Title: Optimized Extraction Method To Remove Humic Acid Interferences from Soil Samples Prior to Microbial Proteome Measurements

Abstract

The microbial composition and their activities in soil environments play a critical role in organic matter transformation and nutrient cycling, perhaps most specifically with respect to impact on plant growth but also more broadly to global impact on carbon and nitrogen-cycling. Liquid chromatography coupled to high performance mass spectrometry provides a powerful approach to characterize soil microbiomes; however, the limited microbial biomass and the presence of abundant interferences in soil samples present major challenges to soil proteome extraction and subsequent MS measurement. To address some of the major issues, we have designed and optimized an experimental method to enhance microbial proteome extraction concomitant with minimizing the soil-borne humic substances co-extraction from soils. Among the range of interferences, humic substances are often the worst in terms of adversely impacting proteome extraction and mass spectrometry measurement. Our approach employs an in-situ detergent-based microbial lysis / TCA precipitation coupled with an additional acidification precipitation step at the peptide level which efficiently removes humic acids. By combing filtration and pH adjustment of the final peptide solution, the remaining humic acids can be differentially precipitated and removed with a membrane filter, thereby leaving much cleaner proteolytic peptide samples for MS measurement. As a result, thismore » modified method is a reliable and straight-forward protein extraction method that efficiently removes soil-borne humic substances without inducing proteome sample loss or reducing or biasing protein identification in mass spectrometry.« less

Authors:

Qian, Chen ^[1]; Hettich, Robert L.  ^[2]

• Search DOE PAGES for author "Hettich, Robert L."
• Search DOE PAGES for ORCID "0000-0001-7708-786X"

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+ Show Author Affiliations

1. Univ. of Tennessee, Knoxville, TN (United States)
2. Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States)

Publication Date:

Wed May 24 00:00:00 EDT 2017

Research Org.:

Oak Ridge National Laboratory (ORNL), Oak Ridge, TN (United States)

Sponsoring Org.:

USDOE Office of Science (SC), Biological and Environmental Research (BER)

OSTI Identifier:

1408036

Grant/Contract Number:  

AC05-00OR22725

Resource Type:

Accepted Manuscript

Journal Name:

Journal of Proteome Research

Additional Journal Information:

Journal Volume: 16; Journal Issue: 7; Journal ID: ISSN 1535-3893

Publisher:

American Chemical Society (ACS)

Country of Publication:

United States

Language:

English

Subject:

59 BASIC BIOLOGICAL SCIENCES; 60 APPLIED LIFE SCIENCES; humic acid removal; proteome extraction; shotgun proteomics; soil metaproteomics