To extract ceramide, DAG and PA, 20 mg of liver was homogenized in cold methanol/phosphate-buffered saline (2:1 v/v) for ceramide and cold chloroform/methanol (2:1 v/v) for DAG and PA. Organic phase were separated from aqueous phase by adding chloroform and water. After centrifugation, the organic layer was collected, dried under nitrogen flow, and reconstituted with 0.1% formic acid in methanol for ceramide, hexane/methylene chloride/ether (95/4.5/0.5 v/v/v) for DAG, and methanol for PA. The internal standards for ceramide, DAG, and PA are C17:0 ceramide (500 ng/ml), 1, 3-dipenta decanoin and phosphatidic acid (C10:0/10:0), respectively. Diacylglycerol was separated from triacylglycerol using preconditioned columns (Waters SepPak Cartridge, WAT020845) and eluted with hexane/ethyl acetate (85/15 v/v) under a low negative pressure. Ceramide and DAG were measured by LC-MS/MS 4000 Q TRAP (Applied Biosystems). Phosphatidic acid was analyzed by Agilent 6490 Triple Quadrupole LC/MS System (Agilent Technologies). Anionic phospholipids (PI, cardiolipin, PS and phosphoinositides) were measured using HPLC combined with suppressed conductivity (Dionex), as previously described54 (link).
Seppak cartridge
Manufactured by Waters Corporation
Sourced in United States, United Kingdom
The SepPak cartridge is a solid-phase extraction device used for sample preparation in analytical chemistry. It consists of a small column packed with various sorbent materials that can selectively retain and concentrate analytes from complex matrices, such as biological fluids or environmental samples, prior to instrumental analysis.
Automatically generated - may contain errors
Lab products found in correlation
Xbridge beh c18 column, by Waters Corporation (12 mentions)
Orbitrap fusion tribrid mass spectrometer, by Thermo Fisher Scientific (10 mentions)
Ultimate 3000 liquid chromatography system, by Thermo Fisher Scientific (9 mentions)
Seppak cartridge, by Thermo Fisher Scientific (5 mentions)
Tmt ten plex reagents, by Thermo Fisher Scientific (4 mentions)
Orbitrap fusion lumos mass spectrometer, by Thermo Fisher Scientific (3 mentions)
Sep pak vac cartridge, by Waters Corporation (3 mentions)
Tmt six plex reagents, by Thermo Fisher Scientific (3 mentions)
Tmtpro sixteen plex reagents, by Thermo Fisher Scientific (3 mentions)
Ultimate 3000, by Thermo Fisher Scientific (2 mentions)
Agilent 6490 triple quadrupole lc ms system, by Agilent Technologies (1 mentions)
Rf 20a, by Shimadzu (1 mentions)
Ascentis rp amide, by Merck Group (1 mentions)
J sphere ods h80 column, by YMC (1 mentions)
Tmt eleven plex reagents, by Thermo Fisher Scientific (1 mentions)
Hydroxylamine, by Merck Group (1 mentions)
Pda detector 2800, by Knauer (1 mentions)
Gf254 plates, by Merck Group (1 mentions)
Sephadex lh 20, by Cytiva (1 mentions)
Hplc system, by Knauer (1 mentions)
41 protocols using seppak cartridge
1
Lipid Extraction and Quantification from Liver Tissue
2
High-Throughput Proteome Fractionation
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For TMT Labeling and high pH reversed‐phase chromatography, the samples were labeled with tandem mass tag (TMT) multiplex reagents according to the manufacturer’s protocol (Thermo Fisher Scientific, Loughborough, UK) and the labeled samples were pooled. The pooled sample was then desalted using a SepPak cartridge according to the manufacturer’s instructions (Waters, Milford, Massachusetts, USA). Eluate from the SepPak cartridge was evaporated to dryness and resuspended in buffer A (20 mM ammonium hydroxide, pH 10) prior to fractionation by high pH reversed‐phase chromatography using an Ultimate 3,000 liquid chromatography system (Thermo Fisher Scientific). In brief, the sample was loaded onto an XBridge BEH C18 Column (130 Å, 3.5 µm, 2.1 mm × 150 mm, Waters, UK) in buffer A and peptides eluted with an increasing gradient of buffer B (20 mM ammonium hydroxide in acetonitrile, pH 10) from 0% to 95% over 60 min. The resulting fractions were evaporated to dryness and resuspended in 1% formic acid prior to analysis by nano‐LC MSMS using an Orbitrap Fusion Tribrid mass spectrometer (Thermo Fisher Scientific).
