Quantitative Proteomic Analysis of the Cell Envelopes and Native Membrane Vesicles Derived from Gram‐Negative Bacteria

Ryszard A. Zielke1, Philip R. Gafken2, Aleksandra E. Sikora1

1 Department of Pharmaceutical Sciences, College of Pharmacy, Oregon State University, Corvallis, Oregon, 2 Proteomics Facility, Fred Hutchinson Cancer Research Center, Seattle, Washington
Publication Name:  Current Protocols in Microbiology
Unit Number:  Unit 1F.3
DOI:  10.1002/9780471729259.mc01f03s34
Online Posting Date:  August, 2014
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library


Proteins localized to the cell envelope and naturally released membrane vesicles (MVs) play diverse functions in physiology and pathogenesis of Gram‐negative bacteria. Study of these proteome fractions is essential for better understanding the basic physiological processes, development of vaccines, and identification of potential drug targets. This unit presents gel‐free quantitative proteomic methods for comprehensive proteomic profiling of the cell envelopes and MVs. The procedure starts with the precipitation of the isolated proteome fractions to remove any potential compounds that may interfere with downstream experimental steps. Subsequently, the proteins are reduced, alkylated, and subjected to trypsin digestion. The trypsinized peptides are labeled using isobaric tagging for relative and absolute quantification (iTRAQ), and analyzed samples are pooled and subjected to rigorous prefractionations by strong cation exchange (SCX) and reversed‐phase (RP) liquid chromatography (LC). Finally, the tandem mass spectrometry (MS/MS) fragmentation enables peptides identification and quantification. Curr. Protoc. Microbiol. 34:1F.3.1‐1F.3.16. © 2014 by John Wiley & Sons, Inc.

Keywords: cell envelope; native membrane vesicles; quantitative proteomics; iTRAQ; 2D‐LC‐MS/MS; bioinformatics

PDF or HTML at Wiley Online Library

Table of Contents

  • Introduction
  • Basic Protocol 1: Protein Precipitation
  • Basic Protocol 2: iTRAQ Labeling
  • Basic Protocol 3: Sample Cleanup
  • Basic Protocol 4: Two‐Dimensional HPLC and Mass Spectroscopy
  • Basic Protocol 5: Protein Identification and Bioinformatics
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
PDF or HTML at Wiley Online Library


Basic Protocol 1: Protein Precipitation

  • Cell envelope, membrane vesicle fractions
  • Protein quantification kit (e.g., 2‐D‐Quant Kit, Bio‐Rad DC Protein Assay Kit)
  • 100% acetone
  • Refrigerated microcentrifuge or microcentrifuge placed in a cold room or a fridge
  • 1.5‐ml acetone‐compatible microcentrifuge tubes (e.g., polypropylene or polymethylpentene test tubes)

Basic Protocol 2: iTRAQ Labeling

  • iTRAQ Reagents Mutliplex Kit (AB Sciex)
  • Sequencing Grade Modified Trypsin (Promega)
  • 100% ethanol
  • 0.1% Trifluoroacetic acid (TFA) solution (see recipe)
  • H 2O (HPLC grade)
  • Centrifugal vacuum concentrator
  • Bench top centrifuge

Basic Protocol 3: Sample Cleanup

  • Peptides labeled with iTRAQ reagents reconstituted in 0.1% TFA solution
  • H 2O (HPLC Grade)
  • Acetonitrile
  • 0.1% trifluoroacetic acid (TFA) solution (see recipe)
  • 0.1% trifluoroacetic acid (TFA) in 70% acetonitrile (see recipe)
  • Sep‐Pak C18 columns (Waters)
  • 15‐ml conical tubes
  • 1.5‐ml microcentrifuge tubes

Basic Protocol 4: Two‐Dimensional HPLC and Mass Spectroscopy

  • Buffer SCX A (see recipe)
  • Buffer SCX B (see recipe)
  • Buffer RP A (see recipe)
  • Buffer RP B (see recipe)
  • 0.1% trifluoroacetic acid (TFA) solution (see recipe)
  • Matrix solution (see recipe)
  • Paradigm MG4 high‐pressure liquid chromatography system (Michrom Bioresources) with Varian ProStar fraction collector for strong cation exchange fractionation
  • Strong cation exchange column (2.1 mm × 150 mm Zorbax 300, Agilent)
  • Nano2D LC high‐pressure liquid chromatography system (AB Sciex) coupled to Dionex Probot MALDI spotter (Thermo Fisher Scientific)
  • NanoLC reverse‐phase column (75 μm × 250 mm) packed with Magic C 18AQ (5‐μm 100 Å resin; Michrom Bioresources)
  • Sep‐Pak C18 cartridges (1 cc/50 mg, Waters)
  • Centrifugal vacuum concentrator
  • Chromatography vials
  • 4800 Proteomics Analyzer (TOF/TOF; AB Sciex)
  • Opti‐TOF LC/MALDI blank insert plate (123 × 81 mm, AB Sciex)

Basic Protocol 5: Protein Identification and Bioinformatics

  • ProteinPilot (AB SCIEX)
  • The proteome of the studied organism in FASTA format
PDF or HTML at Wiley Online Library



