Nectar was collected at times between 14:00 and 16:00 from the leaf midrib using a 2 μL pipette. The foliar nectary and corresponding parts of nectariless samples were cut from leaf midribs. Freeze‐dried nectary and nectariless samples were crushed using a mixer mill (MM 400, RETSCH). To extract metabolites, a 100 mg sample was added to a PE tube with 1.2 mL 70% aqueous methanol and incubated at 4 °C for about 16 h. Then, following centrifugation at 12 000 g for 10 min, the supernatant was filtered before UPLC‐MS/MS analysis. Sample extracts were analysed using an UPLC‐ESI‐MS/MS system (UPLC, Shimadzu; MS, Applied Biosynthesis, 6500 Q TRAP, Applied Biosynthesis). Analysis conditions and metabolite annotations were as previously described (Zhu et al., 2018 ). The clustering heatmap was generated by the R package ‘gplots’.
Ultra performance liquid chromatography (uplc)
Manufactured by Shimadzu
Sourced in Japan, Canada
The UPLC (Ultra Performance Liquid Chromatography) system is an analytical instrument designed for high-performance liquid chromatography (HPLC) applications. It utilizes advanced technology to enable faster separation, higher resolution, and increased sensitivity compared to traditional HPLC systems.
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Lab products found in correlation
Scientz 100f, by Ningbo Scientz Biotechnology (2 mentions)
Scaa 104, by ANPEL (2 mentions)
Triple quad 5500, by AB Sciex (2 mentions)
Microtof 2 mass spectrometer, by Bruker (2 mentions)
Acquity uplc system, by Bruker (2 mentions)
Analyst 1, by AB Sciex (2 mentions)
Lightcycler 480 2 real time system, by Roche (1 mentions)
6500 q trap, by Thermo Fisher Scientific (1 mentions)
Rnaqueous total rna isolation kit, by Thermo Fisher Scientific (1 mentions)
Ion trap time of flight it tof mass spectrometer, by Shimadzu (1 mentions)
Nexera x2, by Shimadzu (1 mentions)
4500 q trap, by Thermo Fisher Scientific (1 mentions)
Nuclease p1, by Fujifilm (1 mentions)
Zorbax sb aq column, by Agilent Technologies (1 mentions)
Acquity csh c18 column, by Waters Corporation (1 mentions)
Dgu 20a5r, by Shimadzu (1 mentions)
4600 q tof mass spectrometer, by AB Sciex (1 mentions)
Peak 2, by AB Sciex (1 mentions)
Beh c18 analytical column, by Waters Corporation (1 mentions)
P 2000 digital polarimeter, by Jasco (1 mentions)
26 protocols using ultra performance liquid chromatography (uplc)
1
Metabolite Extraction and Analysis from Foliar Nectaries
2
Quantitative Real-Time PCR and Metabolomics of Tea Petals
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The quantitative real-time PCR (qRT-PCR) was performed as described before (Zhou et al., 2020 (link)). Briefly, total RNA was extracted from petals by using RNAqueous™ Total RNA Isolation Kit (Thermo, MA, United States). Primers of selected gene members were designed with the Primer Premier 5.0 software and listed in Supplementary Table 4 . β-Actin was used as the reference gene. The fluorescence PCR reagent was the Hieff™ qPCR SYBR Green Master Mix (No Rox) (Yaesen Biotech Co., Ltd., Shanghai, China). The experiment and analysis were carried out on the LightCycler® 480 II Real-Time System (Roche, CA, United States). Metabolome was carried out by using liquid chromatography–mass spectrometry.
The determination of the non-volatile metabolome of tea petals was described as earlier (Zhou et al., 2020 (link)). Briefly, 100 mg powder samples were extracted in 1.0 ml methanol (70%) at 4°C for 24 h, and 5 μl supernatant was injected into ultra-performance liquid chromatography (UPLC, Shimadzu Co., Kyoto, Japan) with a mass system (MS, Applied Biosystems 6500 Q TRAP, MA, United States). Metabolites were identified using MWDB (Metware Database, Metware Biotechnology Co., Ltd., Wuhan, China) and subject to the partial least squares (PLS) discriminant analysis. The significant dissimilarities of metabolites were set as the variable importance (VIP) ≥1 and the fold change ≥2 or ≤0.5.
The determination of the non-volatile metabolome of tea petals was described as earlier (Zhou et al., 2020 (link)). Briefly, 100 mg powder samples were extracted in 1.0 ml methanol (70%) at 4°C for 24 h, and 5 μl supernatant was injected into ultra-performance liquid chromatography (UPLC, Shimadzu Co., Kyoto, Japan) with a mass system (MS, Applied Biosystems 6500 Q TRAP, MA, United States). Metabolites were identified using MWDB (Metware Database, Metware Biotechnology Co., Ltd., Wuhan, China) and subject to the partial least squares (PLS) discriminant analysis. The significant dissimilarities of metabolites were set as the variable importance (VIP) ≥1 and the fold change ≥2 or ≤0.5.
