skip to main content
OSTI.GOV title logo U.S. Department of Energy
Office of Scientific and Technical Information

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:
 [1]; ORCiD logo [2]
  1. Univ. of Tennessee, Knoxville, TN (United States)
  2. Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States)
Publication Date:
Research Org.:
Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States)
Sponsoring Org.:
USDOE Office of Science (SC), Biological and Environmental Research (BER) (SC-23)
OSTI Identifier:
1408036
Grant/Contract Number:
AC05-00OR22725
Resource Type:
Journal Article: 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

Citation Formats

Qian, Chen, and Hettich, Robert L. Optimized Extraction Method To Remove Humic Acid Interferences from Soil Samples Prior to Microbial Proteome Measurements. United States: N. p., 2017. Web. doi:10.1021/acs.jproteome.7b00103.
Qian, Chen, & Hettich, Robert L. Optimized Extraction Method To Remove Humic Acid Interferences from Soil Samples Prior to Microbial Proteome Measurements. United States. doi:10.1021/acs.jproteome.7b00103.
Qian, Chen, and Hettich, Robert L. Wed . "Optimized Extraction Method To Remove Humic Acid Interferences from Soil Samples Prior to Microbial Proteome Measurements". United States. doi:10.1021/acs.jproteome.7b00103. https://www.osti.gov/servlets/purl/1408036.
@article{osti_1408036,
title = {Optimized Extraction Method To Remove Humic Acid Interferences from Soil Samples Prior to Microbial Proteome Measurements},
author = {Qian, Chen and Hettich, Robert L.},
abstractNote = {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, this 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.},
doi = {10.1021/acs.jproteome.7b00103},
journal = {Journal of Proteome Research},
number = 7,
volume = 16,
place = {United States},
year = {Wed May 24 00:00:00 EDT 2017},
month = {Wed May 24 00:00:00 EDT 2017}
}

