Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses were performed using an Ultra-high performance liquid chromatography (UHPLC) system (1,290, Agilent Technologies) with a UPLC HSS T3 column (2.1 mm × 100 mm, 1.7 μm) coupled to Q Exactive (Orbitrap MS, Thermo). The metabolomic procedure, including instruments and regents, metabolite extraction, data preprocessing and annotation, statistical analysis, were descripted detailly in Supplementary Item S1 .
Uplc hss t3 column
Manufactured by Thermo Fisher Scientific
Sourced in United States
The UPLC HSS T3 column is a high-performance liquid chromatography column designed for the separation and analysis of a wide range of analytes. It features a stationary phase composed of sub-2-micron silica particles with a Type-C (trifunctional) bonded phase. This column is suitable for the separation of polar, mid-polar, and non-polar compounds in ultra-high-performance liquid chromatography (UPLC) applications.
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Lab products found in correlation
1290 uhplc system, by Agilent Technologies (10 mentions)
Xcalibur 4, by Thermo Fisher Scientific (8 mentions)
Orbitrap ms, by Thermo Fisher Scientific (7 mentions)
Q exactive, by Thermo Fisher Scientific (5 mentions)
Q exactive orbitrap ms, by Thermo Fisher Scientific (4 mentions)
Q exactive mass spectrometer, by Thermo Fisher Scientific (4 mentions)
Xcalibur, by Thermo Fisher Scientific (3 mentions)
Q exactive hf x mass spectrometer, by Thermo Fisher Scientific (3 mentions)
Uhplc system, by Agilent Technologies (2 mentions)
Qe hfx mass spectrometer, by Thermo Fisher Scientific (2 mentions)
L glutamine 13c5, by Merck Group (2 mentions)
Microcentrifuge tube, by Eppendorf (1 mentions)
Uhplc system, by Thermo Fisher Scientific (1 mentions)
Q exactive hfx mass spectrometer orbitrap ms, by Thermo Fisher Scientific (1 mentions)
Proteome discoverer software, by Thermo Fisher Scientific (1 mentions)
Mascot, by Matrix Science (1 mentions)
Qe mass spectrometer, by Thermo Fisher Scientific (1 mentions)
13c glutamine, by Merck Group (1 mentions)
Orbitrap exploris 120 mass spectrometer, by Thermo Fisher Scientific (1 mentions)
Uplc system, by Thermo Fisher Scientific (1 mentions)
27 protocols using uplc hss t3 column
1
Metabolomic Profiling by LC-MS/MS
2
UHPLC-QE-Orbitrap/MS Analysis of BSE
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First, 50 mg of BSE was dissolved in 1 mL of extract solvent (acetonitrile-methanol-water, 2:2:1, containing internal standard 1 μg/mL) and then centrifugated at 8,000°g at 4°C for 15 min. The resulting supernatants were transferred to LC-MS vials for UHPLC-QE-Orbitrap/MS analysis as previously described (Li et al., 2017 (link)). The analyses were performed using an 1290 UHPLC system (Agilent, Palo Alto, CA, United States) with a UPLC HSS T3 column (2.1 mm × 100 mm, 1.8 μm) coupled to Q Exactive (Orbitrap MS, Thermo, Somerset, NJ, United States). The mobile phase A was 0.1% formic acid in water for positive, and 5 mmol/L ammonium acetate in water for negative, and the mobile phase B was acetonitrile. The elution gradient was set as follows: 0 min, 1% B; 1 min, 1% B; 8 min, 99% B; 10 min, 99% B; 10.1 min, 1% B; and 12 min, 1% B. The flow rate was 0.5 mL/min and the injection volume was 2 μL.
3
Metabolite Profiling by UHPLC-MS/MS
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Ultra-high Pressure Liquid Chromatography-Mass Spectrum/Mass Spectrum (UHPLC-MS/MS) analysis was performed using an UHPLC system (1290, Agilent Technologies) with a UPLC HSS T3 column (2.1 mm × 100 mm, 1.8 μm) coupled to Q Exactive mass spectrometer (Orbitrap MS, Thermo). The mobile phase A was consist of positive (0.1% formic acid in water)and negative modes (5 mmol/L ammonium acetate in water). The mobile phase B was acetonitrile. The LC method used for detailed metabolite profiling with higher resolution was as follows: column temperature 35 °C, flow rate 0.5 mL/min, injected volume 3 μL. The parameters for elution gradient was as follows: 0 ~ 1.0 min, 1% B; 1.0 ~ 8.0 min, 1% ~ 99% B; 8.0 ~ 10.0 min, 99% B; 10.0 ~ 10.1 min, 99% ~ 1% B; 10.1 ~ 12 min, 1% B. The QE mass spectrometer (Orbitrap MS, Thermo) was used to acquire MS/MS spectra data under the control of the acquisition software (Xcalibur 4.0.27, Thermo). In information-dependent acquisition (IDA) mode, the acquisition software continuously evaluated the full scan MS spectrum. The ESI source conditions were set as following: sheath gas flow rate as 45 Arb, Aux gas flow rate as 15Arb, capillary temperature 400 °C, full MS resolution as 70,000, MS/MS resolution as 17,500, collision energy as 20/40/60 eV in NCE mode, spray Voltage as 4.0 kV (positive) or − 3.6 kV (negative), respectively.
