The phenolic profile was produced according to the method described previously by Pu24 (link), with some modification. HPLC analysis was performed on a Shimadzu (Kyoto, Japan) LC-20A system equipped with two pumps (LC-20AB), an injector (SIL-20A), and a detector (SPD-M20A). Separation was performed on a ZORBAX SB-C18 column (I.D. 250 × 4.6 mm, Agilent, USA). The column oven temperature was set to 30 °C. The absorbance was monitored at 253, 279, 284, 310, 324, 327, 355, and 370 nm. The solvent system had a constant flow rate of 0.7 mL/min and a 10 μL injection volume. Solvents such as 0.5% formic acid (eluant B) and methanol (eluant A) were used for the mobile phase. The gradient programs for the samples were as follows: 0–6 min, 90% B; 6–10 min, 90–80% B; 10–11 min, 80–75% B; 11–15 min, 75–70% B; 15–25 min, 70–60% B; 25–32 min, 60–45% B; 32–40 min, 45–0% B; 40–50 min, 0% B; 50–51 min, 0–90% B; 51–60 min, 90% B. The injection volume was maintained at 10 μL, and the constant temperature remained at approximately 30 °C. Detection wavelengths were chosen based on the absorption maximums of the UV spectra of the selected phenolic compounds.
Lc 20ab
Manufactured by Shimadzu
Sourced in Japan, Germany
The LC-20AB is a high-performance liquid chromatography (HPLC) system from Shimadzu. It is designed for analytical and semi-preparative liquid chromatography applications. The LC-20AB features a dual-plunger pump, a built-in degasser, and precise flow and gradient control.
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Lab products found in correlation
Spd m20a, by Shimadzu (10 mentions)
Cto 20ac, by Shimadzu (6 mentions)
Cbm 20a, by Shimadzu (6 mentions)
Sil 20ac ht, by Shimadzu (6 mentions)
Sil 20a, by Shimadzu (5 mentions)
Strata x c18 column, by Phenomenex (5 mentions)
Ultremex scx column, by Phenomenex (5 mentions)
Spd 20a, by Shimadzu (5 mentions)
Lc 20a system, by Shimadzu (4 mentions)
Dgu 20a, by Shimadzu (4 mentions)
Cto 20a, by Shimadzu (4 mentions)
Sil 20ac, by Shimadzu (4 mentions)
Api 4000 triple quadrupole mass spectrometer, by AB Sciex (4 mentions)
High performance liquid chromatography (hplc), by Shimadzu (3 mentions)
Zorbax sb c18 column, by Agilent Technologies (2 mentions)
Dgu 20a5, by Shimadzu (2 mentions)
Q exactive hf x, by Thermo Fisher Scientific (2 mentions)
Lcms 2020, by Shimadzu (2 mentions)
Ghlf plates, by Macherey-Nagel (2 mentions)
Lcms it tof, by Shimadzu (2 mentions)
76 protocols using lc 20ab
1
HPLC Analysis of Phenolic Compounds
2
Reverse-Phase HPLC Separation of Compounds
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The chromatographic separation of compounds was performed on a Shimadzu Prominence LC-20A System (Shimadzu, Kyoto, Japan) equipped with a degasser (DGU-20A5), a binary gradient pump (LC-20AB), and a diode array detection system (SPD-M20A, Shimadzu). The column used was a 5-μm Hypersil ODS C18 column (4.6 mm × 250 mm, i.d.), which was operated at a temperature of 30 °C. Methanol (A)-H2O (B) was used as the mobile phase. The LC gradient program was set as (Tmin/A:B; T0/40:60; T8.0/40:60; T10/50:50; T20/40:60; T26/40:60). The flow rate was 1.0 mL·min−1. The injection volume was 20 μL, and the wavelengths were set at 210 nm and 254 nm.
