Dried lipophilic cell extracts were reconstituted with 200 μL of a methanol:isopropanol:water mixture (65:30:5 by volume)(24 (link)) that contained an internal standard mixture made from the SPLASH Lipidomix standard mixture that included d7-PC(15:0/18:1), d7-PE(15:0/18:1), d7-PG(15:0/18:1), d7-PI(15:0/18:1), d7-PS(15:0/18:1), d7-LPC(18:1), d7-LPE(18:1), d7-TG(15:0/18:1/15:0), d7-DG(15:0/18:1), d7-MG(18:1), d9-SM(d18:1/18:1), d7-CE(18:1), and d7-cholesterol (Avanti Polar Lipids, Alabaster, AL, USA), Cer(d18:1/12:0) (Avanti Polar Lipids), and d27-myristic acid (Sigma-Aldrich, St. Louis, MO, USA). The reconstituted samples were then profiled using an Accela 1250 ultrahigh-performance liquid chromatography (UHPLC) system coupled to a Q-Exactive orbitrap mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA). Details regarding methods and instrumental settings are provided in SI . Quality control (QC) samples were prepared by pooling an equal amount (∼20%) of all processed samples and distributing aliquots into 8 vials after complete mixing. A QC sample was injected at the beginning, end, and after every 10 samples of the run.
Accela 1250
Manufactured by Thermo Fisher Scientific
Sourced in United States, Germany
The Accela 1250 is a high-performance liquid chromatography (HPLC) system designed for a wide range of analytical applications. It features a binary gradient pump, a temperature-controlled autosampler, and a ultraviolet-visible (UV-Vis) detector. The Accela 1250 is capable of delivering solvent flow rates up to 5 mL/min and can operate at pressures up to 400 bar.
Automatically generated - may contain errors
Lab products found in correlation
Proteome discoverer software, by Thermo Fisher Scientific (3 mentions)
Ltq orbitrapexactive mass spectrometer, by Thermo Fisher Scientific (3 mentions)
Kinetex f5 column, by Phenomenex (2 mentions)
Eclipse plus rr hd, by Agilent Technologies (2 mentions)
Msq plus, by Thermo Fisher Scientific (2 mentions)
Q exactive orbitrap mass spectrometer, by Thermo Fisher Scientific (1 mentions)
Cholesterol d7, by Avanti Polar Lipids (1 mentions)
D7 pe, by Avanti Polar Lipids (1 mentions)
D7 lpc, by Avanti Polar Lipids (1 mentions)
Hypersil column, by Thermo Fisher Scientific (1 mentions)
Tracefinder 4, by Thermo Fisher Scientific (1 mentions)
Thermo xcalibur software, by Thermo Fisher Scientific (1 mentions)
Accela lc, by Thermo Fisher Scientific (1 mentions)
Zorbax sb c8, by Agilent Technologies (1 mentions)
Tsq vantage, by Thermo Fisher Scientific (1 mentions)
Atlantis t3, by Waters Corporation (1 mentions)
Tsq vantagetm, by Thermo Fisher Scientific (1 mentions)
Agilent extend c18, by Agilent Technologies (1 mentions)
Xcalibur software, by Thermo Fisher Scientific (1 mentions)
Thermo exactive orbitrap mass spectrometer, by Thermo Fisher Scientific (1 mentions)
21 protocols using accela 1250
1
Lipid Profiling Using UHPLC-MS
2
Phenolic Compounds and Glutathione Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Analysis of the phenolic compounds and glutathione was carried out via liquid chromatography–high-resolution mass spectrometry (LC-HRMS) according to Fia et al. [10 (link)], using an Accela 1250 (Thermo Fisher Scientific, Waltham, MA, USA) coupled with a Linear Trap Quadrupole (LTQ) OrbitrapExactive mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA) equipped with an electrospray ionization (ESI) source in negative mode. Peak assignment was carried out on the basis of the exact mass values of the molecular ions and the cis and trans forms were recognized by comparison of the retention times with the standard sample. The standards were purchased from Sigma-Aldrich (Milan, Italy), except for the quercetin 3-O-glucoside, which was supplied by Analytik GmbH (Rülzheim, Germany). Coumaric and ferulic acids were used as standards for coutaric and fertaric acids due to the lack of reference materials. Data were expressed as mg of phenols/kg of the UG extract or beetroot sample. The phenols recovered (recovery %) from the functionalized beetroot samples were calculated as the percentage of the sum of UG phenols added plus phenols measured in the beetroot puree before the addition.
