For size exclusion chromatography, proteins were first buffer-exchanged into 300 mM ammonium acetate, pH 6.8 with Micro Bio-Spin™ 6 columns (Bio-Rad). 100 pmol protein was injected onto a bioZen™ 1.8 μm SEC-2 column (150 x 4.6 mm) equipped with a security guard column (Phenomenex), and eluted with 300 mM ammonium acetate at a flow rate of 0.2 mL min−1 using an UltiMate™ 3000 RSLC (Thermo Fisher Scientific) coupled to an Exactive™ Plus EMR Orbitrap™ mass spectrometer (Thermo Fisher Scientific) equipped with an ESI source and modified to incorporate a quadrupole mass filter and allow for surface-induced dissociation [19 (link)]. Mass spectra were recorded from 1000 - 10000 m/z at 8750 resolution as defined at 200 m/z. The ion injection time was set to 200 ms. Voltages applied to the transfer optics were optimized to allow for optimal ion transmission. Total ion chromatograms were smoothed (Boxcar; 15 points) and overlaid. Mass spectra were deconvoluted and plotted with UniDec version 4.0.0 beta [20 (link)] using the preset high-resolution native parameters adjusted to sample mass every 1 Da.
Security guard column
Manufactured by Phenomenex
Sourced in United States
The Security Guard column is designed for the separation and analysis of a wide range of compounds. It features a robust, non-polar stationary phase that provides reliable and consistent chromatographic performance. The column is suitable for use in a variety of analytical applications.
Automatically generated - may contain errors
Lab products found in correlation
Sil 10advp autosampler, by Shimadzu (2 mentions)
Breeze 2, by Waters Corporation (2 mentions)
Kinetex c18 column, by Phenomenex (2 mentions)
Exactive plus emr orbitrap mass spectrometer, by Thermo Fisher Scientific (1 mentions)
Micro bio spin 6 column, by Bio-Rad (1 mentions)
Ultimate 3000 rslc, by Thermo Fisher Scientific (1 mentions)
Lc 10, by Shimadzu (1 mentions)
1200 series hplc system, by AB Sciex (1 mentions)
Discovery hs f5 3 hplc column, by Merck Group (1 mentions)
Api 4000 qtrap, by AB Sciex (1 mentions)
1525 binary pump, by Waters Corporation (1 mentions)
2707 autosampler, by Waters Corporation (1 mentions)
Rp c18, by Phenomenex (1 mentions)
Hilic column, by Phenomenex (1 mentions)
Gemini, by Phenomenex (1 mentions)
Spectra system scm1000, by Thermo Fisher Scientific (1 mentions)
Excalibur v 2, by Thermo Fisher Scientific (1 mentions)
Luna c18, by Phenomenex (1 mentions)
Labsolution software, by Shimadzu (1 mentions)
Model lc 20at, by Shimadzu (1 mentions)
18 protocols using security guard column
1
Native Mass Spectrometry of Proteins
2
RP-HPLC Analysis of Glutathione Metabolites
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The levels of the reduced (GSH) and oxidized (GSSG) glutathione, cysteine and cystine in the incubation mixtures were determined using the RP-HPLC method of Dominic et al. [27 (link)] with modifications [28 (link)]. The samples were separated on a 4.6 mm × 250 mm Luna C18 (5 µm) column with a Phenomenex Security Guard column filled with the same packing material. The chromatographic system consisted of LC-10 Atvp Shimadzu Corp. pumps, four channel degassers, column oven, a Shimadzu SIL-10 Advp autosampler and a Shimadzu Corp. SIL-10 SPD-M10Avp-diode array detector; Lab Solution LC software was used to control system operation and facilitate data collection. The standard curves were generated in the supernatant obtained from cellular homogenates in the range from 13 to 75 nM of each compound per ml. All the standard curves generated for the analyte were linear in the investigated concertation range.
