A volume of 1 μL of each derivatised sample was injected splitless by a CTC Combi Pal autosampler (CTC Analytics AG, Zwingen, Switzerland) into an Agilent 6980 GC equipped with a 10 m X 0.18 mm i.d. fused-silica capillary column chemically bonded with 0.18-um DB 5-MS stationary phase (J&W Scientific Folsom, CA). The injector temperature was set to 270°C. Helium was used as the carrier gas at a constant flow rate of 1 mL min-1 through the column. For every analysis, the purge time was set to 60 s at a purge flow rate of 20 mL min-1 and an equilibrium time of 1 min. The column temperature was held initially at 70°C for 2 min, then increased to 320°C at a rate of 30°C min-1, where it was held for 2 min. The column effluent was introduced into the ion source of a Pegasus III time-of-flight mass spectrometer (Leco Corp., St Joseph, MI). The ion source and transfer line temperatures were set to 200°C and 250°C, respectively. Ions were generated by a 70-ev electron beam at a current of 2.0 mA. Masses were acquired in the mass range 50–800 m/z at a rate of 30 spectra s-1. The acceleration voltage was turned on after a solvent delay of 150 s.
Pegasus 3 time of flight mass spectrometer
Manufactured by Leco
Sourced in Sao Tome and Principe, United States
The Pegasus III is a time-of-flight mass spectrometer manufactured by Leco. It is designed to perform high-resolution mass analysis of various samples.
Automatically generated - may contain errors
Lab products found in correlation
0.18 um db 5 ms stationary phase, by Agilent Technologies (2 mentions)
Ctc combipal autosampler, by CTC Analytics (2 mentions)
Simca p v13, by Sartorius (1 mentions)
7890 gas chromatograph, by Leco (1 mentions)
Agilent 6890n gas chromatograph, by Agilent Technologies (1 mentions)
Combipal autosampler, by CTC Analytics (1 mentions)
Bstfa, by Macherey-Nagel (1 mentions)
6890n gas chromatograph, by Agilent Technologies (1 mentions)
Combi pal autosampler, by Agilent Technologies (1 mentions)
6890n gas chromatograph, by Leco (1 mentions)
7 protocols using pegasus 3 time of flight mass spectrometer
1
GC-MS Analysis of Derivatised Samples
2
Fecal Metabolome Analysis via GC-TOF-MS
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Fecal samples were lyophilized, derivatized and analyzed by gas chromatography time-of-flight mass spectrometry (GC-TOF-MS) (Agilent 7890 gas chromatograph and LECO Pegasus III time-of-flight mass spectrometer) as previously described [23 (link),24 (link)]. MS-DIAL software [25 (link)] and FiehnBin base database were used for raw peaks exacting, the data baselines filtering, and calibration of the baseline [26 (link)]. Peaks detected in ≤50% of QC samples or <50% samples of every group were removed, except QC group or RSD>30% in QC samples [27 (link)]. SIMCA-P v13.0 (Umetrics, Umea, Sweden) was used for partial least squares-discriminant analysis (PLS-DA) and orthogonal projections to latent structures-discriminant analysis (OPLS-DA). The first principal component of variable in importance projection (VIP) was obtained to refine the analysis. VIP > 1.5 was first selected as “changed metabolites”. Obtained metabolites were validated by searching in the Kyoto Encyclopedia of Genes and Genomes (KEGG) [28 (link)].
3
Metabolomic Analysis of Leaf Samples
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Frozen leaf samples harvested after 4 h illumination were ground in 2 ml microcentrifuge tubes using stainless steel beads and a Retsch Mixer Mill MM 400 (Retsch, Haan, Germany) under cryogenic conditions. From these samples, metabolites were extracted, derivatized, and analyzed via gas chromatography–time-of-flight mass spectrometry (GC-TOF-MS) analyses as described before (Lisec et al., 2006 (link)). The GC-TOF-MS system consisted of a CTC CombiPAL autosampler (CTC Analytics, Zwingen, Switzerland), an Agilent 6890N gas chromatograph (Agilent Technologies, Santa Clara, CA, USA) and a LECO Pegasus III time-of-flight mass spectrometer running in EI+ mode (Leco Instruments, St. Joseph, MI, USA). Metabolites were identified by comparison with database entries of authentic standards (Kopka et al., 2005 (link); Schauer et al., 2005 (link)) using TagFinder software (Luedemann et al., 2012 ). The peak intensity of a representative fragment was normalized with that of the internal standard ribitol and sample fresh weight (FW) and referred to as relative abundance. The parameters used for the peak annotation are listed in Supplementary Table S1 according to Fernie et al. (2011) (link).
