Db 5ms dg column, by Agilent Technologies - Product details

1

Detailed Metabolite Profiling by GC-MS

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Footprint and fingerprint samples were analysed by GC-MS as described earlier [23] . Briefly, lyophilised intracellular metabolite extracts were prepared for GC-MS analysis by a two-stage derivatisation procedure (methoxyamination followed by trimethylsilylation). GC-MS analysis of the derivatised samples was performed on a 7890A GC System (Agilent Technologies), fitted with a DB-5MS+DG column (Agilent Technologies; 250 m x 30 m, 0.25 m film thickness with 10 m duraguard), coupled to a 5975C Inert XL MSD with Triple-Axis Detector (Agilent Technologies) using the manufacturers software (MSD ChemStation). Metabolite peaks in the raw chromatograms were identified using MSD ChemStation (Agilent Technologies) to search custom libraries based on the Agilent Fiehn GC/MS Metabolomics RTL Library (http://fiehnlab.ucdavis.edu/Metabolite-Library-2007). Metabolite identifications were based on retention times and fragmentation patterns. The data were combined using an in-house Microsoft Excel (Microsoft Corporation) macro and normalized to the retention time locking (RTL) compound (myristic acid d27).

Sellick C.A., Croxford A.S., Maqsood A.R., Stephens G.M., Westerhoff H.V., Goodacre R, & Dickson A.J. (2015). Metabolite profiling of CHO cells: Molecular reflections of bioprocessing effectiveness. Biotechnology journal, 10(9).

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2

GC-APLI-FT-ICR MS Characterization

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Individual standards were separated using a custom-built gas chromatography (GC) and introduced to the APLI source via a GC transfer line heated to 300 °C (more details on the GC-APLI coupling in Figure S-1). This source is now commercially available via Bruker Daltonics, Inc. GC separation was performed using a DB-5 Ms+DG column (30 m × 0.25 mm, 0.25 μm thickness, from Agilent Technologies, Inc., Palo Alto, CA). The GC injection chamber was held at 200 °C and 1 μL of sample was introduced at a 1:20 split ratio. The GC method consisted of a 110–230 °C ramp at a rate of 10 °C/min, followed by a 230–310 °C ramp at a rate of 5 °C/min, and held for 7 min for a total of 35 min. FT-ICR MS spectra were acquired after 5 min, for a total of 25 min, with similar ion transmission conditions to those used during direct-infusion APLI-FT-ICR MS but without averaging and with a shorter collection time of 2 MWord (1 s transient), resulting in an experimental MS resolving power with AMP at m/z 400 of 264 000.

Benigni P., DeBord J.D., Thompson C.J., Gardinali P, & Fernandez-Lima F. (2015). Increasing Polyaromatic Hydrocarbon (PAH) Molecular Coverage during Fossil Oil Analysis by Combining Gas Chromatography and Atmospheric-Pressure Laser Ionization Fourier Transform Ion Cyclotron Resonance Mass Spectrometry (FT-ICR MS). Energy & fuels : an American Chemical Society journal, 30(1), 196-203.

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3

Quantitative Amino Acid Analysis in Bacterial Cells

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During the exponential phase (7.5–8 h), an appropriate amount of culture [OD₆₀₀ × culture volume (ml) = 8] was collected via centrifugation. The cells were washed with 2 ml of 0.2 mol l⁻¹ HCl to remove the CaCO₃ and then further washed with 2 ml saline. The cells were hydrolyzed with 2 ml of 6 mol l⁻¹ HCl at 105°C for 18 h. The samples were filtered through a Cosmonice filter W (0.45 µm; Nacalai Tesque, Kyoto, Japan), mixed with 10 µl of 600 µM cycloleucine, dried, and dissolved in 50 µl acetonitrile. After adding 50 µl of N-(tert-tert-butyldimethylsilyl)-N-methyl-trifluoroacetamide containing 1% tert-butyldimethylchlorosilane, the samples were incubated at 105°C for 1 h for derivatization. mass isotopomer distributions (MIDs) of proteinogenic amino acids were measured using a gas chromatograph/mass spectrometer (Agilent 7890A GC and 5975C Mass Selective Detector; Agilent Technologies, Santa Clara, USA) with a DB-5MS+DG column (Agilent Technologies). The detailed method is described elsewhere.³⁴ (link)