4
Comprehensive LC-MS Metabolite Profiling
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LC-MS analyses were performed using a UHPLC-Q Exactive HF-X system (1290, Agilent Technologies, Shanghai, China) with a UPLC HSS T3 column (100 mm × 2.1 mm i.d., 1.8 µm) coupled to Q Extractive unit (Orbitrap MS, Thermo, Waltham, MA, USA). Mobile phase A was 95% water + 5% acetonitrile (containing 0.1% formic acid), and mobile phase B was 47.5% acetonitrile + 47.5% isopropanol + 5% water (containing 0.1% formic acid). The gradient elution procedure of the mobile phases is according to Xie et al. (2019) (link). The mass spectrum (MS) signals of the sample were scanned in positive and negative ion modes, with a mass range of 70–1,050 m/z. The sheath gas flow rate was set at 50 psi, the auxiliary gas flow rate at 13 psi, the auxiliary gas heating temperature at 425°C, the ion spray voltage in the positive mode at 3,500 V, the ion spray voltage in the negative mode at −3,500 V, the ion-transfer tube temperature at 325°C, and the normalized collision energy at 20–40–60 V cyclic collision energy. The resolution of primary MS was 60,000, and that of secondary MS was 7,500. The data were collected using data-dependent acquisition (DDA) mode.
5
Metabolite Profiling of Leaf Samples
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The extraction of metabolites for leaf samples was performed by UHPLC-QE-MS (Guo et al., 2020 (link)). LC-MS/MS analyses were executed by an UHPLC system (1290, Agilent Technologies) with a UPLC HSS T3 column coupled to Q Exactive (Orbitrap MS, Thermo). The mobile phase proportioning and the elution gradient setting were performed by the method of Zhang et al. (2021) (link). The full scan resolution of ESI source was 70000, and MS/MS used a resolution of 17500. The MS raw data was formatted by ProteoWizard and the conversion results were processed by R package XCMS software (version 3.2). The peak annotation was implemented by OSI-SMMS (version 1.0, Dalian Chem Data Solution Information Technology Co. Ltd.) with in-house MS/MS database. Principal component analysis was performed by R package models for the exposition of relevance among samples. And the metabolites were annotated and classified according to the Kyoto Encyclopedia of Genes and Genomics (KEGG) database.
6
Metabolite Extraction from Chicken Cecal Contents
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The ten chickens’ cecal contents samples (50 mg) were collected in 1.5-mL Eppendorf microcentrifuge tubes, and extract solvent (acetonitrile–methanol–water, 2:2:1, 1000 μL) was then added. The mixture was then vortexed for 30 s, homogenised at 45 Hz for 4 min, sonicated for 5 min in an ice-water bath, and incubated at −20 °C for 1 h prior to centrifugation 12,000 rpm at 4 °C for 15 min. The supernatants (200 μL) were transferred to an injection bottle for UHPLC–MS/MS analysis. UHPLC–MS analysis was performed using a UHPLC system (1290, Agilent Technologies) with a UPLC HSS T3 column (2.1 mm × 100 mm, 1.8 μm) coupled to a Q Exactive benchtop Orbitrap mass spectrometer (Orbitrap MS, Thermo). Samples pooled by mixing an equal aliquot of the supernatants from all samples were used as quality control (QC) samples.
7
Metabolic Profiling of Plant Leaf Tissues
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Five individuals each of the R and WT populations were selected, and the leaf tissue was prepared using a standard method. In brief, 50 mg of sample was ground using liquid nitrogen and homogenized using an extraction solution with an isotopically labeled internal standard mixture. The samples were then incubated for 1 h at −40°C and centrifuged at 12,000 rpm at 4°C for 15 min. The resulting supernatant was transferred to a fresh glass vial for further analysis.
LC-MS/MS analyses were performed using a UHPLC system (Thermo Fisher Scientific, Waltham, MA, United States) with a UPLC HSS T3 column (2.1 mm × 100 mm, 1.8 μm) coupled to a Q Exactive HFX mass spectrometer (Orbitrap MS, Thermo Fisher Scientific, CA, United States). The mobile phase consisted of 5 mmol L–1 ammonium acetate and 5 mmol L–1 acetic acid in water (A) and acetonitrile (B). The auto-sampler temperature was 4°C, and the injection volume was 3 μL. A standard method for QE HFX mass spectrometry was performed, and all parameters were the same as those used in the previous study (Li et al., 2021 ).