3
Multiplexed Quantitative Proteomics
4
Quantification of Urinary 1-Aminopyrene
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Urinary 1-AP levels were measured from 2 h urine samples. One milliliter of 10 M hydrochloric acid was added to each 10 mL urine sample, and the urine was stirred in a 90-°C water bath for 2 h. After adjusting the pH to 7.0–8.0, the supernatant was extracted with Sep-Pak cartridge (C18, 3 mL, 200 mg, Waters, Milford, UK). Before extraction, it was pre-conditioned with 5 mL of methanol and 5 mL of water. After loading the sample, it was washed with 5 mL of 20% methanol in water and then extracted with 4 mL of 100% methanol. The extract was dried with N2 gas and dissolved in 200 μL of methanol. The extract was analyzed by injecting 20 μL into the HPLC system equipped with a fluorescence detector (Shimadzu, RF-20A, Kyoto, Japan). A reverse phase-amide column (Ascentis RP-Amide, 25 cm × 4.6 mm, 5 μm, SUPELCO, Bellefonte, PA, USA) was used. The mobile phase was composed of methanol and 50 mM sodium acetate buffer pH 7.2 (80:20, v/v) at a flow rate of 1.0 mL/min, and the excitation and emission wavelengths were 254 nm and 425 nm, respectively (Fig. 1 ). Urinary 1-AP concentration was corrected using the urinary creatinine level.![]()
Chromatograms for 1-aminopyrene (
5
Radiosynthesis of N-Succinimidyl-4-[18F]Fluorobenzoate
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As described in our previous study[25 (link)], ethyl 4-trimethylammonium triflate benzoate [10 mg, 31.2 μmol, dissolved in 0.2 mL anhydrous acetonitrile (CH3CN)] was added to a reaction flask containing dry 18F and heated at 80 ℃ for 10 min. After cooling, 0.5 mL NaOH (0.5 mol/L) was added and reacted at 90 ℃ for 5 min. After cooling, 0.7 mL HCl (1 mol/L) was added for neutralization. The reaction solution was loaded onto a Waters Sep-Pak cartridge. Next, the cartridge was washed with 2 mL HCl (0.01 mol/L), blown dry with N2 and eluted with 3 mL CH3CN. The elution fraction was mixed with 20 μL of 10% aqueous (CH3)4NOH solution. The mixture was dried at 100 ℃ and then reacted with 12 mg O-(N-succinimidyl)-N, N, N’, N’-tetramethyluronium tetrafluoroborate (dissolved in 0.25 mL CH3CN) at 80 ℃ for 5 min. Then, the mixture was acidified with 3 mL of 5% aqueous acetic acid and purified on a YMC J’Sphere ODS-H80 column with a mobile phase of CH3CN/H2O (55%/45%) followed by final dilution with 6 mL of water. The above solution was loaded onto a Sep-Pak C18 cartridge. The cartridge was then dried and eluted to obtain N-succinimidyl-4-18F-fluorobenzoate (18F-SFB).
6
Urine Proteomics Analysis by TMT Labeling
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Urine samples were analysed at the University of Bristol Proteomics Facility. 190 μl of urine was digested with trypsin (1.25 μg trypsin; 37 °C, overnight), labelled with Tandem Mass Tag (TMT) eleven plex reagents according to the manufacturer’s protocol (Thermo Fisher Scientific, Loughborough, UK) and the labelled samples pooled. The pooled sample was desalted using a SepPak cartridge according to the manufacturer’s instructions (Waters, Milford, Massachusetts, USA). Eluate from the SepPak cartridge was evaporated to dryness and resuspended in 1% formic acid prior to analysis by nano-LC MSMS using an Orbitrap Fusion Lumos mass spectrometer (Thermo Scientific).
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Urine samples were analysed at the University of Bristol Proteomics Facility. 190 μl of urine was digested with trypsin (1.25 μg trypsin; 37 °C, overnight), labelled with Tandem Mass Tag (TMT) eleven plex reagents according to the manufacturer’s protocol (Thermo Fisher Scientific, Loughborough, UK) and the labelled samples pooled. The pooled sample was desalted using a SepPak cartridge according to the manufacturer’s instructions (Waters, Milford, Massachusetts, USA). Eluate from the SepPak cartridge was evaporated to dryness and resuspended in 1% formic acid prior to analysis by nano-LC MSMS using an Orbitrap Fusion Lumos mass spectrometer (Thermo Scientific).
7
Proteomic Quantification via TMT Labeling
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An equal volume of each protein sample was digested overnight at 37 °C with 2.5 µg trypsin, labelled with TMT ten plex reagents according to the manufacturer’s protocol (Thermo Fisher Scientific), and the labelled samples pooled. An aliquot of the pooled sample was evaporated to dryness, resuspended in 5% formic acid and then desalted using a SepPak cartridge according to the manufacturer’s instructions (Waters, USA). Eluate from the SepPak cartridge was again evaporated to dryness and resuspended in buffer A (20 mM ammonium hydroxide, pH 10) prior to fractionation by high pH reversed-phase chromatography using an Ultimate 3000 liquid chromatography system (Thermo Scientific). In brief, the sample was loaded onto an XBridge BEH C18 Column (130 Å, 3.5 µm, 2.1 mm × 150 mm, Waters, UK) in buffer A and peptides eluted with an increasing gradient of buffer B (20 mM ammonium hydroxide in acetonitrile, pH 10) from 0–95% over 60 min. The resulting fractions were evaporated to dryness and resuspended in 1% formic acid prior to analysis by nano-LC MSMS using an Orbitrap Fusion Tribrid mass spectrometer (Thermo Scientific).
