Lc nexera x2 uhplc

Manufactured by AB Sciex

Sourced in United States, Hong Kong

The LC Nexera X2 UHPLC is an ultra-high-performance liquid chromatography system designed for efficient and reliable separation of complex samples. It features a high-pressure pumping system, a low-dispersion flow path, and advanced control and data acquisition capabilities.

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Lab products found in correlation

Qtrap 5500 lc ms ms, by AB Sciex (9 mentions) Multiquant 3, by AB Sciex (9 mentions) Acquity uplc beh amide analytic column, by Waters Corporation (6 mentions) Optima lc ms grade, by Thermo Fisher Scientific (3 mentions) 13c nicotinic acid, by LGC (3 mentions) Masshunter quantitative analysis software, by Agilent Technologies (2 mentions) Qtrap 5500, by AB Sciex (1 mentions) Pre column filter, by Waters Corporation (1 mentions) 7890b 5977b gc ms system, by Agilent Technologies (1 mentions) 7890b 5977b system, by Agilent Technologies (1 mentions) Db 5ms column, by Agilent Technologies (1 mentions)

Spelling variants of this product

Nexera UHPLC (6 citations) Nexera X2 (4 citations)

11 protocols using lc nexera x2 uhplc

UHPLC-MS/MS Metabolite Quantification

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The extracts were analyzed by a Shimadzu LC Nexera X2 UHPLC coupled with a QTRAP 5500 LC MS/MS (AB Sciex). An ACQUITY UPLC UPLC BEH Amide analytic column (2.1 × 50 mm, 1.7 μm, Waters) was used for chromatographic separation. The mobile phase was (A) water with 10 mM ammonium acetate (pH 8.9) and (B) acetonitrile/water (95/5) with 10 mM ammonium acetate (pH 8.2) (All solvents were LC–MS Optima grade from Fisher Scientific). The total run time was 11 min with a flow rate of 0.5 ml/min with an injection volume of 5 μl. The gradient elution is 95–61% B in 6 min, 61–44% B at 8 min, 61–27% B at 8.2 min, and 27–95% B at 9 min. The column was equilibrated with 95% B at the end of each run. The source and collision gas was N₂. The ion source conditions in positive and negative mode were: curtain gas (CUR) = 25 psi, collision gas (CAD) = high, ion spray voltage (IS) = 3800/−3800 V, temperature (TEM) = 500 °C, ion source gas 1 (GS1) = 50 psi, and ion source gas 2 (GS2) = 40 psi. Each metabolite was tuned with standards for optimal transitions (Supplemental Table 1) ¹³C-nicotinic acid (Toronto Research Chemicals) was used as the internal standard. The extracted MRM peaks were integrated using MultiQuant 3.0.2 software (AB Sciex).

Zhu S., Yam M., Wang Y., Linton J.D., Grenell A., Hurley J.B, & Du J. (2018). Impact of euthanasia, dissection and postmortem delay on metabolic profile in mouse retina and RPE/choroid. Experimental eye research, 174, 113-120.

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Quantitative Metabolomic Analysis of RPE-Choroid

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Briefly, isolated RPE-choroid complexes were processed in 80% cold methanol via homogenization and metabolites were analyzed using Shimadzu LC Nexera X2 UHPLC coupled with a QTRAP 5500 LC MS/MS (AB Sciex, Framingham, MA, USA). Chromatographic separation was achieved using ACQUITY UPLC BEH Amide analytic column (2.1×50 mm, 1.7 μm, Waters) with mobile phase A (water with 10 mM ammonium acetate with pH 8.9) and mobile phase B (acetonitrile/water (95/5) with 10 mM ammonium acetate with pH 8.2) (All solvents were LC-MS Optima grade from Fisher Scientific, Pittsburgh, PA, USA). Chromatographic separation was completed in a total run time of 11min with a gradient as previously described 20 (link), 21 (link). Multiple reaction monitoring was performed for 97 transitions listed in Table S3. MultiQuant 3.0.2 software (AB Sciex, Framingham, MA, USA) was used to integrate the extracted peaks and volcano plots were used to analyze the data. Different metabolites and enriched metabolic pathways were identified by MetaboAnalyst software.

Huang J., Gu S., Chen M., Zhang S.J., Jiang Z., Chen X., Jiang C., Liu G., Radu R.A., Sun X., Vollrath D., Du J., Yan B, & Zhao C. (2019). Abnormal mTORC1 signaling leads to retinal pigment epithelium degeneration. Theranostics, 9(4), 1170-1180.

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Targeted Metabolomics by LC-MS and GC-MS

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Targeted metabolomics was performed as described in detail before with liquid chromatography-mass spectrometry and gas chromatography-mass spectrometry.²² (link)^,³⁷ (link) A total of 133 metabolites that cover major metabolic pathways were quantified (see detailed pathways and parameters in Supplementary Table S1). A Shimadzu LC Nexera X2 UHPLC coupled with a QTRAP 5500 liquid chromatography-mass spectrometry (AB Sciex, Hong Kong), and an Agilent 7890B/5977B gas chromatography-mass spectrometer (Agilent Technologies, Santa Clara, CA. USA) were used for metabolite analysis. The data were analyzed by MultiQuant 3.0.2 (AB Sciex) and Agilent MassHunter Quantitative Analysis Software.³⁹ (link)

Saravanan M., Xu R., Roby O., Wang Y., Zhu S., Lu A, & Du J. (2023). Tissue-Specific Sex Difference in Mouse Eye and Brain Metabolome Under Fed and Fasted States. Investigative Ophthalmology & Visual Science, 64(3), 18.

