materials
  • Polyacrylamide gel containing proteins of interest stained with MS-compatible stain: glutaraldehyde-free silver, traditional Coomassie stain (TCS; UNIT 10.6), or colloidal Coomassie stain (CCS)
  • Gel destain solution (see recipe specific for each type of stained gel: colloidal Coomassie destain, glutaraldehyde-free silver destain, or traditional Coomassie destain)
  • 100% acetonitrile
  • 10 mM dithiothreitol (DTT)
  • 55 mM iodoacetamide (prepare fresh immediately prior to use; protect from light)
  • Gel wash solution (see recipe)
  • Gel enzyme solution (see recipe)
  • 25 mM ammonium bicarbonate (NH4HCO3)
  • Gel extraction solution (see recipe)
  • Formic acid (optional)
  • Gloves
  • Glass plate
  • Light box
  • Sharp cutting tool (e.g., razor, scissors, scalpel)
  • 0.6-ml low-binding, siliconized microcentrifuge tubes (Fisher Scientific)
  • Vacuum centrifuge
  • 55°C water bath
  • Additional reagents and equipment for desalting/cleanup prior to MS analysis (Basic Protocol 5)

NOTE: Wear gloves at all times to prevent contamination from proteins such as keratins.

Basic Protocol 2: Preparation of Protein from a Liquid Chromatography Fraction for Mass Spectrometric Analysis

 Materials
  • RP-HPLC fraction containing protein of interest (see Basic Protocols 1 and 5)
  • 100 mM and 1 M ammonium bicarbonate (NH4HCO3), pH 8.5
  • Narrow-range pH paper
  • 100 mM tris(2-carboxyethyl)phosphine hydrochloride (TCEP) stock, or 100 mM dithiothreitol (DTT) stock (prepare fresh immediately prior to use)
  • 100 mM iodoacetamide stock (prepare fresh immediately prior to use)
  • Proteolytic enzyme (e.g., chymotrypsin, trypsin, LysC, or AspN) or chemical (e.g., CNBr/formic acid)
  • 1.0% (v/v) trifluoroacetic (TFA) acid in H2O
  • Low-binding, siliconized microcentrifuge tubes (Fisher Scientific)
  • End-over-end rotator
  • Additional reagents and equipment for desalting/cleanup prior to MS analysis (Basic Protocol 5)

Basic Protocol 3: Preparation of Protein from Affinity Capture for Mass Spectrometric Analysis

 Materials
  • Affinity matrix with protein(s) of interest bound (see UNITS 10.11A & 10.11B)
  • Phosphate buffered saline (PBS) pH 7.4 (see recipe)
  • 0.1 M glycine, pH 2.5
  • 200 mM ammonium bicarbonate (NH4HCO3)
  • 100 mM tris(2-carboxyethyl)phosphine hydrochloride (TCEP) stock, or 100 mM dithiothreitol (DTT) stock (prepare fresh immediately prior to use)
  • 100 mM iodoacetamide stock (prepare fresh immediately prior to use)
  • Proteolytic enzyme (e.g., trypsin, chymotrypsin, AspN, or LysC) or chemical (CNBr/70% formic acid)
  • 1.0% (v/v) trifluoroacetic acid (TFA)
  • Low-binding, siliconized microcentrifuge tubes (Fisher Scientific)
  • End-over-end rotator
  • Additional reagents and equipment for protein assay (UNIT 10.1A) and desalting/cleanup prior to MS analysis (Basic Protocol 5)

Basic Protocol 4: Preparation of Peptide Samples for Mass Spectrometric Analysis Using Phosphopeptide Enrichment Methods