3
Tryptophan Metabolites Analysis by HRMS
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Tryptophan metabolites were
analyzed according to Koper et al.25 (link) Samples
were centrifuged (21700g, 10 min, 4 °C) and
diluted in 0.1% formic acid, followed by filtration using a 0.22 μm
cellulose filter (Phenomenex) and high-resolution mass spectrometry
(HRMS) analysis. A silica modified Luna Polar C18 column (50 ×
2.1 mm, 1.6 μm, Phenomenex) was used for the chromatographic
separation of Trp and tryptophan metabolites. The mobile phases consisted
of water (A) and acetonitrile (B) both with 0.1% v/v of formic acid,
and the following gradient (min/%B) was used: (0/2), (0.50/2), (9.5/70),
and (12/70). The flow rate was 200 μL/min, the column temperature
was 40 °C, and 5 μL was injected. The U-HPLC system (Accela
1250, Thermo Fisher, Bremen, Germany) was interfaced to an Exactive
Orbitrap HRMS (Thermo), and the analytes were detected through a heated
electrospray interface (HESI-II) in positive mode by scanning the
ions listed inTable S1 in the m/z range of 50–400. Analytical
performances, mass spectrometry optimization, and linearity range
were monitored according to Koper et al.25 (link) Each sample was analyzed in duplicate, and the concentrations are
given in micromolars.
analyzed according to Koper et al.25 (link) Samples
were centrifuged (21700g, 10 min, 4 °C) and
diluted in 0.1% formic acid, followed by filtration using a 0.22 μm
cellulose filter (Phenomenex) and high-resolution mass spectrometry
(HRMS) analysis. A silica modified Luna Polar C18 column (50 ×
2.1 mm, 1.6 μm, Phenomenex) was used for the chromatographic
separation of Trp and tryptophan metabolites. The mobile phases consisted
of water (A) and acetonitrile (B) both with 0.1% v/v of formic acid,
and the following gradient (min/%B) was used: (0/2), (0.50/2), (9.5/70),
and (12/70). The flow rate was 200 μL/min, the column temperature
was 40 °C, and 5 μL was injected. The U-HPLC system (Accela
1250, Thermo Fisher, Bremen, Germany) was interfaced to an Exactive
Orbitrap HRMS (Thermo), and the analytes were detected through a heated
electrospray interface (HESI-II) in positive mode by scanning the
ions listed in
performances, mass spectrometry optimization, and linearity range
were monitored according to Koper et al.25 (link) Each sample was analyzed in duplicate, and the concentrations are
given in micromolars.
4
Quantification of Collagen Modifications
5
Phenolic and Glutathione Quantification
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The phenolic composition and glutathione content were measured with liquid chromatography high-resolution mass spectrometry (LC-HRMS) following the method described by Fia et al. [21 (link)]. A chromatograph Accela 1250 (Thermo Fisher Scientific, Waltham, MA, USA), a Kinetex F5 column (2.1 × 100 mm 1.7 μm-Phenomenex (Torrance, CA, USA)) and an LTQ OrbitrapExactive mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA) were used. The quantitative analysis was performed with TraceFinderTM 4.1 software (Thermo Fisher Scientific, Waltham, MA, USA) following an external standard method, using a linear regression from 0.05 to 1 g/L of five standard solutions.
