Header

UZH-Logo

Maintenance Infos

Soil metaproteomics – Comparative evaluation of protein extraction protocols


Keiblinger, Katharina M; Wilhartitz, Inés C; Schneider, Thomas; Roschitzki, Bernd; Schmid, Emanuel; Eberl, Leo; Riedel, Kathrin; Zechmeister-Boltenstern, Sophie (2012). Soil metaproteomics – Comparative evaluation of protein extraction protocols. Soil Biology and Biochemistry, 54(15-10):14-24.

Abstract

Metaproteomics and its potential applications are very promising to study microbial activity in environmental samples and to obtain a deeper understanding of microbial interactions. However, due to the complexity of soil samples the exhaustive extraction of proteins is a major challenge. We compared soil protein extraction protocols in terms of their protein extraction efficiency for two different soil types. Four different protein extraction procedures were applied based on (a) SDS extraction without phenol, (b) NaOH and subsequent phenol extraction, (c) SDS–phenol extraction and (d) SDS–phenol extraction with prior washing steps. To assess the suitability of these methods for the functional analysis of the soil metaproteome, they were applied to a potting soil high in organic matter and a forest soil. Proteins were analyzed by two-dimensional liquid chromatography/tandem mass spectrometry (2D-LC–MS/MS) and the number of unique spectra as well as the number of assigned proteins for each of the respective protocols was compared. In both soil types, extraction with SDS–phenol (c) resulted in “high” numbers of proteins. Moreover, a spiking experiment was conducted to evaluate protein recovery. To this end sterilized forest soil was amended with proteins from pure cultures of Pectobacterium carotovorum and Aspergillus nidulans. The protein recovery in the spiking experiment was almost 50%. Our study demonstrates that a critical evaluation of the extraction protocol is crucial for the quality of the metaproteomics data, especially in highly complex samples like natural soils.

Abstract

Metaproteomics and its potential applications are very promising to study microbial activity in environmental samples and to obtain a deeper understanding of microbial interactions. However, due to the complexity of soil samples the exhaustive extraction of proteins is a major challenge. We compared soil protein extraction protocols in terms of their protein extraction efficiency for two different soil types. Four different protein extraction procedures were applied based on (a) SDS extraction without phenol, (b) NaOH and subsequent phenol extraction, (c) SDS–phenol extraction and (d) SDS–phenol extraction with prior washing steps. To assess the suitability of these methods for the functional analysis of the soil metaproteome, they were applied to a potting soil high in organic matter and a forest soil. Proteins were analyzed by two-dimensional liquid chromatography/tandem mass spectrometry (2D-LC–MS/MS) and the number of unique spectra as well as the number of assigned proteins for each of the respective protocols was compared. In both soil types, extraction with SDS–phenol (c) resulted in “high” numbers of proteins. Moreover, a spiking experiment was conducted to evaluate protein recovery. To this end sterilized forest soil was amended with proteins from pure cultures of Pectobacterium carotovorum and Aspergillus nidulans. The protein recovery in the spiking experiment was almost 50%. Our study demonstrates that a critical evaluation of the extraction protocol is crucial for the quality of the metaproteomics data, especially in highly complex samples like natural soils.

Statistics

Citations

50 citations in Web of Science®
54 citations in Scopus®
Google Scholar™

Altmetrics

Downloads

156 downloads since deposited on 30 Jan 2013
14 downloads since 12 months
Detailed statistics

Additional indexing

Item Type:Journal Article, refereed, original work
Communities & Collections:04 Faculty of Medicine > Functional Genomics Center Zurich
07 Faculty of Science > Department of Plant and Microbial Biology
Dewey Decimal Classification:570 Life sciences; biology
580 Plants (Botany)
610 Medicine & health
Language:English
Date:2012
Deposited On:30 Jan 2013 09:45
Last Modified:23 Aug 2017 01:14
Publisher:Elsevier
ISSN:0038-0717
Free access at:PubMed ID. An embargo period may apply.
Publisher DOI:https://doi.org/10.1016/j.soilbio.2012.05.014
PubMed ID:23125465

Download

Preview Icon on Download
Preview
Content: Accepted Version
Filetype: PDF
Size: 999kB
View at publisher