 Materials
  • IMAC beads (e.g., PHOS-Select Iron Affinity Gel from Sigma)
  • IMAC wash solution (see recipe)
  • Sample containing phosphorylated peptides
  • Low-pH elution solution (see recipe)
  • High-pH elution solution (see recipe)
  • TiO2 beads (GL Science Inc., http://www.glsciences.com/)
  • StageTips packed with C8 disks (Proxeon Biosystems, http://www.proxeon.com/)
  • 1% (w/v) sodium dodecyl sulfate (SDS)
  • TiO2 loading solution (see recipe)
  • TiO2 wash solution (see recipe)
  • Reversed phase resin for processing phosphopeptides (e.g., Oligo R3, Applied Biosystems)
  • 0.1% (v/v) trifluoroacetic acid (TFA)
  • Phosphopeptide RP elution buffer for MALDI-TOF-MS/MS (see recipe)
  • Phosphopeptide RP elution buffer for LC-ESI-MS/MS (see recipe)
  • 100% and 0.1% (v/v) formic acid in H2O
  • 100% and 0.1% (v/v) trifluoroacetic acid (TFA)
  • Low-binding, siliconized microcentrifuge tubes (Fisher Scientific)
  • Orbital shaker
  • Combi-Syringe tips (e.g., Fisher Scientific)
  • Narrow-bore P-20 pipet tips
  • Vacuum centrifuge
  • Sample plate for MALDI

Basic Protocol 5: Peptide Sample Cleanup Prior to MS Analysis, MS Instrument Selection, and Database Searching

 Materials
  • 100% acetonitrile
  • 0.1% (v/v) and 10% (v/v) trifluoroacetic acid (TFA)
  • Sample containing peptides for MS analysis (see previous protocols)
  • RP LC-ESI-MS/MS elution solution (see recipe)
  • RP MALDI-TOF-MS/MS elution solution (see recipe)
  • Low-binding, siliconized microcentrifuge tubes (Fisher Scientific)
  • C18 spin column, cartridge, or pipet tip with sufficient binding capacity for peptides in sample
  • Vacuum manifold (optional)
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Figures

  •  FigureFigure 10.25.1 Bottom-up proteomics workflow. Proteins are enzymatically or chemically cleaved into peptides which are then analyzed in the mass spectrometer. The proteins and peptides for analysis are often derived from separation/purification methods such as gel electrophoresis (UNIT 10.2A), liquid chromatography (UNITS 10.9-10.11A), and affinity chromatography (UNIT 10.11B).
    View Image
  •  FigureFigure 10.25.2 Strategies for the enrichment of phosphopeptides prior to MS analysis. It is possible to perform either IMAC enrichment or TiO2 enrichment alone, or in combination (SIMAC strategy). Following digestion of proteins, enrichment of phosphopeptides using the SIMAC strategy is achieved first by IMAC enrichment. Some phosphorylated peptides may not be captured by IMAC (IMAC unbound) and should therefore be enriched further by TiO2. This sample can be analyzed directly by MS/MS. Singly phosphorylated (singly PO4) peptides are eluted from IMAC (IMAC elution 1), then further enriched by TiO2 and analyzed by MS/MS. Finally, multiply phosphorylated peptides are eluted from IMAC and analyzed directly by MS/MS.
    View Image
  •  FigureFigure 10.25.3 200 µg of whole-tissue extract from mouse myocardium run on pH 4 to 7 two-dimensional gel and visualized with an MS-compatible silver stain. Protein spots that will likely provide reliable protein identification from a single gel (circled), as well as protein spots that will likely need to be pooled from multiple gels (squares), are indicated. Also shown are protein spots (triangles) that may or may not contain sufficient protein for identification, depending upon the MS instrumentation used.
    View Image