3
Targeted Metabolomic Profiling by UPLC-IT-TOF
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The UPLC from Shimadzu (Kyoto, Japan) mentioned above was connected with an Ion Trap-Time of Flight (IT-TOF) mass spectrometer from Shimadzu. The molecules were analysed using an ion accumulation time of 10 msec and the collision energy for MSn was adjusted to 50% in the analysis and the isolation width of precursor ions was 3.0Th. For full-scan MS analyses, the spectra were recorded in the range of m/z 150–500. Data-dependent acquisition was set such that the most abundant ions in full-scan MS would trigger tandem mass spectrometry (MSn, n = 2–3). Data were acquired and processed by LC/MS solution software including a formula predictor to calculate elemental compositions.
4
Comprehensive Sphingolipid Profiling by UPLC-MS
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Sphingolipid extraction was performed as already described in the previous section (par 4.7) except for the addition of alkaline methanolysis step to enhance the recovery of low abundant sphingolipid species. The clear supernatant was injected on a Shimadzu UPLC coupled with a Triple TOF 6600 Sciex (Concord, ON, CA) equipped with Turbo Spray IonDrive. Sphingosines analysis was completed on Acquity BEH C18 column 1.7 μm, 2.1 × 100 mm (Waters, Franklin, MA, USA) by using, as mobile phase (A) 0.2% formic acid 2 mM ammonium formate in water and as mobile phase (B) methanol 0.2% formic acid 1 mM ammonium formate. The flow rate was 0.3 mL/min and the column temperature was 30°C. The elution gradient (%B) was set as follows: 0–10 min (80–99%), 10–15 min (99%), 15–15.2 min (99–80%), 15.2–20 min (80%). Sphingoid long-chain bases were determined by monitoring the high-resolution transitions m/z 380.25 > 264.26 (S1P), 300.28 > 282.27 (Sph), 302.30 > 284.29 (DhSph), and 382.27 > 284.29 (DhS1P), by applying a DP of 50 eV and CE 30 ± 15 eV. Quantitative analysis was corrected for internal standard responses (sphinganine d17:0; m/z 288.28 > 270.27), interpolation with calibration curves and the results were expressed as µM.
5
Freeze-Dried Metabolite Extraction and UPLC-MS/MS Analysis
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Biological samples were freeze-dried by vacuum freeze-dryer (Scientz-100F). The freeze-dried samples were crushed using a mixer mill (MM 400, Retsch) with a zirconia bead for 1.5 min at 30 Hz. One-hundred milligrams of lyophilized powder was dissolved with 1.2ml 70% aqueous methanol, vortexed 30 seconds every 30 minutes for 6 times in total, then placed in a refrigerator at 4°C overnight. Following centrifugation at 12000rpm for 10 min, the extracts were filtrated (SCAA-104, 0.22μm pore size; ANPEL, Shanghai, China, http://www.anpel.com.cn/ ) before Ultra Performance Liquid Chromatography Tandem Mass Spectrometry (UPLC-MS/MS, UPLC, SHIMADZU Nexera X2, https://www.shimadzu.com.cn/ ; MS/MS, Applied Biosystems 4500 QTRAP, http://www.appliedbiosystems.com.cn/ ) analysis.
6
MDA Quantification in Tissue Samples
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To prepare a MDA standard solution, 20 mL of 0.1 M HCl was added to 34 μL of 1,1,3,3-tetramethoxypropane (TMP) followed by incubation at 40°C for 1 h to hydrolyse TMP into MDA. The concentration of MDA in the standard solution was determined by measuring its absorbance at 245 nm (ε = 13,700) and freshly diluted with deionized water to establish a calibration curve.
Tissue samples (100 mg) were homogenized in 0.1 M phosphate buffer (pH 7.4) followed by an addition of 1 M KOH and 0.02 M BHT. After then, they were left to incubation at 60°C for 1 h with continuous shaking at dark. Following acidification with concentrated HCl to pH 2, they were centrifuged at 15000 ×g for 5 min at 4°C. The resulting supernatants were then derivatized with an equal volume of DNPH (1.2 mM) at 50°C for 60 min and protected from light. After derivatization, the sample was allowed to cool down and centrifuged at 15,000 ×g for 7 min at 4°C. The supernatant was transferred to a clean vial, filtered by 45 μm filter, and 20 μL of a resulting solution was injected onto the LC-MS/MS instrument (Shimadzu UPLC, AB-Sciex 4000 QTrap) for chromatographic analysis. The relative standard peak areas of MDA-DNPH were determined and compared with the peak areas of samples to calculate concentration as ng/mL.
Tissue samples (100 mg) were homogenized in 0.1 M phosphate buffer (pH 7.4) followed by an addition of 1 M KOH and 0.02 M BHT. After then, they were left to incubation at 60°C for 1 h with continuous shaking at dark. Following acidification with concentrated HCl to pH 2, they were centrifuged at 15000 ×g for 5 min at 4°C. The resulting supernatants were then derivatized with an equal volume of DNPH (1.2 mM) at 50°C for 60 min and protected from light. After derivatization, the sample was allowed to cool down and centrifuged at 15,000 ×g for 7 min at 4°C. The supernatant was transferred to a clean vial, filtered by 45 μm filter, and 20 μL of a resulting solution was injected onto the LC-MS/MS instrument (Shimadzu UPLC, AB-Sciex 4000 QTrap) for chromatographic analysis. The relative standard peak areas of MDA-DNPH were determined and compared with the peak areas of samples to calculate concentration as ng/mL.
