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Differential Proteomics Based on 2D-Difference In-Gel Electrophoresis and Tandem Mass Spectrometry for the Elucidation of Biological Processes in Antibiotic-Producer Bacterial Strains

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Metabolic Network Reconstruction and Modeling

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1716))

Abstract

Proteomics based on 2D-Difference In Gel Electrophoresis (2D-DIGE) coupled with mass spectrometry (MS) procedures can be considered a “gold standard” to determine quantitatively and comparatively protein abundances in cell extracts from different biological sources/conditions according to a gel-based approach. In particular, 2D-DIGE is used for protein specie separation, detection, and relative quantification, whenever tandem MS is used to obtain peptide sequence information that is managed according to bioinformatic procedures to identify the differentially represented protein species. The proteomic results consist of a dynamic portray of over- and down-represented protein species that, with the integration of gene ontology resources, allow obtaining a comprehensive understanding of the complex network of molecular signaling, regulatory circuits, and biochemical reactions occurring in cellular contexts. For this reason, proteomics has been widely used for studying molecular physiology of Gram-positive bacterial strains producing bioactive metabolites and belonging to actinomycete family. This highlighted the complex relationships linking overall regulatory processes and metabolic pathways to the biosynthesis of interesting bioactive molecules. In this chapter, we provide a detailed description of the procedures adopted to perform a differential proteomic analysis of the actinomycete Microbispora ATCC-PTA-5024, producing the promising NAI-107 lantibiotic. Although each experimental proteomic procedure has to be optimized to face the specific molecular characteristics of the organism under investigation, the protocols here described have also been used with minor modifications for proteomic studies on other bacterial strains, including the actinomycetes Streptomyces coelicolor, S. ambofaciens, Amycolatopsis balhimycina, and the Gram-negative proteobacteria Klebsiella oxytoca and Pseudoalteromonas haloplanktis.

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Author information

Authors and Affiliations

  1. Laboratory of Molecular Microbiology and Biotechnology, STEBICEF Department, University of Palermo, Palermo, Italy

    Giuseppe Gallo

  2. Proteomic and Mass Spectrometry Laboratory, ISPAAM, National Research Council, Naples, Italy

    Andrea Scaloni

Authors
  1. Giuseppe Gallo
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  2. Andrea Scaloni
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Corresponding author

Correspondence to Giuseppe Gallo .

Editor information

Editors and Affiliations

  1. Department of Biology, University of Florence, Sesto Fiorentino, Florence, Italy

    Marco Fondi

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Gallo, G., Scaloni, A. (2018). Differential Proteomics Based on 2D-Difference In-Gel Electrophoresis and Tandem Mass Spectrometry for the Elucidation of Biological Processes in Antibiotic-Producer Bacterial Strains. In: Fondi, M. (eds) Metabolic Network Reconstruction and Modeling. Methods in Molecular Biology, vol 1716. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7528-0_12

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  • DOI: https://doi.org/10.1007/978-1-4939-7528-0_12

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-7527-3

  • Online ISBN: 978-1-4939-7528-0

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