Literature Cited

Literature Cited
   Bendtsen, J.D. , Nielsen, H. , Widdick, D. , Palmer, T. , and Brunak, S. 2005. Prediction of twin‐arginine signal peptides. BMC Bioinform. 6:167.
   Bos, M.P. , Robert, V. , and Tommassen, J. 2007. Biogenesis of the Gram‐negative bacterial outer membrane. Annu. Rev. Microbiol. 61:191‐214.
   Imai, K. , Asakawa, N. , Tsuji, T. , Akazawa, F. , Ino, A. , Sonoyama, M. , and Mitaku, S. 2008. SOSUI‐GramN: High performance prediction for sub‐cellular localization of proteins in gram‐negative bacteria. Bioinformation 2:417‐421.
   Kulp, A. and Kuehn, M.J. 2010. Biological functions and biogenesis of secreted bacterial outer membrane vesicles. Annu. Rev. Microbiol. 64:163‐184.
   Lee, E.Y. , Choi, D.S. , Kim, K.P. , and Gho, Y.S. 2008. Proteomics in gram‐negative bacterial outer membrane vesicles. Mass Spectrom. Rev. 27:535‐555.
   Molloy, M.P. , Herbert, B.R. , Slade, M.B. , Rabilloud, T. , Nouwens, A.S. , Williams, K.L. , and Gooley, A.A. 2000. Proteomic analysis of the Escherichia coli outer membrane. Eur. J. Biochem. 267:2871‐2881.
   Nouwens, A.S. , Cordwell, S.J. , Larsen, M.R. , Molloy, M.P. , Gillings, M. , Willcox, M.D. , and Walsh, B.J. 2000. Complementing genomics with proteomics: the membrane subproteome of Pseudomonas aeruginosa PAO1. Electrophoresis 21:3797‐3809.
   Ong, S.E. , Blagoev, B. , Kratchmarova, I. , Kristensen, D.B. , Steen, H. , Pandey, A. , and Mann, M. 2002. Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics. Mol. Cell Proteomics 1:376‐386.
   Otto, A. , Becher, D. , and Schmidt, F. 2013. Quantitative Proteomics in the field of microbiology. Proteomics 14:547‐565.
   Petersen, T.N. , Brunak, S. , von Heijne, G. , and Nielsen, H. 2011. SignalP 4.0: Discriminating signal peptides from transmembrane regions. Nat. Methods 8:785‐786.
   Phadke, N.D. , Molloy, M.P. , Steinhoff, S.A. , Ulintz, P.J. , Andrews, P.C. , and Maddock, J.R. 2001. Analysis of the outer membrane proteome of Caulobacter crescentus by two‐dimensional electrophoresis and mass spectrometry. Proteomics 1:705‐720.
   Poetsch, A. and Wolters, D. 2008. Bacterial membrane proteomics. Proteomics 8:4100‐4122.
   Ross, P.L. , Huang, Y.N. , Marchese, J.N. , Williamson, B. , Parker, K. , Hattan, S. , Khainovski, N. , Pillai, S. , Dey, S. , Daniels, S. , Purkayastha, S. , Juhasz, P. , Martin, S. , Bartlet‐Jones, M. , He, F. , Jacobson, A. , and Pappin, D.J. 2004. Multiplexed protein quantitation in Saccharomyces cerevisiae using amine‐reactive isobaric tagging reagents. Mol. Cell. Proteomics 3:1154‐1169.
   Sikora, A.E. , Zielke, R.A. , Lawrence, D.A. , Andrews, P.C. , and Sandkvist, M. 2011. Proteomic analysis of the Vibrio cholerae type II secretome reveals new proteins, including three related serine proteases. J. Biol. Chem. 286:16555‐16566.
   Silhavy, T.J. , Kahne, D. , and Walker, S. 2010. The bacterial cell envelope. Cold Spring Harbor Perspect. Biol. 2:a000414.
   Solis, N. and Cordwell, S.J. 2011. Current methodologies for proteomics of bacterial surface‐exposed and cell envelope proteins. Proteomics 11:3169‐3189.
   Tatusov, R.L. , Koonin, E.V. , and Lipman, D.J. 1997. A genomic perspective on protein families. Science 278:631‐637.
   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.
   Wu, S. , Zhu, Z. , Fu, L. , Niu, B. , and Li, W. 2011. WebMGA: a customizable web server for fast metagenomic sequence analysis. BMC Genomics 12:444.
   Yoo, J.S. , Seong, W.K. , Kim, T.S. , Park, Y.K. , Oh, H.B. , and Yoo, C.K. 2007. Comparative proteome analysis of the outer membrane proteins of in vitro‐induced multi‐drug resistant Neisseria gonorrhoeae . Microbiol. Immunol. 51:1171‐1177.
   Yu, C.S. , Lin, C.J. , and Hwang, J.K. 2004. Predicting subcellular localization of proteins for Gram‐negative bacteria by support vector machines based on n‐peptide compositions. Protein Sci. 13:1402‐1406.
   Yu, C.S. , Chen, Y.C. , Lu, C.H. , and Hwang, J.K. 2006. Prediction of protein subcellular localization. Proteins 64:643‐651.
   Yu, N.Y. , Wagner, J.R. , Laird, M.R. , Melli, G. , Rey, S. , Lo, R. , Dao, P. , Sahinalp, S.C. , Ester, M. , Foster, L.J. , and Brinkman, F.S. 2010. PSORTb 3.0: Improved protein subcellular localization prediction with refined localization subcategories and predictive capabilities for all prokaryotes. Bioinformatics 26:1608‐1615.
   Zielke, R.A. , Wierzbicki, I.H ., Weber, J.V. , Gafken, P.R. , and Sikora, A.E. 2014. Quantitative proteomics of the Neisseria gonorrhoeae cell envelope and membrane vesicles for the discovery of potential therapeutic targets. Mol. Cell. Proteomics 13:1299‐1317.
PDF or HTML at Wiley Online Library