Yamamoto J., Chumsakul O., Toya Y., Morimoto T., Liu S., Masuda K., Kageyama Y., Hirasawa T., Matsuda F., Ogasawara N., Shimizu H., Yoshida K.I., Oshima T, & Ishikawa S. (2022). Constitutive expression of the global regulator AbrB restores the growth defect of a genome-reduced Bacillus subtilis strain and improves its metabolite production. DNA Research: An International Journal for Rapid Publication of Reports on Genes and Genomes, 29(3), dsac015.

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4

GC-MS/MS Analysis of Metabolites

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GC-MS (GCMS-QP2010 Ultra, Shimadzu, Kyoto, Japan) and GC-MS/MS (GCMS-TQ8040, Shimadzu) equipped with DB-5MS+DG column ((30 m ×0.25 mm ID ×0.25 μm), Agilent Technologies, Santa Clara, CA, USA) were used.^{8 (link))} Analysis conditions were as follows: constant flow rate of helium at 1.0 mL/min; ion source temperature, 230°C; electron impact ionization, 70 eV; injection volume, 1 μL; injection, pulsed split (split ratio, 1 : 10); oven temperature, 60°C for 3.5 min, increased at a rate of 10°C/min to 325°C, and maintained at that temperature for 10 min or 70°C for 2 min, increased at a rate of 3°C/min to 280°C, and maintained at that temperature for 5 min for the analysis of TMS- and TBDMS-derivatives, respectively. Argon (200 kPa) was used as collision gas for the MS/MS analysis. Collision energy was optimized using Smart MRM (Shimadzu, Kyoto, Japan). The obtained raw MS and MS/MS spectra were deposited in MassBank.^{13 (link))}

Okahashi N., Kawana S., Iida J., Shimizu H, & Matsuda F. (2019). Fragmentation of Dicarboxylic and Tricarboxylic Acids in the Krebs Cycle Using GC-EI-MS and GC-EI-MS/MS. Mass Spectrometry, 8(1), A0073.

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5

GC-MS Metabolite Analysis Protocol

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Dried samples were washed three times with methanol and derivatized first with 20 μl methoxyamine hydrochloride (Merck, 89803) solution in pyridine (Merck, 270970) (20 mg ml⁻¹) for 16 h and then with 20 μl BSTFA + 1% TMCS silylation reagent (Thermo Fisher Scientific, TS-38831) for 30 min. Metabolite analysis was performed by GC–MS using an Agilent 7890B-7000C GC-triple-quadrupole MS. Splitless injection (injection temperature 250 °C) onto a 30 m + 10 m × 0.25 mm DB-5MS + DG column (Agilent J&W) was used, using helium as the carrier gas, in electron ionization mode. The initial oven temperature was 70 °C (2 min), followed by temperature gradients to 295 °C at 12.5 °C min⁻¹, then to 320 °C at 25 °C min⁻¹ (held for 3 min). Data analysis was performed using our in-house-developed software MANIC (v.3.0), based on the software package GAVIN^{74 (link)}. Label incorporation was calculated by subtracting the natural abundance of stable isotopes from the observed amounts. Metabolites were identified and quantified in comparison to authentic standards and scyllo-Inositol as an internal standard (Sigma, I8132).

Papalazarou V., Newman A.C., Huerta-Uribe A., Legrave N.M., Falcone M., Zhang T., McGarry L., Athineos D., Shanks E., Blyth K., Vousden K.H, & Maddocks O.D. (2023). Phenotypic profiling of solute carriers characterizes serine transport in cancer. Nature Metabolism, 5(12), 2148-2168.