3
HPLC-MS/MS Quantification of 2-HG
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HPLC-MS/MS analysis was performed (as described recently)22 (link) using an Agilent (Boeblingen, Germany) 1200 Series HPLC system coupled to an API 4000 QTRAP (AB SCIEX, Darmstadt, Germany) mass spectrometer operating in negative ionization mode. A Discovery HS F5-3 HPLC column (15 cm × 2.1 mm, 3 µm; Supelco, Bellefonte, PA, USA) equipped with a Security Guard column (C18, Phenomenex, Aschaffenburg, Germany) was used. Gradient elution was performed as described in Supplementary Table S5 with phase A consisting of 0.1% formic acid (FA) in water (v/v) and 100% acetonitrile (ACN) as mobile phase B. The column was kept at 30 °C, and an injection volume of 5 μL was used. Turbo ion spray was operated employing the following parameters: gas 1 and 2: 50 psi and curtain gas: 10 psi. The ion spray voltage was set to −4500 V, the declustering potential to −40.0 V, the entrance potential to −10.0 V, the collision exit potential to −5 V, and the collision energy to −24 V. Detection was performed in multiple reaction monitoring (MRM) mode using the following ion transitions: m/z 147.07 (M - H)− to m/z 84.80 for 2-HG and m/z 150.07 to m/z 87.80 for the deuterated internal standard. Quantification was achieved using a calibration curve constructed from the area ratio of 2-HG to the stable isotope-labelled standard.
4
Quantitative Analysis of Metabolites
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The method of analysis of α-KGM and TCA metabolites published previously was used [16 (link),18 (link)]. The HPLC configuration consisted of a Waters 2489 UV/VIS detector, a Waters 1525 binary pump, a Waters 2707 autosampler with a cooled platform (4 °C), a C18 analytical column, YMC, Triart, 250 × 3.0 mm, 3 μm with a Phenomenex Security guard column (C18, 4 × 2 mm). The columns were kept at room temperature, the injection volume was 15 μL at an isocratic flow rate of the mobile phase (20 mM KH2PO4, pH 2.9) of 0.45 mL/min. Metabolites were determined at a wavelength of 210 nm. Sample analysis was controlled using Breeze2 software (Waters Corporation) installed on a Dell computer.
Identification and quantification of individual metabolites was performed against appropriate standards of known concentration. The levels of their content in blood plasma were calculated (µM), and in tissue samples they were recalculated per milligram of wet weight.
Identification and quantification of individual metabolites was performed against appropriate standards of known concentration. The levels of their content in blood plasma were calculated (µM), and in tissue samples they were recalculated per milligram of wet weight.
5
Reversed-phase LC-MS Gradient Protocol
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For reversed-phase chromatography a Kinetex C18 column (100 mm × 2.1 mm, 2.6 μm particles) with a SecurityGuard column (2.1 mm × 2 mm, 2 μm particles) was used (Phenomenex, Torrance, CA, USA). The column temperature was set at 35°C and the autosampler temperature was 5°C. The gradient elution solvents were A; 95:5 water-acetonitrile (v/v), containing 5 mM ammonium formate and 0.1% formic acid (v/v), and B; 95:5 acetonitrile-water, containing 5 mM ammonium formate and 0.1% formic acid. The gradient (A:B, v/v) was as follows: an isobaric period at 98:2 for 5 min, followed by a linear gradient from 98:2 to 2:98 in 25 min, then held at 2:98 for 5 min, followed by a linear gradient change from 2:98 to 98:2 in 1 min, then held at 98:2 for 5 min. The flow rate was 0.2 mL/min.
6
HPLC Analysis of CoA and Acetyl-CoA
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For our most effective HPLC analysis procedure we used Waters instruments (Milford, MA, USA), 1525 binary pump, 2707 autosampler with pre-cooled platform (4 °C), and a 2489 UV/VIS detector. Data collection and data analysis were aided by Breeze2 software (Waters Corp, Milford, MA, USA) installed on a Dell computer. The wavelength for UV detection was set at 259 nm. Separation of CoA from acetyl-CoA was achieved with an ESA Inc. (now Dionex & Thermo Fisher Scientific Company, Bedford, MA, USA) RP-C18, 150 × 3 mm, 3 µm, 120 Å (PN# 70-0636) analytical column equipped with a Phenomenex Security Guard column (Torrance, CA, USA), cartridge C18, 4 × 2 mm, (PN# AJ0-4286). The columns were maintained at room temperature, the flow rate was 0.5 mL/min, and the injection volume was 30 µL. Under these conditions CoA and acetyl-CoA elute at 3.8 and 7.8 min, respectively. The HPLC run for both metabolites is completed within 12 min without any additional time for column re-equilibration between the HPLC runs.