7
Quantification of RNA Modifications
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For analysis of internal RNA modifications, 50–100 ng RNA was digested with 1 unit of Nuclease P1 (Wako) in 50 μL buffer containing 10 mM ammonium acetate (pH 5.3) at 60 °C for 2 h and then at 42 °C overnight, followed by the addition of 5.5 μL of 1 M fresh NH4HCO3 and 1 unit of shrimp alkaline phosphatase (rSAP, New England Biolab). The mixture was incubated at 37 °C for an additional 3 h. Digested samples were filtered through 0.22-mm syringe filters before UPLC-MS/MS analysis. The nucleosides were separated by UPLC (Shimadzu) equipped with a ZORBAX SB-Aq column (Agilent) and detected by MS/MS using a Triple Quad 5500 (AB SCIEX) mass spectrometer in positive ion mode by multiple reaction monitoring. The MS parameters were optimized for m2A detection. Nucleosides were quantified using the nucleoside-to-base ion mass transitions of m/z 268.0 to 136.0 (A), m/z 282.0 to 150.0 (m6A), m/z 282.0 to 150.0 (m1A), m/z 282.0 to 150.0 (m2A), m/z 282.0 to 150.0 (m8A), m/z 244.0 to 112.0 (C), and m/z 284.0 to 152.0 (G). Standard curves were generated by running a concentration series of pure commercial nucleosides (m2A and m8A from Carbosynth while others from Sigma-Aldrich). Concentrations of nucleosides and the m2A/A ratio in samples were calculated by fitting the signal intensities to the standard curves.
8
Lipidomic Analysis of Serum Samples
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Sera (25 µl) were extracted by methanol/chloroform mixture42 (link). The addition of butylated hydroxytoluene (BHT) during sample preparation avoided unspecific oxidation. LC–MS/MS consisted of a Shimadzu UPLC coupled with a Triple TOF 6600 Sciex (Concord, ON, CA) equipped with Turbo Spray IonDrive. All samples were analyzed in duplicate in positive mode with electrospray ionization. Spectra were contemporarily acquired by full-mass scan from m/z 200–1500 and top-20 data-dependent acquisition from m/z 50–1500. Declustering potential was fixed to 50 eV, and the collision energy was 35 ± 15 eV. The chromatographic separation was reached on a reverse-phase Acquity CSH C18 column 1.7 μm, 2.1 × 100 mm (Waters, Franklin, MA, USA) equipped with a precolumn by a gradient between (A) water/acetonitrile (60:40) and (B) 2-propanol/acetonitrile (90:10), both containing 10-mM ammonium acetate and 0.1% of formic acid42 (link).
9
UPLC-QTOF/MS Analysis of Metabolites
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The Shimadzu UPLC ((Shimadzu, Kyoto, Japan) system consists of an online degasser (DGU-20A5R), an auto-sampler (SIL-30AC), two pumps (LC-30AD), and a column oven (CTO-30aHE). Chromatographic separation was performed on a Waters BEHC18 analytical column (2.1 × 100 mm, 1.7 μm, Waters, Milford, MA, USA) at 40 °C. The mobile phase consisted of 0.2% formic acid and acetonitrile. The linear gradient elution with a constant flow rate of 0.2 mL/min was 10%~10%~40%~95%~10% acetonitrile at 0~1~10~13~15 min. The sample solution and mixed working solutions of 5 µL were injected into the UPLC system by the auto-sampler.
TOF/MS measurements in negative ion mode were performed on a 4600 Q-TOF mass spectrometer (AB Sciex, Concord, CA, USA) equipped with an electrospray ionization (ESI) source with the following parameters: Ion source gas 1 (GS1) at 50 psi, ion source gas 1 (GS1) (N2) at 50 psi, curtain gas at 35 psi, temperature at 500 °C, and ionspray voltage floating at −4500 V. The mass range was set to m/z 100–800. The system was operated under Analyst 1.6 and Peak 2.0 (AB Sciex, Concord, CA, USA) and used an APCI negative calibration solution to calibrate the instrument’s mass accuracy in real-time.
TOF/MS measurements in negative ion mode were performed on a 4600 Q-TOF mass spectrometer (AB Sciex, Concord, CA, USA) equipped with an electrospray ionization (ESI) source with the following parameters: Ion source gas 1 (GS1) at 50 psi, ion source gas 1 (GS1) (N2) at 50 psi, curtain gas at 35 psi, temperature at 500 °C, and ionspray voltage floating at −4500 V. The mass range was set to m/z 100–800. The system was operated under Analyst 1.6 and Peak 2.0 (AB Sciex, Concord, CA, USA) and used an APCI negative calibration solution to calibrate the instrument’s mass accuracy in real-time.
10
Characterization of Isolated Natural Products
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