Journal Article:
Free Publicly Available Full Text
Publisher's Version of Record

Save / Share:
  • Proteomics is the study of functions and regulation of biological systems based on the analysis of the protein expression profile, and there is a general agreement that soil proteomics may be a tool for better soil management. Because of the ability of soils to stabilize extracellular proteins by various mechanisms, development of soil proteomics needs an assessment of the efficiency of protein extraction from various soil types. We evaluated the possibility of extraction of soil microbial proteome by inoculating Cupriavidus metallidurans CH34, which has a known proteome, into sterile sand, kaolinite, montmorillonite and a mixture of sand, kaolinite, montmorillonite, goethitemore » and humic acids. One hour after inoculation, the viability of C. metallidurans was determined by the colony-forming units method (CFU), the amount of extracted proteins was determined by the Bradford method and the bacterial proteome was analysed by the two-dimensional gel electrophoresis technique (2D-GE). The bacterial number was 2.5 x 10{sup 6} CFU g{sup -1} of soil in all microcosms, whereas the total extracted protein content varied from 98.1 to 1268 {micro}g g{sup -1} in the various microcosms, but was undetectable in the inoculated montmorillonite. The number of protein spots from the bacterial culture and the inoculated microcosms varied between 317 and 591, with 54 variable spots among the pure culture and the microcosms. No protein spots were detected in the 2D-GE from the montmorillonite microcosm. The 2D-GE of artificial soil microcosms showed a protein pattern that was different from those of pure culture and sand and kaolinite microcosms. The results confirm the importance of clay-specific surface area and CEC in protein adsorption as montmorillonite alone had the largest sorptive capacity, and show that the artificial soil used also had a large sorptive capacity for microbial proteins. Globally, the results indicate that the extraction of proteins from soils is strongly influenced by the clay type and organic matter content, and that poor protein extraction efficiency may reduce the potential of soil proteomics.« less
  • Many methods of microbial proteome characterizations require large quantities of cellular biomass (> 1-2 g) for sample preparation and protein identification. Our experimental approach differs from traditional techniques by providing the ability to identify the proteomic state of a microbe from a few milligrams of starting cellular material. The small-scale, guanidine-lysis method minimizes sample loss by achieving cellular lysis and protein digestion in a single tube experiment. For this experimental approach, the freshwater microbe Shewanella oneidensis MR-1 and the purple non-sulfur bacterium Rhodopseudomonas palustris CGA0010 were used as model organisms for technology development and evaluation. A 2-D LC-MS/MS comparison betweenmore » a standard sonication lysis method and the small-scale guanidine-lysis techniques demonstrates that the guanidine-lysis method is more efficient with smaller sample amounts of cell pellet (i.e. down to 1 mg). The described methodology would enable deep proteome measurements from a few milliliters of confluent bacterial cultures. We also report a new protocol for efficient lysis from small amounts of natural biofilm samples for deep proteome measurements, which should greatly enhance the emerging field of microbial community proteomics. This straightforward sample boiling protocol is complementary to the small-scale guanidine-lysis technique, is amenable for small sample quantities, and requires no special reagents that might complicate the MS measurements.« less
  • Dissolved organic matter (DOM) has been shown to form a stable complex with napropamide [2({alpha}-naphthoxy)-N,N-diethyl propionamide] and to facilitate its transport through soil columns. Liquid-liquid extraction of organics is a common method to transfer napropamide from water into an organic phase for gas chromatography analysis. A study was conducted to determine the effect of Aldrich humic acid, soil-derived dissolved organic matter, electrical conductivity, and hydrogen ion activity on the ability of hexane to extract napropamide from solutions and from soil extracts. The electrical conductivity from solutions and from soil extracts. The electrical conductivity of Aldrich humic acid solutions were adjustedmore » to 0.01, 0.97, and 1.69 dS m{sup {minus}1} by adding NaCl and CaCl{sub 2}, and pH was adjusted using HCl and NaOH. Electrical conductivity had no effect on extraction efficiency. In the absence of DOM pH had no effect on extraction efficiency. In the absence of DOM pH had no effect on extraction efficiency. Extraction efficiency decreased with increasing DOM concentration. Maximum reduction in extraction efficiency occurred in the presence of DOM when solution pH was near neutrality. A maximum extraction efficiency of 100% was observed in the absence of DOM and a minimum of 68% when napropamide was added to DOM solutions at pH 8.2 and then lowered to pH 5.6. Management practices such as liming and allowing napropamide to dry on the soil may increase environmental transport. Also quantification of napropamide in environmental samples can be affected by DOM.« less
  • A novel approach has been developed to separate potential semivolatile interferences from volatile analytes prior to purge-and-trap analysis. This technique involves a closed system with solid-phase extraction which provides a simple, inexpensive method with minimal loss of volatile components. In addition, the separation method minimizes the potential adsorption of unwanted volatile species from the surrounding air during the cleanup procedure and reduces the radioactive level in actual tank waste samples. This cleanup method has been applied to radioactive, hazardous mixed waste samples containing normal paraffin hydrocarbon (NPH) from a Hanford single-shell tank. NPH, present at the percent level in actualmore » tank wastes either as a native component or introduced as a hydrostatic fluid, interferes with the purge-and-trap gas chromatography/mass spectrometry (GC/MS) analysis of volatile components. Validation of the procedure was performed by analyzing blank spikes and actual, spiked single-shell tank waste samples. The NPH removal from actual waste samples and blank samples was better than 99%. This methodology could be easily extended to a sample matrix, such as water, sorbents, or soils, that contains excessive quantities of normal paraffin hydrocarbons. 9 refs., 8 figs., 4 tabs.« less
  • Experiments were carried out to evaluate the effectiveness of a 29 mg/L solution of humic acid to enhance the removal of six aromatic hydrocarbons (benzene, toluene, p-xylene, ethyltoluene, sec-butylbenzene, and tetramethylbenzene) from a sandy material. None of the compounds were completely removed from the material. Nonetheless, the compounds with the highest water solubility, benzene and toluene, were removed effectively; less than 1 mass % was retained with use of either the humic acid solution or water. For the less soluble organic compounds, removal was more difficult and was enhanced by the humic acid solution compared to water. Mass percent retainedmore » with humic acid was as follows: p-xylene, 1.4% (24% less than water); 3-ethyltoluene, 6.4% (40% less); sec-butylbenzene, 39% (14% less); and tetramethylbenzene, 43% (14% less). The positive effect of humic acid on the removal of these organics may arise from the aggregation of the humic acid molecules to form membranes and/or micelles, having hydrophilic exteriors and hydrophobic interiors. Partitioning of the hydrophobic organics from the bulk solution into the hydrophobic interior of these humic acid structures can account for their enhanced removal from the sandy material.« less