8
TMTpro Isobaric Labeling of Peptides
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Digestion was halted by addition of 0.3% v/v trifluoroacetic acid (TFA), and peptides were desalted on Waters Sep-Pak® Vac cartridges (Waters™ Cat No: WAT054955) with a wash of 1 mL 0.1% TFA followed by elution in 0.6 mL of 70% acetonitrile 0.1% formic acid (FA). Peptides were dried by speed vacuum and resuspended 50 mM triethylammonium bicarbonate. Peptide concentrations were checked by Pierce Quantitative colorimetric assay (Cat No: 23275). The same amount of peptide from each sample was then labeled for 2 hours at room temperature, with 0.5 mg of Tandem Mass Tag Pro (TMTpro) reagent (16-plex kit, manufactures instructions Thermo Fisher Scientific, TMTpro™ Isobaric Label Reagent Set; Cat No: 44520, lot no. VI310352) (18 (link)). Labelling reactions were quenched with 0.3% hydroxylamine (v/v) at room temperature for 15 min. Labeled peptides were then mixed and dried by speed vacuum. The TMT-labeled peptide mix was desalted to remove excess label using a 100 mg Waters SepPak cartridge, eluted in 70% acetonitrile, 0.1% formic acid and lyophilized to dryness.
9
Tandem Mass Tag Proteomics Fractionation
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Aliquots of 25 μg of each sample were digested with trypsin (2.5 μg trypsin per 100 μg protein; 37°C, overnight), labelled with Tandem Mass Tag (TMT) 10 plex reagents according to the manufacturer’s protocol (Thermo Fisher Scientific, Loughborough, United Kingdom) and the labelled samples pooled. These were evaporated to dryness, resuspended in 5% (vol/vol) formic acid and desalted using a SepPak cartridge according to the manufacturer’s instructions (Waters, Milford, Massachusetts, United States). Eluate from the SepPak cartridge was again evaporated to dryness and resuspended in buffer A (20 mM ammonium hydroxide, pH 10) prior to fractionation by high pH, reversed-phase chromatography using an Ultimate 3,000 liquid chromatography system (Thermo Scientific). In brief, the sample was loaded onto an XBridge BEH C18 Column (130 Å, 3.5 μm, 2.1 mm × 150 mm, Waters, United Kingdom) in buffer A and peptides eluted with an increasing gradient of buffer B (20 mM ammonium hydroxide in acetonitrile, pH 10) from 0 to 95% over 60 min. The resulting fractions were evaporated to dryness and resuspended in 1% (v/v) formic acid prior to analysis by nano-LC MS/MS using an Orbitrap Fusion Lumos mass spectrometer (Thermo Fisher Scientific, United Kingdom).
10
Quantitative Proteomics Using TMT Labeling
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An equal amount (TMT1 = 40 μg, TMT2 = 40 μg, TMT3 = 35 μg) of each sample was digested with trypsin (2.5 μg trypsin per 100 μg protein; 37°C, overnight), labelled with Tandem Mass Tag (TMT) 6 or 10 plex reagents according to the manufacturer's protocol (Thermo Fisher Scientific) and the labelled samples pooled.
100 μg aliquots of pooled samples were evaporated to dryness, resuspended in 5% formic acid and then desalted using a SepPak cartridge according to the manufacturer's instructions (Waters). Eluates from the SepPak cartridge was again evaporated to dryness and resuspended in buffer C (20 mM ammonium hydroxide, pH 10) prior to fractionation by high pH reversed-phase chromatography using an Ultimate 3000 liquid chromatography system (Thermo Scientific). In brief, samples were loaded onto an XBridge BEH C18 Column (130 Å, 3.5 μm, 2.1 mm × 150 mm, Waters) in buffer C and peptides eluted with increasing gradient of buffer D (20 mM ammonium hydroxide in acetonitrile, pH 10) from 0 to 95% over 60 min. The resulting fractions were evaporated to dryness and resuspended in 1% formic acid prior to analysis by nano-LC–MS/MS using an Orbitrap Fusion Tribrid mass spectrometer (Thermo Scientific).
100 μg aliquots of pooled samples were evaporated to dryness, resuspended in 5% formic acid and then desalted using a SepPak cartridge according to the manufacturer's instructions (Waters). Eluates from the SepPak cartridge was again evaporated to dryness and resuspended in buffer C (20 mM ammonium hydroxide, pH 10) prior to fractionation by high pH reversed-phase chromatography using an Ultimate 3000 liquid chromatography system (Thermo Scientific). In brief, samples were loaded onto an XBridge BEH C18 Column (130 Å, 3.5 μm, 2.1 mm × 150 mm, Waters) in buffer C and peptides eluted with increasing gradient of buffer D (20 mM ammonium hydroxide in acetonitrile, pH 10) from 0 to 95% over 60 min. The resulting fractions were evaporated to dryness and resuspended in 1% formic acid prior to analysis by nano-LC–MS/MS using an Orbitrap Fusion Tribrid mass spectrometer (Thermo Scientific).
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