Abstract
In addition to standard gel-based proteomic approaches, gel-free approaches using isobaric label reagents, such as Tandem Mass Tags (TMT), provide a straightforward method for studying adaptations in microbial proteomes to changing environmental conditions. This approach does not have the known difficulties of 2-D gel electrophoresis with proteins of extreme biochemical properties. The workflow described here was designed to study adaptive responses in bacteria and has been applied to study the response of meningococci to iron limitation. The supplemental use of western blotting allows the confirmation of certain changes in protein abundance identified within the TMT study.
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References
Thompson A, Schäfer J, Kuhn K et al (2003) Tandem mass tags: A novel quantification strategy for comparative analysis of complex protein mixtures by MS/MS. Anal Chem 75:1895–1904.
Dayon L, Hainard A, Licker V et al (2008) Relative quantification of proteins in human cerebrospinal fluids by MS/MS using 6-plex isobaric tags. Anal Chem 80:2921–2931.
Ross PL, Huang YN, Marchese JN et al (2004) Multiplexed protein quantitation in Saccharomyces cerevisiae using amine-reactive isobaric tagging reagents. Mol Cell Proteomics 3:1154–1169.
Pettersson A, Poolman JT, van der Ley P et al (1997) Response of Neisseria meningitidis to iron limitation. Antonie van Leeuwenhoek 71:129–136.
van Ulsen P, Kuhn K, Prinz T et al (2009) Identification of proteins of Neisseria meningitidis induced under iron-limiting conditions using the isobaric tandem mass tag (TMT) labeling approach. Proteomics 9:1771–1781.
Byers HL, Campbell J, van Ulsen P et al (2009) Candidate verification of iron-regulated Neisseria meningitidis proteins using isotopic versions of tandem mass tags (TMT) and single reaction monitoring. J Proteomics 73:231–239.
Keller A, Nesvizhskii AI, Kolker E et al (2002) Empirical statistical model to estimate the accuracy of peptide identifications made by MS/MS and database search. Anal Chem 74:5383–5392.
Nesvizhskii AI, Keller A, Kolker E et al (2003) A statistical model for identifying proteins by tandem mass spectrometry. Anal Chem 75:4646–4658.
Tettelin H, Saunders N, Heidelberg J et al (2000) Complete genome sequence of Neisseria meningitidis serogroup B strain MC58. Science 287:1809–1815.
Grifantini R, Sebastian S, Frigimelica E et al (2003) Identification of iron-activated and – repressed Fur-dependent genes by transcriptome analysis of Neisseria meningitidis group B. Proc Natl Acad Sci USA 100:9542–9547.
Fujiki Y, Hubbard AL, Fowler S et al (1982) Isolation of intracellular membranes by means of sodium carbonate treatment: application to endoplasmic reticulum. J Cell Biol 93:97–102.
Fujiki Y, Fowler S, Shio H et al (1982) Polypeptide and phospholipid composition of the membrane of rat liver peroxisomes: comparison with endoplasmic reticulum and mitochondrial membranes. J Cell Biol 93:103–110.
Acknowledgments
Work in the lab of J.T. is supported by the Netherlands Organization for Health Research and Development (ZonMw).
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Kuhn, K., Baumann, C., Tommassen, J., Prinz, T. (2012). TMT Labelling for the Quantitative Analysis of Adaptive Responses in the Meningococcal Proteome. In: Christodoulides, M. (eds) Neisseria meningitidis. Methods in Molecular Biology, vol 799. Humana, Totowa, NJ. https://doi.org/10.1007/978-1-61779-346-2_8
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DOI: https://doi.org/10.1007/978-1-61779-346-2_8
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