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

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Abstract

The microbial composition and their activities in soil environments play a critical role in organic matter transformation and nutrient 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 proteome extraction and subsequent MS measurement. To this end, we have designed an experimental method to improve microbial proteome measurement by removing the soil-borne humic substances coextraction from soils. Our approach employs an in situ detergent-based microbial lysis/TCA precipitation coupled to an additional cleanup step involving acidified precipitation and filtering at the peptide level to remove most of the humic acid interferences prior to proteolytic peptide measurement. The novelty of this approach is an integration to exploit two different characteristics of humic acids: (1) Humic acids are insoluble in acidic solution but should not be removed at the protein level, as undesirable protein removal may also occur. Rather it is better to leave the humics acids in the samples until the peptide level, at which point the significant differential solubility of humic acids versus peptides at low pH can be exploited very efficiently. (2) Most of the humic acids have larger molecule weights than the peptides. Therefore, filtering a pH 2 to 3 peptide solution with a 10 kDa filter will remove most of the humic acids. This method is easily interfaced with normal proteolytic processing approaches and provides a reliable and straightforward protein extraction method that efficiently removes soil-borne humic substances without inducing proteome sample loss or biasing protein identification in mass spectrometry. In general, this humic acid removal step is universal and can be adopted by any workflow to effectively remove humic acids to avoid them negatively competing with peptides for binding with reversed-phase resin or ionization in the electrospray.

Original languageEnglish
Pages (from-to)2537-2546
Number of pages10
JournalJournal of Proteome Research
Volume16
Issue number7
DOIs
StatePublished - Jul 7 2017

Funding

We thank Dr. Karuna Chourey for assisting the bacteria inoculation and Dr. Hongmei Chen for assisting the UV measurement. Stipend support for C.Q. was provided by the University of Tennessee-Knoxville Genome Science and Technology Program. Funding for this research was provided by the U.S. DOE-BER, Genome Sciences Program. Oak Ridge National Laboratory is managed by UT-Battelle, LLC, for the U.S. Department of Energy.

FundersFunder number
U.S. DOE-BER
University of Tennessee-Knoxville Genome Science and Technology Program
U.S. Department of Energy
Oak Ridge National Laboratory

    Keywords

    • humic acid removal
    • proteome extraction
    • shotgun proteomics
    • soil metaproteomics

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