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6

GC-MS Analysis of Polar Metabolites

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Polar metabolites were derivatized and analysed by GC-MS (Agilent 7890B-5977A) and identification and abundance of individual metabolites was estimated as previously described ^{113 (link)}. In brief, dried metabolite samples were washed with methanol (twice), and derivatized overnight at RT with methoxyamine (20 mg/mL in pyridine, Sigma) followed by addition of BSTFA + 1% TMCS (Sigma) for > 1 hr at RT. GC-MS was performed using splitless injection (injection temperature 270°C) onto a 30 m + 10 m × 0.25 mm DB-5MS+DG column (Agilent J&W), with helium carrier gas, in electron impact ionization mode. The oven temperature was initially 70°C (2 min), followed by a temperature increase to 295°C at 12.5°C/min and subsequently to 320°C at 25°C/min (held for 3 min). GAVIN software ^{114 (link)} was used for metabolite identification and quantification by comparison to the retention times, mass spectra, and responses of known amounts of authentic standards.

Vowinckel J., Hartl J., Marx H., Kerick M., Runggatscher K., Keller M.A., Mülleder M., Day J., Weber M., Rinnerthaler M., Yu J.S., Aulakh S., Lehmann A., Mattanovich D., Timmermann B., Zhang N., Dunn C.D., MacRae J.I., Breitenbach M, & Ralser M. (2021). The metabolic growth limitations of petite cells lacking the mitochondrial genome. Nature metabolism, 3(11), 1521-1535.

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7

GC-MS Metabolite Profiling Protocol

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Metabolite analysis was performed by GC-MS using an Agilent 7890B-5977A system. Splitless injection (injection temperature 270°C) onto a 30 m + 10 m × 0.25 mm DB-5MS + DG column (Agilent J&W) was used, using helium as the carrier gas, in electron ionization mode. The initial oven temperature was 80°C (2 min), followed by temperature gradients to 140°C at 30°C/min, to 250°C at 5°C/min and then to 320°C at 15°C/min (held for 6 min). Metabolites were identified by comparison with the retention times and mass spectrum of authentic standards using the MassHunter Workstation software (B.06.00 SP01, Agilent Technologies). Abundance was calculated by comparison to responses of known amounts of standards.

Blassberg R., Macrae J.I., Briscoe J, & Jacob J. (2015). Reduced cholesterol levels impair Smoothened activation in Smith–Lemli–Opitz syndrome. Human Molecular Genetics, 25(4), 693-705.

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8

GC-MS Analysis of Metabolite Derivatization

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For GC-MS analysis, 150 µl of the aqueous phase were dried down in a vial insert, washed twice with 40 µl MeOH and dried again. Samples were then derivatised by methoximation (20 µl of 20 mg/mL methoxyamine in pyridine, RT overnight) before addition of 20 µl of N,O-bis(trimetylsilyl)trifluoroacetamide (BSTFA) + 1% trimethylchlorosilane (TMCS) (Sigma, 33148) for ≥1 h. Metabolite analysis was performed by GC-MS using an Agilent 7890B-5977A system. Splitless injection (injection temperature 270°C) onto a 30 m + 10 m × 0.25 mm DB-5MS+DG column (Agilent J&W) was used, with a helium carrier gas, using electron impact ionization (EI) mode. Oven temperature was initially 70 °C (2 min), followed by a temperature increase to 295 °C at 12.5 °C/min and subsequently to 320 °C at 25 °C/min (held for 3 min). MassHunter Workstation software (B.06.00 SP01, Agilent Technologies) was used for metabolite identification and quantification by comparison to the retention times, mass spectra, and responses of known amounts of authentic standards. Fractional labelling of individual metabolites was calculated as the fraction of carbons in the metabolite pool that were ¹³C atoms after correction for natural abundance.

Fets L., Driscoll P.C., Grimm F., Jain A., Nunes P.M., Gounis M., Doglioni G., Papageorgiou G., Ragan T.J., Campos S., Silva dos Santos M., MacRae J.I., O’Reilly N., Wright A.J., Benes C.H., Courtney K.D., House D, & Anastasiou D. (2018). MCT2 mediates concentration-dependent inhibition of glutamine metabolism by MOG. Nature chemical biology, 14(11), 1032-1042.

