Membrane Proteomics in Gram-Positive Bacteria: Two Complementary Approaches to Target the Hydrophobic Species of Proteins

  • Susanne SieversEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1841)


This protocol represents a detailed instruction how to prepare protein samples in order to raise mass spectrometry-based identification and quantification rates with respect to the challenging class of membrane proteins. This will increase comprehensiveness of global proteome studies on the one hand but could also be of interest for researchers targeting specific membrane proteins or membrane protein sequences on the other hand. The protocol is a composite of two parts, one focusing on the identification of protein sequences exterior to a cellular membrane (loops of integral membrane proteins, peripheral membrane proteins), and the other part targeting primarily protein domains spanning the lipid bilayer. The feasibility of the protocol, as it is described here, was originally shown for the gram-positive pathogenic bacterium Staphylococcus aureus but should be applicable to any kind of membrane protein.

Key words

Integral membrane proteins Membrane-spanning domains Membrane protein loops Membrane shaving Proteinase K cleavage Chymotryptic digestion 



This work was supported by the German Research Foundation Grant SFB/TR34.


  1. 1.
    Fagerberg L, Jonasson K, Heijne v G et al (2010) Prediction of the human membrane proteome. Proteomics 10:1141–1149CrossRefGoogle Scholar
  2. 2.
    Blonder J, Goshe MB, Moore RJ et al (2002) Enrichment of integral membrane proteins for proteomic analysis using liquid chromatography-tandem mass spectrometry. J Proteome Res 1:351–360CrossRefPubMedGoogle Scholar
  3. 3.
    Fischer F, Wolters D, Rögner M, Poetsch A (2006) Toward the complete membrane proteome: high coverage of integral membrane proteins through transmembrane peptide detection. Mol Cell Proteomics 5:444–453CrossRefPubMedGoogle Scholar
  4. 4.
    Gilmore JM, Washburn MP (2010) Advances in shotgun proteomics and the analysis of membrane proteomes. J Proteome 73:2078–2091CrossRefGoogle Scholar
  5. 5.
    Helbig AO, Heck AJ, Slijper M (2010) Exploring the membrane proteome--challenges and analytical strategies. J Proteome 73:868–878CrossRefGoogle Scholar
  6. 6.
    Speers AE, Wu CC (2007) Proteomics of integral membrane proteins--theory and application. Chem Rev 107:3687–3714CrossRefPubMedGoogle Scholar
  7. 7.
    Cech NB, Enke CG (2000) Relating electrospray ionization response to nonpolar character of small peptides. Anal Chem 72:2717–2723CrossRefPubMedGoogle Scholar
  8. 8.
    Tabb DL, Huang Y, Wysocki VH, Yates JR 3rd (2004) Influence of basic residue content on fragment ion peak intensities in low-energy collision-induced dissociation spectra of peptides. Anal Chem 76:1243–1248CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Fujiki Y, Hubbard AL, Fowler S, Lazarow PB (1982) Isolation of intracellular membranes by means of sodium carbonate treatment: application to endoplasmic reticulum. J Cell Biol 93:97–102CrossRefPubMedGoogle Scholar
  10. 10.
    Eymann C, Dreisbach A, Albrecht D et al (2004) A comprehensive proteome map of growing Bacillus subtilis cells. Proteomics 4:2849–2876CrossRefPubMedGoogle Scholar
  11. 11.
    Speers AE, Blackler AR, Wu CC (2007) Shotgun analysis of integral membrane proteins facilitated by elevated temperature. Anal Chem 79:4613–4620CrossRefPubMedGoogle Scholar
  12. 12.
    Wolff S, Hahne H, Hecker M, Becher D (2008) Complementary analysis of the vegetative membrane proteome of the human pathogen Staphylococcus aureus. Mol Cell Proteomics 7:1460–1468CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Yu Y, Gilar M, Lee PJ et al (2003) Enzyme-friendly, mass spectrometry-compatible surfactant for in-solution enzymatic digestion of proteins. Anal Chem 75:6023–6028CrossRefPubMedGoogle Scholar
  14. 14.
    Masuda T, Tomita M, Ishihama Y (2008) Phase transfer surfactant-aided trypsin digestion for membrane proteome analysis. J Proteome Res 7:731–740CrossRefPubMedGoogle Scholar
  15. 15.
    Masuda T, Saito N, Tomita M, Ishihama Y (2009) Unbiased quantitation of Escherichia coli membrane proteome using phase transfer surfactants. Mol Cell Proteomics 8:2770–2777CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Blackler AR, Speers AE, Ladinsky MS, Wu CC (2008) A shotgun proteomic method for the identification of membrane-embedded proteins and peptides. J Proteome Res 7:3028–3034CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Whitelegge JP (2013) Integral membrane proteins and bilayer proteomics. Anal Chem 85:2558–2568CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Taylor SW, Fahy E, Zhang B et al (2003) Characterization of the human heart mitochondrial proteome. Nat Biotechnol 21:281–286CrossRefPubMedGoogle Scholar
  19. 19.
    Graham RL, Pollock CE, O'Loughlin SN et al (2007) Multidimensional analysis of the insoluble sub-proteome of Oceanobacillus iheyensis HTE831, an alkaliphilic and halotolerant deep-sea bacterium isolated from the Iheya ridge. Proteomics 7:82–91CrossRefPubMedGoogle Scholar
  20. 20.
    Candiano G, Bruschi M, Musante L et al (2004) Blue silver: a very sensitive colloidal Coomassie G-250 staining for proteome analysis. Electrophoresis 25:1327–1333CrossRefPubMedGoogle Scholar
  21. 21.
    Lassek C, Burghartz M, Chaves-Moreno D et al (2015) A metaproteomics approach to elucidate host and pathogen protein expression during catheter-associated urinary tract infections. Mol Cell Proteomics 14:989–1008CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    Schook W, Puszkin S, Bloom W et al (1979) Mechanochemical properties of brain clathrin: interactions with actin and alpha-actinin and polymerization into basketlike structures or filaments. Proc Natl Acad Sci U S A 76:116–120CrossRefPubMedPubMedCentralGoogle Scholar
  23. 23.
    Okamoto T, Schwab RB, Scherer PE, Lisanti MP (2001) Analysis of the association of proteins with membranes. In: Current Protocols in Cell Biology. John Wiley & Sons, Inc., Hoboken., Chapter 5, unit 5.4, pp 1–17Google Scholar
  24. 24.
    Rombouts I, Lagrain B, Brunnbauer M et al (2013) Improved identification of wheat gluten proteins through alkylation of cysteine residues and peptide-based mass spectrometry. Sci Rep 3:2279CrossRefPubMedPubMedCentralGoogle Scholar

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© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Department of Microbial Physiology and Molecular Biology, Institute for MicrobiologyUniversity of GreifswaldGreifswaldGermany

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