4
GC-TOFMS Analysis of Derivatized Samples
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A volume of 1 μl of each derivatised sample was injected splitless by a CTC Combi Pal autosampler (CTC Analytics AG, Zwingen, Switzerland) into an Agilent 6980 GC equipped with a 10 m X 0.18 mm i.d. fused-silica capillary column chemically bonded with 0.18-um DB 5-MS stationary phase (J&W Scientific Folsom, CA). The injector temperature was set to 270°C. Helium was used as the carrier gas at a constant flow rate of 1 mL min-1 through the column. For every analysis, the purge time was set to 60 s at a purge flow rate of 20 mL min-1 and an equilibrium time of 1 min. The column temperature was held initially at 70°C for 2 min, then increased to 320°C at a rate of 30°C min-1, where it was held for 2 min. The column effluent was introduced into the ion source of a Pegasus III time-of-flight mass spectrometer (Leco Corp., St Joseph, MI). The ion source and transfer line temperatures were set to 200°C and 250°C, respectively. Ions were generated by a 70-ev electron beam at a current of 2.0 mA. Masses were acquired in the mass range 50–800 m/z at a rate of 30 spectra s-1. The acceleration voltage was turned on after a solvent delay of 150 s. The detector voltage was 1670 V.
5
Metabolic Profiling via GC-TOF-MS
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Metabolite extraction, and chromatography data processing were performed according to the methods described previously 36 . In brief, the freeze-dried sample (10 mg) was mixed with 360µL methanol-mix that includes 13 C 6 -sorbitol as an internal standard, followed by 200 µL chloroform and 400 µL water that are added for phase separation. Solvents were of highest available purity (Merck, Darmstadt, Germany). Samples were vortexed and agitated 15 min at 70°C after adding the methanol-mix, incubated 5 min at 37°C after chloroform addition, and thoroughly vortexed after water addition. Phase separation was induced by centrifugation. The upper polar phase (80 µL) was dried. Each sample was chemically derivatized using methoxyamine hydrochloride in pyridine and BSTFA (Macherey-Nagel, Düren, Germany) as described 36 , including n-alkanes for retention index calculation. The derivatized samples (1 µL) were analyzed by splitless-and 1:30 split-injection modes with a 6890N gas chromatograph (Agilent, Santa Clara, CA, USA) connected to a Pegasus III time-of-flight mass spectrometer (Leco Instruments, St. Joseph, MI, USA). Metabolite annotation was performed by matching of mass spectra and retention index information to the Golm Metabolome Database using TagFinder software [36] [37] [38] .
6
Metabolic Profiling of Reproductive Tissues
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Anther, pistil and pollinated pistil samples (120 mg) with four biological replicates were shock frozen in liquid nitrogen and a fraction enriched for polar primary metabolites was prepared and processed as described previously (Siahpoosh et al. 2012) . Gas chromatography coupled to electron impact ionization time-of-flight mass spectrometry (GC/EI-TOF-MS) was performed using an Agilent 6890N24 gas chromatograph hyphenated to a Pegasus III time-of-flight mass spectrometer (LECO, Mönchengladbach, Germany) as described by Wagner et al. (2003) . Chromatograms were acquired and processed by CHROMATOF software 1.00, Pegasus driver 1.61 (Leco, http://www.leco.de; accessed 25 April 2015). Selective peak heights representing arbitrary mass spectral ion currents were normalized to the sample fresh weight and U-13 C-sorbitol, which was added upon metabolite extraction as an internal standard. Data processing was performed using the TagFinder software (Luedemann et al. 2008) . Metabolites were identified under manual supervision using TagFinder, the NIST08 software (www.nist.gov/srd/mslist.htm; accessed 19 April 2015) and the mass spectral and retention time index (RI) reference collection of the Golm Metabolome Database (GMD; Kopka et al. 2005; Hummel et al. 2010) .
7
Metabolite Analysis of Plant Leaves
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Metabolite analysis was performed in the same leaves used for gas exchange measurements. Leaves were sampled at midday and immediately frozen in liquid nitrogen for subsequent analysis. Leaf powder (approx. 50 mg) obtained by quick grinding under liquid nitrogen was extracted, derivatized and subsequently analyzed by gas chromatography-time of flight-mass spectrometry (GC-TOF-MS) as previously described [50] (link). The GC-TOF-MS system was composed of a CTC CombiPAL autosampler, an Agilent 6890N gas chromatograph and a LECO Pegasus III time-of-flight mass spectrometer running in Electron Ionization (EI)+ mode. Metabolites were manually annotated by comparison with database entries of standards with the aid of TagFinder software [51] (link). Metabolite data is reported following recommended standards [52] (link) with the parameters used for peak annotation being provided in Table S1. We collected three to six replicates per treatment and cultivar.
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