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9

GC-MS Analysis of Metabolite Derivatization

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For GC-MS analysis, 150 µl of the aqueous phase were dried down in a vial insert, washed twice with 40 µl MeOH and dried again. Samples were then derivatised by methoximation (20 µl of 20 mg/mL methoxyamine in pyridine, RT overnight) before addition of 20 µl of N,O-bis(trimetylsilyl)trifluoroacetamide (BSTFA) + 1% trimethylchlorosilane (TMCS) (Sigma, 33148) for ≥1 h. Metabolite analysis was performed by GC-MS using an Agilent 7890B-5977A system. Splitless injection (injection temperature 270°C) onto a 30 m + 10 m × 0.25 mm DB-5MS+DG column (Agilent J&W) was used, with a helium carrier gas, using electron impact ionization (EI) mode. Oven temperature was initially 70 °C (2 min), followed by a temperature increase to 295 °C at 12.5 °C/min and subsequently to 320 °C at 25 °C/min (held for 3 min). MassHunter Workstation software (B.06.00 SP01, Agilent Technologies) was used for metabolite identification and quantification by comparison to the retention times, mass spectra, and responses of known amounts of authentic standards. Fractional labelling of individual metabolites was calculated as the fraction of carbons in the metabolite pool that were ¹³C atoms after correction for natural abundance.

Fets L., Driscoll P.C., Grimm F., Jain A., Nunes P.M., Gounis M., Doglioni G., Papageorgiou G., Ragan T.J., Campos S., Silva dos Santos M., MacRae J.I., O’Reilly N., Wright A.J., Benes C.H., Courtney K.D., House D, & Anastasiou D. (2018). MCT2 mediates concentration-dependent inhibition of glutamine metabolism by MOG. Nature chemical biology, 14(11), 1032-1042.

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10

GC-MS Protocol for Metabolite Analysis

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For GC–MS analysis, 150 µl of the aqueous phase was dried down in a vial insert, before washing twice with 40 µl MeOH and drying again. Samples were methoximated (20 µl of 20 mg/ml methoxyamine in pyridine, RT overnight) before derivatising with 20 µl of N,O-bis(trimetylsilyl)trifluoroacetamide + 1% trimethylchlorosilane (Sigma, 33148) for ≥1 h. An Agilent 7890B-5977A GC–MS system was use to perform metabolite analysis. Splitless injection (injection temperature 270 °C) onto a 30 m + 10 m × 0.25 mm DB-5MS + DG column (Agilent J&W) was used, using helium carrier gas, in electron-impact ionization (EI) mode. Initial oven temperature was 70 °C (2 min) with a subsequent increase to 295 °C at 12.5 °C/min, then to 320 °C at 25 °C/min (before holding for 3 min). Metabolite identification and quantification was performed using MassHunter Workstation software (B.06.00 SP01, Agilent Technologies) or MANIC software, an in-house developed adaptation of the GAVIN package^{58 (link)}, by comparison to the retention times, mass spectra and responses of known amounts of authentic standards. Fractional labelling of individual metabolites is reported after correction for natural abundance.

Fets L., Bevan N., Nunes P.M., Campos S., dos Santos M.S., Sherriff E., MacRae J.I., House D, & Anastasiou D. (2022). MOG analogues to explore the MCT2 pharmacophore, α-ketoglutarate biology and cellular effects of N-oxalylglycine. Communications Biology, 5, 877.

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Db 5ms dg column

Lab products found in correlation

12 protocols using db 5ms dg column

Detailed Metabolite Profiling by GC-MS

GC-APLI-FT-ICR MS Characterization

Quantitative Amino Acid Analysis in Bacterial Cells

GC-MS/MS Analysis of Metabolites

GC-MS Metabolite Analysis Protocol

GC-MS Analysis of Polar Metabolites

GC-MS Metabolite Profiling Protocol

GC-MS Analysis of Metabolite Derivatization

GC-MS Analysis of Metabolite Derivatization

GC-MS Protocol for Metabolite Analysis

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