Sample analysis was performed using the Thermo Accela UHPLC system (Thermo Fisher Scientific, San Jose, California, United States). Chromatographic separation was performed on a maintained reverse-phase column Waters HSS T3-C18 (2.1 × 100 mm, 1.8 µm). The mobile phase was a mixture of methanol (A) and 0.1% formic acid in water (B). The following elution gradient was used: 0–5 min, 3%–10% A; 5–25 min, 10%–40% A; 25–35 min, 40%–60% A; 35–45 min, 60%–80% A; 45–50 min, 80%–95% A; 50–60 min, 95% A. The flow rate was set to 0.3 ml/min, and the injection volume was 1 µL.
Hss t3 c18
Manufactured by Waters Corporation
Sourced in United Kingdom, Japan, Ireland, United States
The HSS T3 C18 is a high-performance liquid chromatography (HPLC) column designed for the separation and analysis of a wide range of organic compounds. The column features a C18 stationary phase that provides efficient and reliable separation of analytes.
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Lab products found in correlation
Thermo accela uhplc system, by Thermo Fisher Scientific (1 mentions)
Pst c18, by Waters Corporation (1 mentions)
Synapt g2 hdms mass spectrometer, by Waters Corporation (1 mentions)
Glu1 fibrinopeptide b, by Waters Corporation (1 mentions)
Masslynx version 4, by Waters Corporation (1 mentions)
Nanoacquity uplc system, by Waters Corporation (1 mentions)
Synapt g2 si hdms mass spectrometer, by Waters Corporation (1 mentions)
Lc30 system, by Shimadzu (1 mentions)
Acquity ultra performance liquid chromatography, by Waters Corporation (1 mentions)
Hss t3 c18 column, by Waters Corporation (1 mentions)
8 protocols using hss t3 c18
1
Reverse-Phase UHPLC for Compound Separation
2
Peptide Separation and Characterization
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Peptides were loaded on reversed-phase trap column PST C18, 100 Å, 5 μm, 180 μm × 20 mm (Waters Corporation, UK) with a flow rate of 15 μL/min using loading buffer of 0.1% formic acid and subsequently separated on HSS-T3 C18 1.8 μm, 75 μm × 250 mm analytical column (Waters Corporation, UK) in 90 min linear gradient (A: 0.1% formic acid, B: 100% CH3CN, and 0.1% formic acid) at a flow rate of 300 nl per min.
The nano-LC was coupled with HDMS Synapt G2 mass spectrometer (Waters Corporation, UK). Data were acquired using Masslynx version 4.1 software (Waters Corporation, UK) in positive ion mode. LC-MS data were collected using data independent acquisition (DIA) mode MSE in combination with online ion mobility separation. The trap collision energy of mass spectrometer was ramped from 18 to 40 eV for high-energy scans in MSE mode. The trap and transfer collision energy for high-energy scans in HDMS mode was ramped from 4 to 5 eV and from 27 to 50 eV. For both analyses, the mass range was set to 50–2,000 Da with a scan time set to 0.9 seconds. A reference compound [Glu1]-fibrinopeptide B (Waters Corporation, UK) was infused continuously (500 fmol/μL at flow rate 500 nL per min) and scanned every 1 minute for online mass spectrometer calibration purpose. The samples were run in triplicate.
The nano-LC was coupled with HDMS Synapt G2 mass spectrometer (Waters Corporation, UK). Data were acquired using Masslynx version 4.1 software (Waters Corporation, UK) in positive ion mode. LC-MS data were collected using data independent acquisition (DIA) mode MSE in combination with online ion mobility separation. The trap collision energy of mass spectrometer was ramped from 18 to 40 eV for high-energy scans in MSE mode. The trap and transfer collision energy for high-energy scans in HDMS mode was ramped from 4 to 5 eV and from 27 to 50 eV. For both analyses, the mass range was set to 50–2,000 Da with a scan time set to 0.9 seconds. A reference compound [Glu1]-fibrinopeptide B (Waters Corporation, UK) was infused continuously (500 fmol/μL at flow rate 500 nL per min) and scanned every 1 minute for online mass spectrometer calibration purpose. The samples were run in triplicate.
3
Proteomic Analysis of Streptomyces Catechol Response
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For the analysis of the expression of biosynthetic enzymes in response to catechol, we applied natural product proteomining34 (link). Samples were prepared from Streptomyces surface-grown cultures grown with or without catechol. Desalted peptide solutions were injected into Waters nanoAcquity UPLC system equipped with a Waters HSS T3 C18 (1.8 μm, 100 Å, 75 μm × 250 mm). A gradient from 1% to 40% acetonitrile in water (with added 0.1% FA) over 110 min was applied. Online MS/MS analysis was done using a Waters Synapt G2-Si HDMS mass spectrometer with a UDMSE method set up as described previously55 (link). [Glu1]-fibrinopeptide B was used as a lock mass compound and sampled every 30 s. Raw data from all samples were first analysed using the vender software ProteinLynx Global SERVER (PLGS, version 3.0.3, waters, USA). The resulting dataset was imported in ISOQuant version 1.855 (link) for label-free quantification and log2 fold changes were calculated. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE56 (link) partner repository with the dataset identifiers PXD029669, PXD030319, and PXD030484.