6
UPLC Analysis of Organic Compounds
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The samples were analyzed on a Thermo UPLC system (Accela 1250, Thermo). UPLC separation was accomplished on an octadecylsilane (ODS) Hypersil column (2.1 mm × 250 mm, 1.9 μm, Thermo). The mobile phase, consisting of ACN and 0.1% formic acid in water, was used at a flow rate of 0.44 mL/min. The gradient elution program was: 5%–15% ACN (B) (0–1 min), 15% B (1–4 min), 15%–25% B (4–5 min), 25%–40% B (5–6 min), 40%–55% B (6–8 min), 55%–100% B (8–9 min), 100% B (9–11 min), 100%–5% B (11–12 min), and 5% B (12–14 min). The injection volume was 5 μL, and the detection wavelength was 254 nm. The column temperature was maintained at 45 °C using a temperature controller.
7
Phenolic and Glutathione Quantification
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The phenolic composition and glutathione content were measured with liquid chromatography-high-resolution mass spectrometry (LC-HRMS) following Fia et al. [22 (link)], using a chromatograph Accela 1250 (Thermo Fisher Scientific, Waltham, MA, USA) and a Kinetex F5 column (2.1 × 100 mm 1.7 µm—Phenomenex (Torrance, CA, USA)). An LTQ OrbitrapExactive mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA) was used. The quantitative analysis was performed with the TraceFinder™ 4.1 software (Thermo Fisher Scientific, Waltham, MA, USA) with an external standard method, using a linear regression from 0.05 to 1 g/L of five standard solutions.
8
Collagen Hydroxylation Analysis by Mass Spectrometry
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Mass spectrometric analysis of 3Hyp content within collagen α-chains was performed as previously described [5] (link), [18] (link). Collagen α-chains and CNBr peptides were cut from SDS-PAGE gels and subjected to in-gel trypsin digestion. Electrospray mass spectrometry was carried out on the tryptic peptides using an LTQ XL linear quadrapole ion-trap mass spectrometer equipped with in-line Accela 1250 liquid chromatography and automated sample injection (ThermoFisher Scientific). Thermo Xcalibur software and Proteome Discoverer software (ThermoFisher Scientific) were used for peptide identification. Tryptic peptides were also identified manually by calculating the possible MS/MS ions and matching these to the actual MS/MS spectrum. Hydroxyl differences were determined manually by averaging the full scan MS over several minutes to include all the posttranslational variations of a given peptide. Protein sequences used for MS analysis were obtained from the Ensembl genome database.
9
Liquid Chromatographic Analysis of Samples
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The liquid chromatographic system was an Accela LC (Thermo Scientific, Bremen, Germany) equipped with degasser, Accela 1250 pump, autosampler thermostated at 10°C and a heated column compartment. The column employed was a Zorbax SB-C8, 2.1 x 50 mm and 1.8 µm particle size from Agilent Technologies (Böblingen, Germany) thermostated at 25°C. The mobile phases were 1 mM NH4OAc/0.01% FA in water (A) and 0.01% FA in acetonitrile (B). The gradient program was as follows: 100% A for 0.5 min, then decreased linearly to 20% in 7.0 min, and finally decreased linearly to 0% in 0.5 min and held at 0% for 1.5 min followed by an increase to the initial concentration of 100% A in 0.1 min. Equilibrium time was 2.4 min resulting in a total run time of 12 min. The flow rate was set constant at 250 µL · min−1 and the injection volume was 20 µL.
10
Quantifying NADP+ and NADPH from Cell Pellets
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The protocol for extraction of NADP+ and NADPH from cell pellets was adapted from the works of Canelas et al., (2009) (link). One milliliter samples of 24 h grown cultures were centrifuged and the cell pellets were extracted in 1 mL of 5 mM ammonium acetate (pH 8.0) for 3 min at 85 °C with intermediate mixing. After centrifugation, supernatants were separated on a silica-based C18 column (Waters Atlantis T3, 2.1×150 mm2, 3 µm). The HPLC system (Thermo Accela 1250) was coupled to a triple quadrupole MS system (Thermo TSQ Vantage) via a heated ESI ion source for quantification. As described by Ortmayr et al., (2014) an absolute quantification of NADPH is limited by its rapid oxidation and the lack of an isotopically labeled standard to compensate for losses during sample preparation. However relative quantification can be achieved reproducibly. NADPH was also measured in the same samples with the enzymatic cycling assay as described by Ask et al. (2013) (link) showing the same relative NADPH levels.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!