LC/MS analysis (metabolomics soil)

Extraction of the metabolome and LC-MS analysis: Method 1. Extracellular metabolites were extracted from 2 g of wet soil from each sample (with five technical replicates) using 10 ml of deionized water. The samples were shaken on a mechanical shaker for 12 hours at 4°C, then centrifuged at 2,500 x g for 15 minutes at 4°C, and finally filtered through 0.2 µm polyethersulfone membrane filters (Pall Corporation, New York, NY, USA). Polar metabolites were completely dried using a rapid vacuum concentrator, then chemically derivatized and analyzed by gas chromatography-mass spectrometry (GC-MS) (https://www.mdpi.com/2076-2607/10/8/1653].

Method 2. Biocrust soil water samples (1.5 mL) were lyophilized and resuspended in methanol (200 μL) containing internal standards (2–10 μg/mL) and filtered through 96-well Millipore filter plates (0.2 μm PVDF) by centrifuging at 1500 × g for 2 min. Samples were analyzed using normal-phase LC/MS with a ZIC-pHILIC column (150 × 2.1 mm, 3.5 μm 200 Å, Merck Sequant, Darmstadt, Germany) using an Agilent 1290 series UHPLC (Agilent Technologies, Santa Clara, California, USA)(https://www.nature.com/articles/s41467-017-02356-9).

Method 3. (Soil non-targeted metabolomic detection and analysis) Twelve rhizosphere soil samples were sent to Majorbio (Shanghai, China) on dry ice for metabolite extraction, detection, and analysis. In detail, 1,000-mg soil aliquot of each sample was homogenized with 1,000 µL methanol/water (4:1, v/v) solution, including 0.02 mg/mL internal standard (L-2-chlorophenylamine acid) for 6 min at −10 °C and 50 kHz using a Geno-grinder 2,000 (SPEX, Metuchen, NJ, USA) and spun down for 30 min at 5 °C and 40 kHz. After resting for 30 min at −20 °C, each material was centrifuged at 13,000 g (relative centrifugal force) for 15 min at 4 °C, then the supernatant was transferred and concentrated by a Termovap Sample Concentrator (DC-24, Anpel Laboratory Technologies, Shanghai, China). The dry residue was derivatized by adding 50 µL acetonitrile/water (1:1), homogenized for 30 s at 5 °C and 40 kHz, and centrifuged at 13,000 g (relative centrifugal force) for 10 min at 4 °C. Finally, the supernatant was subjected to LC-tandem MS (MS/MS) analysis; 20 µL supernatant of each sample was mixed for the quality control sample. The UHPLC-Triple TOF system (AB Sciex, Foster City, CA, USA) equipped with an ACQUITY UPLC HSS T3 (100 mm × 2.1 mm i.d., 1.8 µm; Waters, Milford, CT, USA) was applied to chromatographic separating the metabolites with two mobile phases, phase A (95% water and 5% acetonitrile (with 0.1% formic acid)) and phase B (5% water (with 0.1% formic acid), 47.5% acetonitrile, and 47.5% isopropanol). The whole system was integrated to a quadrupole time-of-flight mass spectrometer (Triple TOFTM 5600th; AB Sciex, Foster City, CA, USA) equipped with an electrospray ionization source and operated in positive and negative modes doi: [10.7717/peerj.13254](https://doi.org/10.7717%2Fpeerj.13254).