Videos

Literature Cited

Literature Cited
    Arrell, D.K., Elliott, S.T., Kane, L.A., Guo, Y., Ko, Y.H., Pedersen, P.L., Robinson, J., Murata, M., Murphy, A.M., Marban, E. and Van Eyk, J.E. 2006. Proteomic analysis of pharmacological preconditioning: Novel protein targets converge to mitochondrial metabolism pathways. Circ. Res. 99:706-714.
    Candiano, G., Bruschi, M., Musante, L., Santucci, L., Ghiggeri, G.M., Carnemolla, B., Orecchia, P., Zardi, L. and Righetti, P.G. 2004. Blue silver: A very sensitive colloidal Coomassie G-250 staining for proteome analysis. Electrophoresis 25:1327-1333.
    Chrambach, A., Reisfeld, R.A., Wyckoff, M., and Zaccari, J. 1967. A procedure for rapid and sensitive staining of protein fractionated by polyacrylamide gel electrophoresis. Anal. Biochem. 20:150-154.
    Gorg, A., Weiss, W., and Dunn, M.J. 2004. Current two-dimensional electrophoresis technology for proteomics. Proteomics 4:3665-3685.
    Laemmli, U.K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680-685.
    Larsen, M.R., Thingholm, T.E., Jensen, O.N., Roepstorff, P., and Jorgensen, T.J. 2005. Highly selective enrichment of phosphorylated peptides from peptide mixtures using titanium dioxide microcolumns. Mol. Cell. Proteomics 4:873-886.
    Macek, B., Mann, M., and Olsen, J.V. 2009. Global and site-specific quantitative phosphoproteomics: Principles and applications. Annu. Rev. Pharmacol. Toxicol. 49:199-221.
    Mikesh, L.M., Ueberheide, B., Chi, A., Coon, J.J., Syka, J.E., Shabanowitz, J., and Hunt, D.F. 2006. The utility of ETD mass spectrometry in proteomic analysis. Biochim. Biophys. Acta 1764:1811-1822.
    Porath, J. 1992. Immobilized metal ion affinity chromatography. Protein Expr. Purif. 3:263-281.
    Schagger, H. and von Jagow, G. 1991. Blue native electrophoresis for isolation of membrane protein complexes in enzymatically active form. Anal. Biochem. 199:223-231.
    Shevchenko, A., Wilm, M., Vorm, O., and Mann, M. 1996. Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels. Anal. Chem. 68:850-858.
    Sleno, L. and Volmer, D.A. 2004. Ion activation methods for tandem mass spectrometry. J. Mass Spectrom. 39:1091-1112.
    Smith, J.C. and Figeys, D. 2008. Recent developments in mass spectrometry-based quantitative phosphoproteomics. Biochem. Cell. Biol. 86:137-148.
    Thingholm, T.E. and Larsen, M.R. 2009. The use of titanium dioxide micro-columns to selectively isolate phosphopeptides from proteolytic digests. Methods Mol. Biol. 527:57-66.
    Thingholm, T.E., Jorgensen, T.J., Jensen, O.N., and Larsen, M.R. 2006. Highly selective enrichment of phosphorylated peptides using titanium dioxide. Nat. Protoc. 1:1929-1935.
    Thingholm, T.E., Jensen, O.N., Robinson, P.J., and Larsen, M.R. 2008. SIMAC (sequential elution from IMAC), a phosphoproteomics strategy for the rapid separation of monophosphorylated from multiply phosphorylated peptides. Mol. Cell. Proteomics 7:661-671.
    Thingholm, T.E., Jensen, O.N., and Larsen, M.R. 2009. Enrichment and separation of mono- and multiply phosphorylated peptides using sequential elution from IMAC prior to mass spectrometric analysis. Methods Mol. Biol. 527:67-78.
    Zubarev, R.A. 2004. Electron-capture dissociation tandem mass spectrometry. Curr. Opin. Biotechnol. 15:12-16.
 Key References
    Thingholm et al., 2008. See above.

This is a landmark paper in which both IMAC and TiO2 are combined in a sequential manner to allow expanded phosphopeptide enrichment.

    Shevchenko et al., 1996. See above.

This is the most commonly used silver staining and peptide extraction method for MS compatible samples.

 Internet Resources
    http://ca.expasy.org/ch2d/

Two-dimensional polyacrylamide gel electrophoresis database.

    http://ca.expasy.org/tools

Database of proteomic analysis tools which include cleavage site predictions, modification predictions, topology prediction, and visualization software, among others.

    http://www.ionsource.com/

Database of useful protocols for preparing proteomic samples, as well as tutorials and principles of chromatography and mass spectrometry.

    http://www.unimod.org

Comprehensive database of protein modifications and their masses for mass spectrometry.

    http://www.cbs.dtu.dk/services/NetPhos/

Predicts sites of phosphorylation on eukaryotic proteins.

    http://www.proteomecenter.org/course.php

Excellent repository of lecture notes for proteomics, mass spectrometry, and bioinformatics courses designed for beginners, taught at the Institute for Systems Biology at the Seattle Proteome Center.

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