DOI: 10.5281/zenodo.7519320
title: EMERGE 2016 Autochamber Sites FT-ICRMS

description: METHODS:   Water soluble metabolites were extracted from peat by adding 7 mL of autoclaved milliQ water to 1g of peat in a sterile 15 mL Eppendorf tube. Tubes were vortexed twice for 30 seconds, and then the peat-water mixture was sonicated for 2 hours at 22˚C. Samples were then centrifuged to separate the supernatant, which served as the water extract.   Water extracts were first purified using solid phase extraction (SPE) to remove contaminants (i.e., salts) according to Dittmar et al., 2008.Briefly, water extracts were acidified to pH 2 using 1M HCL. Then, extracts were filtered through a 3 mL Bond Elut PPE cartridge (Aligient) that was previously activated using methanol. Cartridges were washed 3 mL of a 0.01 M HCl solution for five times, then dried using filtered air. Finally, extracts were eluted using 1.5 mL of methanol and stored at -80 °C until used.   Purified extracts were analyzed by direct injection using a 12 Tesla Bruker FTICR mass spectrometer located at the Pacific Northwest National Laboratory (PNNL). Positive and negative charged molecular ions were generated using a Bruker electrospray ionization (ESI) source. The instrument stability was optimized using A Suwannee River Fulvic Acid standard (SRFA), obtained from the International Humic Substance Society (IHCC). Potential carry-over between samples was monitored by injecting HPLC grade methanol. The instrument was flushed between samples using a combination of milliQ water and methanol. In order to account for variations in carbon concentrations in different samples the ion accumulation (IAT) was varied between 0.03 and 0.05 s. A total of 144 individual scans per sample were collected, averaged, and calibrated using an organic matter homologous series separated by 14 Da (CH2). Mass accuracy was < 1 ppm for single charged ions measured across a m/z range of 100–1,200 m/z, the mass resolution was ~240K at 341 m/z and the transient was 0.8 s. Raw spectra collected per sample was transformed into a list of m/z values using the FT-MS peak picker module within the BrukerDaltonik version 4.2 software using a signal to noise ratio of 7 and absolute intensity threshold of 100 (default). Formularity software was used to assign putative chemical formulae following Tfaily et al., 201838.COLUMN DEFINITIONS:Columns B-J : elemental composition of compoundColumn K : Biochemical Class assigned as in Tfaily et al 2017Column O: Formularity assigned molecular formulaColumn P-Q : O:C and H:C ratios from the formula, used to determine biochemical classColumn R: Nominal Oxidation state of Carbon (NOSC)Column S: Gibb's Free Energy (GFE)Column T: Double Bond Equivalents (DBE)Column U : DBE minus oxygen (DBE_O)Column V : Aromaticity Index (AI)Column W : Modified Aromaticity Index (AI_mod)Column X : DBE minus AI (DBE_AI)Columns Y-AY : normalized peak intensities per sampleThe spectral files are provided in the FTICRMS.neg.tar.gz, each file within is named for the sample it came from.FUNDING:This research is a contribution of the EMERGE Biology Integration Institute, funded by the National Science Foundation, Biology Integration Institutes Program, Award # 2022070.We thank the Swedish Polar Research Secretariat and SITES for the support of the work done at the Abisko Scientific Research Station. SITES is supported by the Swedish Research Council's grant 4.3-2021-00164.This study was also funded by the Genomic Science Program of the United States Department of Energy Office of Biological and Environmental Research, grant #s DE-SC0010580 and DE-SC0016440.