LC-MS/MS analyses were performed using a UHPLC system (Thermo Fisher Scientific, Waltham, MA, United States) with a UPLC HSS T3 column (2.1 mm × 100 mm, 1.8 μm) coupled to a Q Exactive HFX mass spectrometer (Orbitrap MS, Thermo Fisher Scientific, CA, United States). The mobile phase consisted of 5 mmol L–1 ammonium acetate and 5 mmol L–1 acetic acid in water (A) and acetonitrile (B). The auto-sampler temperature was 4°C, and the injection volume was 3 μL. A standard method for QE HFX mass spectrometry was performed, and all parameters were the same as those used in the previous study (Li et al., 2021 ).
8
Quantitative Peptide Analysis by LC-MS/MS
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Five milligrams of peptide powder were weighed into EP tubes. After the addition of extracted solvent (acetonitrile–methanol–water, 2 : 2 : 1, containing internal 1 μg mL−1 standard), the samples were vortexed for 30 s, homogenized at 45 Hz for 4 min, and sonicated for 5 min in an ice-water bath. The quality control (QC) sample was prepared by mixing equal volumes of aliquots of the supernatants from all of the samples. LC-MS/MS analysis was performed by using an UHPLC system (1290, Agilent Technologies) with a UPLC HSS T3 column (2.1 mm × 100 mm, 1.8 μm) coupled to an Q Exactive (Orbitrap MS, Thermo) apparatus. The raw data were converted to mzXML format using ProteoWizard and processed by using MAPS software (version 1.0). The optimization conditions of the UHPLC-QE-MS mass spectrometer are shown in Table 1 . After mass spectrometry scanning, Proteome Discoverer software (PD) (version 1.4.0.288, Thermo Fisher Scientific) was used to screen the mass spectra. After PD extraction, the spectra were examined using Mascot (version 2.3.2, Matrix Science). Peptide sequences were obtained after analysis of the Mascot search results and the first screening of the mass spectra.36 (link)
9
UHPLC-MS/MS Metabolite Profiling Protocol
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LC-MS/MS analyses were performed using a UHPLC system (1290, Agilent Technologies) with a UPLC HSS T3 column (2.1 mm × 100 mm, 1.8 μm) coupled to Q Exactive mass spectrometer (Orbitrap MS, Thermo). The mobile phase A was 0.1% formic acid in water for positive mode and 5 mmol/L ammonium acetate in water for negative mode, and the mobile phase B was acetonitrile. The elution gradient was set as follows: 0∼1.0 min, 1% B; 1.0∼8.0 min, 1%∼99% B; 8.0∼10.0 min, 99% B; 10.0∼10.1 min, 99%∼1% B; 10.1∼12 min, 1% B. The flow rate was 0.5 mL/min. The injected volume was 2 μL. The QE mass spectrometer was used for its ability to acquire MS/MS spectra on information-dependent acquisition (IDA) mode in the control of the acquisition software (Xcalibur 4.0.27, Thermo). In this mode, the acquisition software continuously evaluates the full scan MS spectrum. The ESI source conditions were set as follows: sheath gas flow rate as 45 Arb, aux gas flow rate as 15 Arb, capillary temperature 400°C, full MS resolution as 70000, MS/MS resolution as 17500, collision energy as 20/40/60 eV in NCE mode, and spray voltage as 4.0 kV (positive) or −3.6 kV (negative), respectively.
10
Metabolite Profiling of BALF Samples
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The BALF supernatants were thawed at 4 °C on ice and 100 µL was mixed with 300 µL of methanol, vortexed for 30s, and sonicated for 10 minutes in an ice-water bath, followed by incubation for 1 h at −20 °C to precipitate proteins. After centrifugation (12,000 rpm, 15 min, 4 °C) the resulting supernatants were stored at −80 °C until analysis by ultra-high-performance liquid chromatography coupled with Q-exactive hybrid Orbitrap mass spectrometry (UHPLC–QE Orbitrap/MS)24 (link). A quality control (QC) sample was injected once for every eight samples, which was prepared by combining equal volume aliquots of all supernatants from all samples. LC–MS/MS analyses was performed using a UHPLC system (1290, Agilent Technologies) with a UPLC HSS T3 column (2.1 mm×100 mm, 1.8 μm) coupled to Q Exactive (Orbitrap MS, Thermo) as described elsewhere25 (link). Obtained MS raw data files were converted to the mzML format using ProteoWizard and processed by R package XCMS (v.3.2) as described25 (link). For this, a data matrix was generated consisting of the retention time (RT), mass-to-charge ratio (m/z) values, and peak intensity. After XCMS data processing, peak annotation was performed with OSI-SMMS (v.1.0, Dalian Chem Data Solution Information Technology Co. Ltd.) with the use of an in-house MS/MS database.
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