Detailed protocols for the quantification of metabolites in the FHS have been previously published. [23 (link)–25 (link)] Briefly, the metabolome was assessed in FHS Offspring on plasma samples collected following an overnight fast and stored at −80°C (see Supplementary Methods ). Plasma metabolites were analyzed using liquid chromatography-tandem mass spectrometry (LC-MS). LC-MS data were acquired using either an AB SCIEX 4000 QTRAP triple quadrupole mass spectrometer (positively charged polar compounds and lipids) or an AB SCIEX 5500 QTRAP triple quadrupole mass spectrometer (negatively charged polar compounds). Polar, positively charged metabolites were separated using hydrophobic interaction liquid chromatography (HILIC) and analyzed using multiple reaction monitoring (MRM) in the positive ion mode. Polar, negatively charged compounds, including central and polar phosphorylated metabolites, were separated using a Luna NH2 column (150 × 2 mm, Luna NH2, Phenomenex) and analyzed using MRM in the negative ion mode. Lipids were separated on a Prosphere C4 HPLC column and underwent full scan MS analysis in the positive ion mode. MultiQuant software (Version 1.2, AB SCIEX) was used for automated peak integration and manual review of data quality prior to statistical analysis. Quality control and normalization of metabolite levels are detailed in Supplementary Methods .
Luna nh2
Manufactured by Phenomenex
Sourced in United States
The Luna NH2 is a reversed-phase high-performance liquid chromatography (HPLC) column designed for the separation and analysis of a wide range of polar and moderately polar analytes. It features a silica-based stationary phase with amino (NH2) functional groups, which provide a unique selectivity for the separation of these compounds.
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Lab products found in correlation
Ultimate 3000 rslc, by Thermo Fisher Scientific (6 mentions)
Q exactive mass spectrometer, by Thermo Fisher Scientific (6 mentions)
Ultimate 3000, by Thermo Fisher Scientific (1 mentions)
Triple quad 6500 mass spectrometer, by AB Sciex (1 mentions)
U 13c6 glucose, by Cambridge Isotopes (1 mentions)
Novaseq 6000 sequencing platform, by Illumina (1 mentions)
Ms grade water, by Thermo Fisher Scientific (1 mentions)
Nexera lc 30a, by Shimadzu (1 mentions)
6500 qtrap ms, by AB Sciex (1 mentions)
Kinetex c18, by Phenomenex (1 mentions)
Superscript double stranded cdna synthesis kit, by Thermo Fisher Scientific (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
6540 uhd q tof, by Agilent Technologies (1 mentions)
Model lc 20, by Shimadzu (1 mentions)
Qtrap 5500, by AB Sciex (1 mentions)
4000 qtrap triple quadrupole mass spectrometer, by AB Sciex (1 mentions)
5500 qtrap triple quadrupole mass spectrometer, by AB Sciex (1 mentions)
Multiquant software, by AB Sciex (1 mentions)
Synergi hydro rp, by Phenomenex (1 mentions)
1260 infinity, by Agilent Technologies (1 mentions)
17 protocols using luna nh2
1
Quantification of Metabolites in the FHS
2
Targeted Metabolomic Profiling of Cells
3
Hippocampal Metabolite Profiling by LC-MS
4
Quantification of EdC and EdU Metabolites
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HeLa or 143B cells were incubated either with 10 µM EdU or 10 µM EdC for 4 h or without any treatment (control). In the case of hypotonic treatment, the HeLa cells were treated according to Koberna et al. [28 (link)]: the cells were quickly rinsed with 1 × KHB buffer and incubated in a hypotonic solution containing 1 × KHB and 0.4 mM EdCTP for 10 min. Then, the hypotonic solution was aspirated and the culture medium was added to the samples for 15 min. The procedure of cell extraction was adapted from Bennett et al. [29 (link)] and partly adjusted. EdC, EdU and their mono-, di- and triphosphates were analysed by the liquid chromatography system UltiMate 3000 (ThermoFisher Scientific) coupled with a Triple Quad 6500 mass spectrometer (Sciex). The chromatographic separations were performed at 35°C on a Luna NH2 (100 × 2.0 mm, 3 µm; Phenomenex) (for a detailed description, see the electronic supplementary material, S5).
