Environmental Proteomics: Extraction and Identification of Protein in Soil

Solaiman, Zakaria; Kashem, Mohammed A.; Matsumoto, Izuru

doi:10.1007/978-3-540-70865-0_10

Cited by 2 publications

(2 citation statements)

References 10 publications

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“…Proteins released in soil can potentially be absorbed to soil particles and soil organic matter, thus hampering their recovery. Furthermore, humic materials that are normally found in soil have been shown to degrade proteins, and may cause peptide signal suppression in LC−MS measurements. To achieve success in application of metaproteomics to soil microbes, it is imperative to have a method that can effectively lyse the microbial cells directly in the soil matrix and also quantitatively extract the proteins from the sample without significant losses or contaminants that interfere with downstream processing and analysis of the sample …”

Section: Introductionmentioning

confidence: 99%

Direct Cellular Lysis/Protein Extraction Protocol for Soil Metaproteomics

et al. 2010

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We present a novel direct protocol for deep proteome characterization of microorganisms in soil. The method employs thermally assisted detergent-based cellular lysis (SDS) of soil samples, followed by TCA precipitation for proteome extraction/cleanup prior to liquid chromatography-mass spectrometric characterization. This approach was developed and optimized using different soils inoculated with genome-sequenced bacteria (Gram-negative Pseudomonas putida or Gram-positive Arthrobacter chlorophenolicus). Direct soil protein extraction was compared to protein extraction from cells isolated from the soil matrix prior to lysis (indirect method). Each approach resulted in identification of greater than 500 unique proteins, with a wide range in molecular mass and functional categories. To our knowledge, this SDS-TCA approach enables the deepest proteome characterizations of microbes in soil to date, without significant biases in protein size, localization, or functional category compared to pure cultures. This protocol should provide a powerful tool for ecological studies of soil microbial communities.

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Section: Introductionmentioning

confidence: 99%

Direct Cellular Lysis/Protein Extraction Protocol for Soil Metaproteomics

et al. 2010

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“…Protein solubilization and cell lysis are critical factors for effective proteomic analysis (80,81,99). The compatibility of downstream analysis should also be considered (69,81); for example, if mass spectrometry is to be used, proteins should be resuspended in mass spectrometry-compatible buffers and should be at correct concentrations for effective identification.…”

Section: Metaproteomicsmentioning

confidence: 99%

Proteomic investigations of vinyl chloride-assimilating bacteria

Chuang¹

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Vinyl chloride (VC) is a common groundwater pollutant and known human carcinogen that is commonly produced from the incomplete reductive dechlorination of tetrachloroethene and trichloroethene. A possible approach to clean-up VC-contaminated sites is with microorganisms that grow on VC and/or ethene as a carbon and energy source. However, little is known about the biochemical pathways involved in VCassimilation within these strains and their distribution and activity in situ in the environment. This work uses mass-spectrometry-based proteomics to contribute to the understanding of these microbial communities in both pure cultures and in the environment. The biochemical pathways of VC and ethene oxidation in Nocardioides sp. strain JS614 were studied using proteins identified with a peptide mass fingerprinting approach. New insights into a previously proposed pathway were made using mass spectrometry (MS)-based protein identifications, and potential protein biomarkers for the presence and activity of VC-assimilating bacteria in the environment were identified. Techniques to extract and identify proteins from various environmental samples such as activated sludge, sediments, soils, and water samples were developed based on preliminary experiments with protein extraction from strain JS614. These techniques were extended to 14 ethene-enrichment cultures derived from VC-contaminated groundwater to detect the presence and diversity of VC-and/or ethene-assimilating bacteria with previously identified protein biomarkers. VC-assimilating organisms can evolve under laboratory conditions from bacteria that grow on ethene but very little is known about the molecular changes involved. Tandem mass spectrometry and spectral counting were applied to three variants of ethene-grown Mycobacterium strain JS623 (a wild type and two VC-adapted variants) resulting in the identification of 174 differentially expressed proteins. The results of this v ABSTRACT Vinyl chloride (VC) is a common groundwater pollutant and known human carcinogen that is commonly produced from the incomplete reductive dechlorination of tetrachloroethene and trichloroethene. A possible approach to clean-up VC-contaminated sites is with microorganisms that grow on VC and/or ethene as a carbon and energy source. However, little is known about the biochemical pathways involved in VCassimilation within these strains and their distribution and activity in situ in the environment. This work uses mass-spectrometry-based proteomics to contribute to the understanding of these microbial communities in both pure cultures and in the environment. The biochemical pathways of VC and ethene oxidation in Nocardioides sp. strain JS614 were studied using proteins identified with a peptide mass fingerprinting approach. New insights into a previously proposed pathway were made using mass spectrometry (MS)-based protein identifications, and potential protein biomarkers for the presence and activity of VC-assimilating bacteria in the environment were identified. Techniques to extract a...

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Environmental Proteomics: Extraction and Identification of Protein in Soil

Cited by 2 publications

References 10 publications

Direct Cellular Lysis/Protein Extraction Protocol for Soil Metaproteomics

Direct Cellular Lysis/Protein Extraction Protocol for Soil Metaproteomics

Proteomic investigations of vinyl chloride-assimilating bacteria

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