Method 4. The 45 collected soil samples, and 5 blank samples containing no soil, were lyophilized on an Edwards Super Modulyo freeze-drier (SciQuip Ltd., Shropshire, UK) for 7 d. Subsequently, these were ground in a Retsch MM200 stainless steel ball mill (Retsch GmbH, Haan, Germany) at a frequency of 20 Hz to aid recovery of metabolites from the microbial biomass (Fiehn et al., 2002; Wang et al., 2015). The samples were then stored in individual sterile glass vials at −80 °C to minimize changes in metabolites (Wellerdiek et al., 2009). The soils were extracted using 3:3:2 (v/v/v) acetonitrile-isopropanol-water, vortexed for 15 s, shaken at 4 °C for 5 min, centrifuged at 1400 rpm for 2 min, and dried using a CentriVap Benchtop Centrifugal Concentrator (Labconco Corp., Kansas City, MO) (Barupal et al., 2019; Fu et al., 2019). Non-targeted primary metabolism analysis was performed using a Gerstel Automated Linear Exchange-Cold Injection System (ALEX-CIS) with Agilent gas chromatograph (GC) and Leco Pegasus IV Time Of Flight (TOF) MS at the UC Davis West Coast Metabolomics Facility using the method of Fiehn (2016). Briefly, 0.5 μl of each sample was injected onto a Rtx-5Sil MS capillary column (30 m length × 0.25 m i.d with 10 m integrated guard column; 0.25 μm 95% dimethylsiloxane/5% diphenylpolysiloxane coating; Restek Corp., Bellefonte, PA). Using a He mobile phase, the GC thermal programme was 50 °C for 1 min, ramped to 330 °C at 20 °C min−1 and finally held at 330 °C for 5 min. Upon elution, samples were injected into a Pegasus IV GC-time of flight mass spectrometer (Leco Corp., St Joseph, MI), using mass resolution of 17 spectra s−1, from 80 to 500 Da, at −70 eV ionization energy and 1800 V detector voltage with a 230 °C transfer line and 250 °C ion source (https://www.sciencedirect.com/science/article/pii/S0038071720300559). Method 5 (3 methods in the same paper): Soil Sample Extractions for NMR and MS Analyses Extraction in DMSO Antarctic soil (2 g) was suspended in 10 mL of DMSO, sonicated at 37 kHz, pulse, 100 pw, 30 min, 30–60 °C (Elmasonic P 30 H, Elma Schmidbauer GmbH, Singen, Germany). Each sample was centrifuged (Beckman Avanti J-20 Centrifuge, Beckman Coulter, Indianapolis, IN, USA) at 20,000× g for 15 min, 4 °C. The supernatant was collected and freeze-dried (Christ Alpha 1-2 LD plus, Martin Christ Gefriertrocknungsanlagen GmbH, Osterode am Harz, Germany). The extraction yield was calculated for each sample. Lyophilized samples were stored at −20 °C. Extraction in H2O/CH3OH 1:1 Antarctic soil (2 g) was suspended in 10 mL of MilliQ water/CH3OH 1:1 with 50 µL NaN3 0.3 M to obtain NaN3 1.5 mM (0.01%), sonicated and centrifuged as described above. The supernatant was collected and CH3OH evaporated under reduced pressure at 40 °C (Heidolph Rotary Evaporator, Laborota 4000, Heidolph Instruments GmbH & Co. KG, Schwabach, Germany). The water phase was transferred in flask and freeze-dried as above. The extraction yield was calculated for each sample. Lyophilized samples were stored at −20 °C. Extraction in H2O/CH3CN 7:3 Antarctic soil (2 g) was suspended in 10 mL of MilliQ water/CH3CN 7:3 with 50 µL NaN3 0.3 M to obtain NaN3 1.5 mM (0.01%) sonicated at 37 kHz, pulse, 100 pw, 60 min, 30–60 °C, and centrifuged as described above. The extraction was repeated with 10 mL of fresh extraction solution. The supernatants were transferred in a flask and freeze-dried as above. The extraction yield was calculated for each sample. Lyophilized samples were stored at −20 °C (https://www.mdpi.com/1422-0067/24/15/12340). Method 5 (+ ammonium bicarbonate) 5 g of lyophilized soil were placed in a 25 ml centrifuge tube equipped with a Teflon-lined screw cap and 4 ml of 0.25 M aqueous ammonium bicarbonate, 10 ml methanol, and 5 ml chloroform containing the internal standards for NLFA and PLFA analysis (trinonadecanoin glyceride, Nu-Chek Prep, Inc., Elysian, MN, USA; 19:0 phosphatidylcholine, Avanti Polar Lipids, Alabaster, AL), were added. The contents of the centrifuge tubes were then sonicated 10 min in a water bath sonicator at room temperature, incubated at room temperature with end-over-end mixing for 2 h, and centrifuged 10 min at 1710 G. The liquid phase was transferred to a 30 ml test tube with a Teflon-lined screw cap, 5 ml each of water and chloroform were added, and the sample mixed by vigorous shaking. After centrifuging for 10 min at 1710 G, 2 layers were formed. The top layer (aqueous phase) contained the water-soluble metabolites while the bottom layer (organic phase) contained the lipids. Each phase was transferred into a separate test tube and evaporated in vacuo using a centrifugal evaporator. The organic phase was evaporated to dryness for NLFA and PLFA analyses while the aqueous phase was evaporated to approximately one-quarter of the original volume for metabolomic analysis. Samples were stored at −80 °C until further processing (https://www.sciencedirect.com/science/article/pii/S0929139318309958?casa_token=Ke9s59BsPbMAAAAA:m0W7-gJGUad-_iMOcfnUumQW_JYGiiW9exCDR7bVKgGh0JTUPbzVJq_X2I94sHOyuGtfys5Ftw). Method 7. methanol–chloroform–water method [DOI: 10.1002/etc.2418].

commons-research / microcosmics

LC/MS analysis (metabolomics soil) #7