5
Intracellular Metabolite Profiling using Labeled Glucose
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For metabolomic analysis, cells were incubated in medium containing [U-13C6] glucose (Cambridge Isotope Laboratories CML1396, Tewksbury, MA, USA) for 24 h. To extract intracellular metabolites, cells grown in 6-well plate were briefly rinsed with 2 ml of ice-cold 150 mM ammonium acetate (pH = 7.3), before addition of 1 ml of ice-cold 80% methanol. Cells were scraped and transferred into Eppendorf tubes and then 5 nM D/L-norvaline was added. After vortexing at maximum velocity, samples were spun at 20,000g for 5 min at 4 °C. Supernatant was then moved into a glass vial, dried using speedvac centrifuge, and reconstituted in 50 μl 70% acetonitrile. 5 μl of each sample was injected onto a Luna NH2 (150 mm × 2 mm, Phenomenex, Torrance, CA, USA) column. Samples were analyzed with an UltiMate 3000RSLC (Thermo Scientific, Waltham, MA, USA) coupled to a Q Exactive mass spectrometer (Thermo Scientific, Waltham, MA, USA). The Q Exactive was run with polarity switching (+3.00 kV/−2.25 kV) in full scan mode with an m/z range of 70–1050. Separation was achieved using 5 mM NH4AcO (pH 9.9) and ACN. The gradient started with 15% NH4AcO and reached 90% over 18 min, followed by an isocratic step for 9 min and reversal to the initial 15% NH4AcO for 7 min.
6
Transcriptomic and Metabolomic Profiling of Osteoclastogenesis
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The mBMDMs (≈1 × 106 cells per sample) cultured with osteoclastogenic medium with/without rapamycin for 4 days were subjected to transcriptome sequencing and targeted metabolomic analysis. Briefly, for transcriptomic analysis, cells were lysed with the Invitrogen TRIzol Reagent to extract cDNA. The cDNA library was constructed using the SuperScript double‐stranded cDNA synthesis kit (Invitrogen, USA) protocol. Illumina NovaSeq 6000 sequencing platform (Shanghai Applied Protein Technology) was performed for transcriptomic analysis. For targeted metabolomic analysis, samples were mixed with methanol, internal lipid standards and methyl tert‐butyl ether (Aladdin, USA). The mixture was adequately vortexed, sonicated and then kept for 30 min. After that, MS‐grade water (Thermo Fisher, USA) was added, and the mixture was vortexed and centrifuged. The upper organic solvent layer was obtained and dried under nitrogen. For LC‐MS analysis, the samples were re‐dissolved in IPA/CAN (9:1, v/v) solvent and then centrifuged. The analysis was performed on a UHPLC system (Nexera LC‐30A, Shimadzu) coupled with QTRAP MS (6500+, Sciex). The analytes were separated on HILIC (Phenomenex, Luna NH2, 2.0 mm ×100 mm, 3 µm) and C18 column (Phenomenex, Kinetex C18, 2.1 × 100 mm, 2.6 µm).
7
Multiplatform Metabolomic Credentialing Protocol
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The data shown herein were obtained
from an Agilent 6540 UHD QTOF interfaced with an Agilent 1260 Capillary
LC. The column used for separation was a Phenomenex Luna NH2 (150 mm × 1 mm, 3 μm). HILIC solvents were A, 95% water
in acetonitrile with 10 mM ammonium acetate/10 mM ammonium hydroxide
(pH 9.8), and B, 95% acetonitrile in water. HILIC was performed at
45 μL/min with the following linear gradient (minutes, %B):
0, 100%; 5, 100%; 45, 0%; 50, 0%; 51, 100%; 60, 100%. For all experiments,
5 μL of extract was injected. MS parameters were as follows:
gas, 300 °C 9 L/min; nebulizer, 35 psi 1000 V; sheath gas, 350
°C 11 L/min; capillary, 3500 V; fragmentor, 175 V; scan rate,
1 scan/s.
