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 (NH 4HCO 3)
  • 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 ( 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 protocol 11 and protocol 55)
  • 100 mM and 1 M ammonium bicarbonate (NH 4HCO 3), 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 H 2O
  • Low‐binding, siliconized microcentrifuge tubes (Fisher Scientific)
  • End‐over‐end rotator
  • Additional reagents and equipment for desalting/cleanup prior to MS analysis ( 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 10.11& 10.11)
  • Phosphate buffered saline (PBS) pH 7.4 (see recipe)
  • 0.1 M glycine, pH 2.5
  • 200 mM ammonium bicarbonate (NH 4HCO 3)
  • 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 10.1) and desalting/cleanup prior to MS analysis ( 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)
  • TiO 2 beads (GL Science Inc., http://www.glsciences.com/)
  • StageTips packed with C 8 disks (Proxeon Biosystems, http://www.proxeon.com/)
  • 1% (w/v) sodium dodecyl sulfate (SDS)
  • TiO 2 loading solution (see recipe)
  • TiO 2 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 H 2O
  • 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)
  • C 18 spin column, cartridge, or pipet tip with sufficient binding capacity for peptides in sample
  • Vacuum manifold (optional)
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Figures

Videos

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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