3
Quantitative Proteomics of MS275-Treated S180 Cells
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The S180 cells were treated with 5 μM MS275 or 0.4% (v/v) DMSO as control group for 48-h incubation and the sufficient samples were submitted for iTRAQ quantitative proteomics analysis (BGI, Shenzhen, China). Protein extraction, SDS-PAGE purification, protein digestion and peptide quantification was dealt as reference [1 (link)]. The peptide samples were respectively labeled using the iTRAQ Reagent-4plex Multiplex Kit (AB SCIEX) according to the manufacturer's instructions. The labeled peptide fractionation was carried out by using Shimadzu LC-20 AB liquid phase system with 5 μm 4.6 × 250 mm Gemini C18 column and followed by HPLC (Thermo UltiMate 3000 UHPLC). The nanoliter liquid phase separation end was directly connected to the mass spectrometer with a tandem mass spectrometer Q-Exactive HF X (Thermo Fisher Scientific, San Jose, CA). The MS/MS data were searched against the Mascot database (uniprot-human 20151227.fasta) for peptide identification and quantification. The searching result of peptides was filtered by FDR p value with a cut off of 0.05. Based on statistical dispersion of the dataset, ratio of > 1.5 or < 0.667 was used as a strict significance cutoff to acquire a short list of the differentially distributed proteins as indicated in the data legends.
4
SCX Chromatography Fractionation and Desalting
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SCX chromatography was performed using the Shimadzu LC-20AB HPLC pump system according to published methods32 (link). Ultimately, 20 fractions were obtained after screening, which were desalted by Strata X C18 column (Phenomenex) and vacuum-dried.
5
Fractionation and Mass Spectrometry of iTRAQ Peptides
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iTRAQ-labeled peptides were fractionated through SCX chromatography on an Ultremex SCX column using an LC-20AB high-performance liquid chromatography (HPLC) system (Shimadzu, Kyoto, Japan), and a total of 13 fractions were collected. Each fraction was desalted and dried for subsequent tandem MS analysis. All fractions were analyzed on a Q-Exactive mass spectrometer (Thermo Fisher Scientific, CA, USA) coupled to an LC-20AD nanoflow HPLC instrument (Shimadzu, Kyoto, Japan).
6
Quantification of PA Concentrations by LC-MS/MS
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The determination of PA concentrations after incubation with microsomes or supersomes was performed by LC-MS/MS as described earlier by Kaltner et al. (2019) and Enge et al. (2021) [16 (link),57 (link)]. Briefly, a 50 × 2.1 mm Kinetex 2.6 μm Core-Shell EVO C18 100 Å column (Phenomenex, Aschaffenburg, Germany) protected by a SecurityGuard ULTRA EVO C18 2.1 mm guard column (Phenomenex, Aschaffenburg, Germany) was used for chromatographic separation on a Shimadzu Prominence HPLC device (LC-20AB, SIL-20AC HT, CTO-20AC, CBM-20A, Shimadzu, Duisburg, Germany). Column oven temperature was maintained at 30 °C, the flow rate was consistently hold at 0.4 mL/min and the injection volume was 10 µL. The HPLC system was coupled to an API4000 triple quadrupole MS (Sciex, Darmstadt, Germany) which was operated in positive electro spray ionisation (ESI) mode with the following parameters: ionisation voltage: 2500 V; nebuliser gas: 50 psi; heating gas: 50 psi; curtain gas: 30 psi; temperature: 600 °C; collision gas: level 7. The selected PA analytes were determined in multiple reaction monitoring (MRM) mode and quantified by external calibration standards ranging from 10 to 125 nmol/mL in DMEM. The PA content was always normalized to the content at t = 0 h.
7
Shimadzu HPLC-MS/MS Protocol for Quantification
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A Shimadzu high-performance liquid chromatography (HPLC) apparatus including binary pumps, a degasser, an autosampler, a column oven and a control unit (LC-20AB, SIL-20AC HT, CTO-20AC, CBM-20A, Duisburg, Germany) was used for all measurements. The HPLC was coupled to an API4000 triple quadrupole mass spectrometer (MS) provided by Sciex (Darmstadt, Germany). The MS ion source parameters were set as follows: ESI + ionization voltage, 4.200 V; nebulizer gas, 50 psi; heating gas, 50 psi; curtain gas, 35 psi; temperature, 550 °C; collision gas level, 7. The MS parameters used are summarized in Online Resource 1 . Data acquisition and processing were conducted with Sciex Analyst (Version 1.6.2) and MultiQuant software (Version 3.0.1).