To demonstrate the wide applicability of our credentialing
approach to other metabolomic platforms, we also analyzed our samples
and subsequently validated correct credentialing with multiple chromatographic
and mass spectrometrometric technologies. In addition to the Agilent
QTOF, we credentialed data from the Thermo QE, the AB SCIEX TripleTOF,
and the LECO Pegasus GC-HRT. Chromatographic methods we credentialed
include reversed-phase LC and HILIC. Effective parameters for credentialing
each of these experimental platforms are listed in theSupporting Information Table S-3.
from an Agilent 6540 UHD QTOF interfaced with an Agilent 1260 Capillary
LC. The column used for separation was a Phenomenex Luna NH2 (150 mm × 1 mm, 3 μm). HILIC solvents were A, 95% water
in acetonitrile with 10 mM ammonium acetate/10 mM ammonium hydroxide
(pH 9.8), and B, 95% acetonitrile in water. HILIC was performed at
45 μL/min with the following linear gradient (minutes, %B):
0, 100%; 5, 100%; 45, 0%; 50, 0%; 51, 100%; 60, 100%. For all experiments,
5 μL of extract was injected. MS parameters were as follows:
gas, 300 °C 9 L/min; nebulizer, 35 psi 1000 V; sheath gas, 350
°C 11 L/min; capillary, 3500 V; fragmentor, 175 V; scan rate,
1 scan/s.
To demonstrate the wide applicability of our credentialing
approach to other metabolomic platforms, we also analyzed our samples
and subsequently validated correct credentialing with multiple chromatographic
and mass spectrometrometric technologies. In addition to the Agilent
QTOF, we credentialed data from the Thermo QE, the AB SCIEX TripleTOF,
and the LECO Pegasus GC-HRT. Chromatographic methods we credentialed
include reversed-phase LC and HILIC. Effective parameters for credentialing
each of these experimental platforms are listed in the
8
Quantifying Trehalose and Mannitol Deposition
9
Quantifying Nucleotide Levels in HeLa Cells
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The HeLa cells were incubated with 10 μM EdU or 8 nM FdU or without any inhibitor (control) for 2 hours. Next, the cells were harvested and quenched according to the published protocol [18 (link)]. Lyophilized cell extracts were dissolved in MeOH/H2O (vol. 1:1, 200 μL), centrifuged (10,000 x g, 5 min.) and transferred to glass vials. All samples were analysed by HPLC (UHPLC Dionex Ultimate 3000 RS, Thermo Fisher Scientific, MA, USA) using the previously published HILIC method on Luna NH2 (Phenomenex) columns under alkaline separation conditions [19 (link)]. For the detection of selected nucleotides, the adopted method using the triple quadrupole mass spectrometer 5500 QTrap (AB Sciex, CA, USA) working in polarity switching multiple reaction monitoring mode was applied [20 (link)]. The data were acquired using Analyst 1.6.2 software and processed by MultiQuant 2.1.1. (AB Sciex, CA, USA) and Microsoft Excel. The experiments were conducted in triplicate.
10
Trehalose Quantification in Inhaler Components
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The deposition of trehalose at the capsule, inhaler, adaptor and each part of the MSLI was determined using a UPLC system (Model Code CHA D16CHA036G; Waters, Milford, MA, USA) using RI detection. The configuration consisted of an Acquity series of quaternary solvent manager, column manager, RI detector, sample manager-FTN, and Empower® software. An amino acid column (Phenomenex Luna NH2, 3 μm, 100 Å, 150 × 2 mm) was used. The mobile phase was a mixture of water (30%) and acetonitrile (70%). A volume of 300 μL dispersion sample was mixed with 700 μL acetonitrile before HPLC analysis. The calibration curve for trehalose was linear in the concentration range of 0.05–0.2 mg/mL (R2 = 0.999, n = 3).
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