7
HILIC Chromatography for Metabolite Analysis
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For HILIC chromatography a Kinetex HILIC column (100 mm × 2.1 mm, 2.6 μm particles) with a SecurityGuard column (2.1 mm × 2 mm, 2 μm particles) was used (Phenomenex, Torrance, CA, USA). The column temperature was set at 35°C and the autosampler temperature was 5°C. The gradient elution solvents were A; 95:5 water-acetonitrile (v/v), containing 5 mM ammonium formate and 0.1% formic acid (v/v), and B; 95:5 acetonitrile-water, containing 5 mM ammonium formate and 0.1% formic acid. The gradient (A:B, v/v) was as follows: an isobaric period at 5:95 for 5 min, followed by a linear gradient from 5:95 to 50:50 in 25 min, then held at 50:50 for 5 min, followed by a linear gradient change from 50:50 to 5:95 in 1 min, then held at 5:95 for 5 min. The flow rate was 0.2 mL/min.
8
HPLC Separation and Quantification of Organic Compounds
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In total, 10 µL of the supernatant were injected onto the SecurityGuard™ column (KJ0-4282) with a (4.0 mm × 3.0 mm) guard cartridge before separation using a Phenomenex Gemini® C18 analytical column (5 µm packing, 150 mm × 4.6 mm). Gradient starting conditions of 5% MeCN/H2O were held for 1 min before development into 50% MeCN/H2O over 3 min, followed by development into 95% MeCN/H2O over 3 min. Approximately 95% MeCN/H2O was held for 1 min before equilibration returned to starting conditions over 1 min. Starting conditions were held for 1 min, followed by another 2 min post-run. Flow rates were kept constant at 1 mL/ min. The column temperature was kept constant at 30 °C. UV absorbance was detected at 220 nm, 254 nm, and 210 nm throughout the run.
9
HPLC Analysis of Pharmaceutical Compounds
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Analyses were performed using an HPLC Thermo Fisher Scientific liquid chromatography system (Model: Spectra System P2000) coupled to a photodiode array detector (PDA) Model: Spectra System UV6000LP. Mobile phase was directly on-line degassed by using a Spectra System SCM1000 (Thermo Fisher Scientific, Waltham, MA, USA). Excalibur v.2.0 Software (Thermo Fisher Scientific, Waltham, MA, USA) was used to collect and analyze the data. The Luna C18 (250 × 4.6 mm, 5 μm particle size; Phenomenex, Torrance, CA, USA) packing column connected to a security guard column (4.0 × 3.0 mm, 5 μm particle size; Phenomenex, Torrance, CA, USA) were used to separate drugs and internal standard (IS). The column and security guard column were thermostated at 25 °C (± 1 °C) using a Jetstream2 Plus column oven during the analysis. Drugs and IS were detected at the maximum wavelengths of 283 nm (ciprofloxacin), 369 nm (sulfasalazine), 260 nm (methyl-p-hydroxybenzoate), 247 nm (cortisone), respectively, following the validated method reported in reference [6 (link)].
10
HPLC Analysis of Unknown Compounds
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HPLC system (Model-LC-20AT, Shimadzu Corporation, Kyoto, Japan) equipped with low pressure quaternary gradient pump, column oven, autosampler, and PDA detector was used for analysis. Chromatography data was processed by LabSolution software (Shimadzu, Kyoto, Japan). Analysis was performed on Reverse phase C18 ACE® (250 × 4.6 mm i.d., particle size 5 μm) column coupled with security guard column (4 × 3 mm, Phenomenex®) at room temperature. The mobile phase was composed of 0.1 % phosphoric acid (solvent A) and methanol (solvent B) in gradient elution mode as follows: 0–1 min 85% of solvent A; 1–4 min linear gradient to 70% of solvent A; 4–5 min 70% of solvent A; 5–6 min linear gradient to 85% of solvent A; 6–20 min 85% of solvent A to re-equilibrate the system. The mobile phase was degassed before pumping into the HPLC system at a flow rate of 1.0 mL/min. Injection volume of 50 μL was used. The wavelength monitored was 280 nm.
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