Single-throughput Complementary High-resolution Analytical Techniques for Characterizing Complex Natural Organic Matter Mixtures

Summary

This protocol describes a single throughput for complementary analytical and omics techniques culminating in a fully-paired characterization of natural organic matter and microbial proteomics in different ecosystems. This approach permits robust comparisons for identifying metabolic pathways and transformations important for describing greenhouse gas production and predicting responses to environmental change.

Abstract

Natural organic matter (NOM) is composed of a highly complex mixture of thousands of organic compounds which, historically, proved difficult to characterize. However, to understand the thermodynamic and kinetic controls on greenhouse gas (carbon dioxide [CO₂] and methane [CH₄]) production resulting from the decomposition of NOM, a molecular-level characterization coupled with microbial proteome analyses is necessary. Further, climate and environmental changes are expected to perturb natural ecosystems, potentially upsetting complex interactions that influence both the supply of organic matter substrates and the microorganisms performing the transformations. A detailed molecular characterization of the organic matter, microbial proteomics, and the pathways and transformations by which organic matter is decomposed will be necessary to predict the direction and magnitude of the effects of environmental changes. This article describes a methodological throughput for comprehensive metabolite characterization in a single sample by direct injection Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS), gas chromatography mass spectrometry (GC-MS), nuclear magnetic resonance (NMR) spectroscopy, liquid chromatography mass spectrometry (LC-MS), and proteomics analysis. This approach results in a fully-paired dataset which improves statistical confidence for inferring pathways of organic matter decomposition, the resulting CO₂ and CH₄ production rates, and their responses to environmental perturbation. Herein we present results of applying this method to NOM samples collected from peatlands; however, the protocol is applicable to any NOM sample (e.g., peat, forested soils, marine sediments, etc.).

Introduction

Globally, wetlands are estimated to contain 529 Pg of carbon (C), mostly as organic C buried in peat deposits¹. Currently, such peatlands act as a net C sink, sequestering 29 Tg C y^-1 in North America alone¹. However, environmental disturbance such as draining, fires, drought, and warmer temperatures can offset this C sink by increasing organic matter decomposition resulting in increased C losses via greenhouse gas (carbon dioxide [CO₂] and methane [CH₄]) production^1,2. Climate change may contribute to C loss if warmer te....

Protocol

1. Sequential Extraction of Organic Matter from Soil, Sediments, or Peat

1. Collect soil, sediments, or peat via coring and divide cores according to the hypothesis being tested (e.g.,depth). Store samples in polytetrafluoroethylene coated containers and freeze at -80 °C for storage prior to analysis.
   NOTE: Approximately 25 mg C is needed for this protocol. For peat (typically 45% C), 50 mg of dried peat is required. Larger amounts of sample may be needed for low organic samples like mineral or forested uplands soils depending on the C content ( up to 5 g). Because extraction with organic solvents will pull any polyethylene glyco....

Results

We performed the described complementary analysis protocol and compared peat with depth in the S1 bog in the Spruce and Peatlands Response Under Changing Environments (SPRUCE) site in Minnesota, USA. These results are compared to those from a permafrost bog and fen from northern Sweden to show how sites may vary in metabolite and enzyme activities. We identified 3,312 enzymes in the proteomics analysis. An analysis of the enzymes activities with depth reveals that the number of enzymes declines sharply between 15 cm and .......

Discussion

The single-throughput, fully-coupled analysis stream used to characterize metabolites and the proteome provides insights into the pathways by which C cycling is occurring in a complex ecosystem. Soil and peat are heterogeneous matrices, and therefore, one of the critical steps of this method occurs in the earliest steps in ensuring that the starting peat or soil material is highly homogenous. It is preferable to grind the sample well as aggregates can reduce extraction efficiency. This is a particular problem for aggrega.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
methoxyamine hydrochloride	Sigma Aldrich	226904	derivitization agent
5 mm triple resonance salt-tolerant cold probe	Bruker		instrumentation
capillary GC column HP-5MS column (30 m × 0.25 mm × 0.25 μm)	Agilent	AG19091S-433	instrumentation
reversed phase charged surface hybrid column (3.0 mm × 150 mm × 1.7 μm particle size)	ThermoFisher		instrumentation
2 mL glass vials	VWR International	46610-722	sample vials
autosampler vials	VWR International	97055-324; 9467671	sample vials
Chloroform	VWR International	JT9174-3	solvent
Ethanol	VWR International	BDH67002.400	solvent
methanol	VWR International	BDH85681.400	solvent
pyridine	VWR International	BDH67007.400	solvent
2,2-dimethyl-2-silapentane-5-sulfonate-d6	Sigma Aldrich	178837	standard
C8-C24 fatty acid methyl ester	Sigma Aldrich	CRM18918	standard
N-methyl-N- (trimethylsilyl)trifluoroacetamide	Sigma Aldrich	24589-78-4	standard
Suwanee River Fulvic Acid standard	International Humic Substances Society	2S101F	standard
trimethylchlorosilane	Sigma Aldrich	89595	standard
Tuning Solution	Agilent
FTICR-MS analysis software	Bruker	Compass DataAnalysis 4.1
Formularity Software	Pacific Northwest National Laboratory	Formularity	available for download at: https://omics.pnl.gov/software/formularity
GC-MS	Agilent	Agilent GC 7890A with MSD 5975C
silica-based sorbent	Phenomenex (Torrance, CA)	Strata C18-E (PN 8E-S001-DAK)
NMR TUBE 3MM 8 150 CS5	VWR International	KT897820-0008	NMR tube
Varian Direct Drive 600-MHz NMR spectrometer	Varian Inova	Varian Direct Drive 600-MHz	NMR spectrometer
Chenomx NMR Suite 8.3	Chenomx	Chenomx NMR Suite	NMR software
ultra-performance liquid chromatograph	waters	Aquity UPLC H	liquid chromatograph
Velos-ETD Orbitrap mass spectrometer	ThermoFisher	Thermo Scientific LTQ Orbitrap Velos	mass spectrometer