4
HPLC Analysis of Organic Compounds
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HPLC was performed according to a published method [16 (link)]. The injection was 2 μL. The column was 10 cm long HSS T3 C18 (Waters, 1.8 µm, 2.1 mm. The gradient was: 100% of A (water, 0.04% acetic acid) at 0 min, 95% of B (acetonitrile, 0.04% acetic acid) at 11 min, 95% of B at 12 min, 5% of B at 12.1 min (till 15 min). The flow rate was 0.40 mL/min and temperature was 40 °C.
5
UHPLC-MS/MS Analysis of Compounds
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The UHPLC analysis was conducted utilizing an LC30 system (Shimadzu Co., Tokyo, Japan) equipped with an HSS T3 C18 (100 mm × 2.1 mm × 1.8 μm) column sourced from Waters Co. (Milford, MA, USA). The mobile phases consisted of solvent A (0.1 % formic acid in water) and solvent B (acetonitrile). The elution program was optimized based on a previous protocol (Chen et al., 2021 (link)): 0–2 min, 5.0 % B; 2–5 min, 5 %-10 % B; 5–14 min, 10 %-40 % B; 14–24 min, 40 %-100 % B; 24–26.5 min, 100 %-100 % B; 26.5–27 min, 100 %-5% B. A re-equilibration step was carried out for three minutes at 5 % B. The system operated at a flow rate of 0.3 mL/min, with an injection volume of 5 µL and a column temperature of 40 °C.
Mass spectrometry (SCIEX Co., Framingham, MA, USA) was performed in positive ion mode, with a mass range of 50–1000 m/z. The following settings were used for data collection: declustering potential at 50 V, collision energy at 10 V, and a temperature of 450 °C, curtain gas 35 psi, nebulizer gas (nitrogen) pressure at 2 bar, capillary voltage at 4,200 V (L. Zhang et al., 2023b). The MS/MS data collection was carried out in IDA mode, where the top 10 ions of each spectrum were fragmented using collision-induced dissociative energy of 35 ± 10 eV (Li, Zhou, et al., 2023).
Mass spectrometry (SCIEX Co., Framingham, MA, USA) was performed in positive ion mode, with a mass range of 50–1000 m/z. The following settings were used for data collection: declustering potential at 50 V, collision energy at 10 V, and a temperature of 450 °C, curtain gas 35 psi, nebulizer gas (nitrogen) pressure at 2 bar, capillary voltage at 4,200 V (L. Zhang et al., 2023b). The MS/MS data collection was carried out in IDA mode, where the top 10 ions of each spectrum were fragmented using collision-induced dissociative energy of 35 ± 10 eV (Li, Zhou, et al., 2023).
6
UPLC-TOF-MS Metabolite Separation Protocol
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UPLC conditions: Sample separation was achieved using a Waters HSS T3-C18 (1.8 μm, 3.0 mm × 50 mm, Waters, Wexford, Ireland) at 40 °C. The flow rate was 0.5 mL/min. The injection volume was 3 μL for each run. The mobile phase was composed of 90% methanol in water (A) and Isopropanol:Acetonitrile = 1:1 (B) with a gradient program: 0–10 min, 0–11% B; 10–18 min, 100% B. The detection wavelength was 254 nm.
TOF-MS conditions were consistent withSection 4.4.1 .
TOF-MS conditions were consistent with
7
Phenolic Profiling of Cloudberry Extract
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Preparation of extract from freeze-dried cloudberries followed the same procedure as for the cell culture studies. The final cloudberry extract was dissolved in 20% methanol, and filtered with 0.2 μm syringe filters prior to analysis. The phenolic profile of berry extract was determined according to the method described earlier [48 (link)] using Waters ACQUITY ultra performance liquid chromatography (UPLC) coupled with eλ photodiode array (PDA) and fluorescence (FLR) detectors. The column used was a Waters HSS T3 C18, 1.7 μm, 2.1 × 150 mm, with 5 μL injection volume, and separation was achieved using a gradient program with water/0.5% formic acid and acetonitrile/0.5% formic acid with a constant flow rate of 0.5 mL/min. Samples were analysed in triplicate with double injections. Identification of phenolic compounds was based on their UV spectra, and phenolics were clustered into seven subclasses: ellagic acids, 365 nm and ellagitannins as ellagic acids, 280 nm; anthocyanins as cyanidin-3-glucosides, 520 nm; flavonols as rutin, 365 nm; hydroxycinnamates as chlorogenic acid, 320 nm; hydroxybenzoates as gallic acid, 280 nm; catechins and flavan-3-ols as procyanidin B2, FLR.
8
Optimization of UHPLC Separation for Ginsenosides
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To separate 26 ginsenosides, four kinds of C18 UHPLC columns such as kinetex (2.1 x 100 mm, 2.6 μm, Phenomenex, Torrance, CA, USA), Luna (2.0 x 100 mm, 3 μm, Phenomenex, Torrance, CA, USA), BEH (2.1 x100 mm, 1.7 μm, Waters, Milford, MA, USA), and HSS T3 C18 (2.1 x 150 mm, 1.8 μm, Waters, Milford, MA, USA) were compared with 0.1% formic acid in water (mobile phase A) and methanol (mobile phase B) as mobile phase. The gradient program used for the comparing was as follows: 0-1 min (50% B); 1-45 min (50-95% B); 45-50 min (95% B); 50-50.1 min (95-50% B); 50.1-60 min (50% B). In addition, shorter HSS T3 C18 column (2.1 x 50 mm, 1.8 μm, Waters, Milford, MA, USA) was further tested to minimize analytical time, using the following gradient program: 0-0.1 min (30% B); 0.1-1 min (30-50% B);
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