8
High-pH Reversed-Phase Peptide Separation
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The peptides were separated on a Shimadzu LC-20AB HPLC pump system coupled with a high pH RP column. The peptides were reconstituted with buffer A (5% ACN, 95% H2O, pH adjusted to 9.8 with ammonia) to 2 mL and loaded onto a column containing 5-μm particles (Phenomenex). The peptides were separated at a flow rate of 1 mL/min with a gradient of 5% buffer B (5% H2O, 95% ACN, pH adjusted to 9.8 with ammonia) for 10 min, 5%–35% buffer B for 40 min, and 35%–95% buffer B for 1 min. The system was then maintained at 95% buffer B for 3 min and decreased to 5% within 1 min before equilibration for 10 min. Elution was monitored by measuring the absorbance at 214 nm, and fractions were collected every 1 min. The eluted peptides were pooled as 20 fractions and vacuum-dried. More details about the HPLC analysis, mass spectrometry detection, and bioinformatics analysis are given in the Supplementary Materials.
9
Lipid Extraction and Quantification
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Plasma was sonicated and extracted with chloroform/methanol/Tris-HCl 50 mmol/L pH 7.5 (2 : 1 : 1, vol/vol) containing internal standards ([H2]8 AEA 5 pmol; [H2]5 2-AG, [H2]5 PEA, and [H2]4 OEA 50 pmol each) for EC quantification as well as 1,2-heptadecanoin (Larodan AB, Malmo, Sweden) for DAG measurement. The lipid-containing organic phase was dried down, weighed, and prepurified by open-bed chromatography on silica gel with 99 : 1, 90 : 10, and 50 : 50 (v/v) chloroform/methanol. The 90 : 10 fraction was used for EC and N-acylethanolamine quantification by LC-APCI-MS (LCMS-2020, Shimadzu) as previously reported [54 (link)]. DAG levels were measured by LC-MS-MS using an LC20AB coupled to a hybrid detector IT-TOF (Shimadzu Corporation, Kyoto, Japan) equipped with an ESI interface [55 (link)].
10
Physicochemical Characterization of Nanomaterials
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Electrospray Ionisation Mass Spectrometry (ESI-MS) analysis was done using a Perkin Elmer Sciex API3000 Instrument (Applied Biosystem/MDS Sciex, Toronto, ON, Canada) with Analytes 1.4 software. Analytical RP-HPLC analysis was done on a Shimadzu (Kyoto, Japan) instrument (DGU-20A5, LC-20AB, SIL-20ACHT, SPD-M10AVP) with a flow rate of 1 mL/min and detection at 214 nm. Analytical HPLC analysis was done with a 0%–100% gradient of analytical grade solvent A (0.1% TFA in water) to solvent B (90% MeCN, 10% water, 0.1% TFA) over 50 min and a Vydac analytical C18 column (218TP54; 5 µm, 4.6 mm × 250 mm). Preparative RP-HPLC analysis was done with a Shimadzu Instrument (Tokyo, Japan) at a flow rate of 20 mL/min and a Vydac C18 column, with detection at 230 nm. Particle size was measured by dynamic light scattering (DLS; Malvern Zetasizer Nano Series with DTS software). Transmission electron microscopy (TEM; HT7700 Exalens, HITACHI Ltd., Tokyo, Japan) was performed at the Australian Microscopy & Microanalysis Research Facility, Centre for Microscopy and Microanalysis, The University of Queensland (UQ). Element microanalysis (EA) was performed in the School of Chemistry and Molecular Biosciences, UQ, using a FLASH 2000 instrument (Thermo Fisher Scientific, Waltham, MA, USA).
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