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Antibiotics pp 281-289 | Cite as

Sample Preparation for Mass-Spectrometry Based Absolute Protein Quantification in Antibiotic Stress Research

  • Florian Bonn
  • Sandra Maass
  • Dörte Becher
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1520)

Abstract

Absolute protein quantification is an essential tool for system biology approaches and elucidation of stoichiometry of multi-protein complexes. In this chapter, a universal protocol for gel free absolute protein quantification in bacterial systems is described, which can be used for sample preparation prior to miscellaneous mass-spectrometry-based quantification workflows like AQUA, Hi3, and emPAI. In addition, a focus has been set to the specific challenges in antibiotic stress research.

Key words

Gel free proteomics Sample preparation Absolute protein quantification In-solution digestion AQUA QconCAT Hi3 emPAI Antibiotic stress response 

References

  1. 1.
    Wenzel M, Bandow JE (2011) Proteomic signatures in antibiotic research. Proteomics 11(15):3256–3268CrossRefPubMedGoogle Scholar
  2. 2.
    Vranakis I, Goniotakis I, Psaroulaki A et al (2014) Proteome studies of bacterial antibiotic resistance mechanisms. J Proteomics 97:88–99CrossRefPubMedGoogle Scholar
  3. 3.
    Otto A, Becher D, Schmidt F (2014) Quantitative proteomics in the field of microbiology. Proteomics 14(4-5):547–565CrossRefPubMedGoogle Scholar
  4. 4.
    Maass S, Becher D (2016) Absolute protein quantification in bacterial systems. J Proteomics 136:222–233CrossRefPubMedGoogle Scholar
  5. 5.
    Gerber SA, Rush J, Stemman O et al (2003) Absolute quantification of proteins and phosphoproteins from cell lysates by tandem MS. Proc Natl Acad Sci 100(12):6940–6945CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Pratt JM, Simpson DM, Doherty MK et al (2006) Multiplexed absolute quantification for proteomics using concatenated signature peptides encoded by QconCAT genes. Nat Protoc 1(2):1029–1043CrossRefPubMedGoogle Scholar
  7. 7.
    Maass S, Sievers S, Zühlke D et al (2011) Efficient, global-scale quantification of absolute protein amounts by integration of targeted mass spectrometry and two-dimensional gel-based proteomics. Anal Chem 83(7):2677–2684CrossRefPubMedGoogle Scholar
  8. 8.
    Ishihama Y, Odat Y, Tabata T et al (2005) Exponentially modified protein abundance index (emPAI) for estimation of absolute protein amount in proteomics by the number of sequenced peptides per protein. Mol Cell Proteomics 4(9):1265–1272CrossRefPubMedGoogle Scholar
  9. 10.
    Schwanhäusser B, Busse D, Li N et al (2011) Global quantification of mammalian gene expression control. Nature 473(7347):337–342CrossRefPubMedGoogle Scholar
  10. 11.
    Silva JC, Gorenstein Marc V, Guo-Zhong L et al (2006) Absolute quantification of proteins by LCMSE. Mol Cell Proteomics 5(1):144–156CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2017

Authors and Affiliations

  1. 1.Department for Microbial Proteomics, Institute for MicrobiologyErnst-Moritz-Arndt University of GreifswaldGreifswaldGermany

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