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HaCaT Metabolite Extraction and LCMS Analysis

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HaCaT cells were extracted for metabolites as previously described with little modification (Grenell et al, 2019 (link)). Briefly, the extracts were analyzed using Shimadzu LC Nexera X2 UHPLC coupled with a QTRAP 5500 LC MS/MS (AB Sciex). Chromatographic separation was performed with ACQUITY UPLC UPLC BEH Amide analytic column. The mobile phase was performed in buffer A (10 mM ammonium acetate in water, pH 8.8) and buffer B (10 mM ammonium acetate in acetonitrile/water (95/5), pH 8.2). The gradient elution was 95–61% buffer B in 7 min, 61–44% buffer B at 9 min, 61–27% buffer B at 9.2 min, and 27–95% buffer B at 10 min. The column was equilibrated with 95% buffer B at the end. 13C‐nicotinic acid (Toronto Research Chemicals) was added as the internal standard. MultiQuant 3.0.2 software (AB Sciex) was used to integrate the extracted MRM peaks.

Chen F., Ni C., Wang X., Cheng R., Pan C., Wang Y., Liang J., Zhang J., Cheng J., Chin Y.E., Zhou Y., Wang Z., Guo Y., Chen S., Htun S., Mathes E.F., de Alba Campomanes A.G., Slavotinek A.M., Zhang S., Li M, & Yao Z. (2022). S1P defects cause a new entity of cataract, alopecia, oral mucosal disorder, and psoriasis‐like syndrome. EMBO Molecular Medicine, 14(5), e14904.

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Plasma Purine Metabolite Analysis

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Purine metabolite analysis was done as previously described.^{34 (link)} Briefly, 20 μL of plasma was combined with 80 μL of methanol on ice for 15 minutes to precipitate proteins. The plasma/methanol mixture was centrifuged at 13,300 rpm at 4°C for 15 minutes, followed by lyophilization of the supernatant. To reconstitute the metabolites, 200 μL of 5 mM ammonium acetate in 95% water, 5% acetonitrile, and 0.5% acetic acid was passed through a 0.45-μm PDVF filter. The extracts were analyzed by a Shimadzu LC Nexera X2 UHPLC coupled with a QTRAP 5500 LC MS/MS (AB Sciex). An ACQUITY UPLC BEH Amide analytic column (2.1 X 50 mm, 1.7 μm, Waters) was used for chromatographic separation. The extracted MRM peaks were integrated using MultiQuant 3.0.2 software (AB Sciex).^{34 (link)}

Schmidt H.M., Wood K.C., Lewis S.E., Hahn S.A., Williams X.M., McMahon B., Baust J.J., Yuan S., Bachman T.N., Wang Y., Oh J.Y., Ghosh S., Ofori-Acquah S.F., Lebensburger J.D., Patel R.P., Du J., Vitturi D.A., Kelley E.E, & Straub A.C. (2020). Xanthine Oxidase Drives Hemolysis and Vascular Malfunction in Sickle Cell Disease. Arteriosclerosis, thrombosis, and vascular biology, 41(2), 769-782.

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Targeted Metabolomics Analysis by LC-MS/MS

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LC-MS/MS was performed as previously reported (Huang et al., 2019 (link)). The extracts were analyzed by a Shimadzu LC Nexera X2 UHPLC coupled with a QTRAP 5500 LC MS/MS (AB Sciex). Chromatographic separation was performed by using an ACQUITY UPLC BEH Amide analytic column (2.1 X 50 mm, 1.7 μm, Waters) with a guard column and 0.2 μm pre-column filter (Waters). The mobile phase was (A) water with 10 mM ammonium acetate (pH 8.9) and (B) acetonitrile/water (95/5) with 10 mM ammonium acetate (pH 8.2) (All solvents were LC–MS Optima grade from Fisher Scientific). The gradient elution is 95–61% B in 6 min, 61–44% B at 8 min, 61–27% B at 8.2 min, and 27–95% B at 9 min. The column was equilibrated with 95% B at the end of each run. The source and collision gas were N2. We targeted 84 key metabolites in central carbon metabolism, and each metabolite was tuned for optimal transitions (Table S1). MultiQuant 3.0.2 software (AB Sciex) was applied to extract ions for peak area under the curve. All samples were analyzed in one batch.

Huang J., Schaefer J., Wang Y., Gioia L., Pei Y., Shi X., Waris S., Zhao C., Nguyen J, & Du J. (2020). Metabolic signature of eyelid basal cell carcinoma. Experimental eye research, 198, 108140.

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Targeted Metabolite Profiling of BMDMs

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BMDMs were extracted for metabolites. The extracts were analyzed using Shimadzu LC Nexera X2 UHPLC coupled with a QTRAP 5500 LC MS/MS (AB Sciex). ACQUITY UPLC UPLC BEH Amide analytic column was used for chromatographic separation. The mobile phase was performed in buffer solution A (10 mM ammonium acetate in water, pH 8.8) and buffer solution B (10 mM ammonium acetate in acetonitrile/water (95/5), pH 8.2). The gradient elution was 95–61% buffer solution B in 7 min, 61–44% buffer solution B in 9 min, 61–27% buffer solution B in 9.2 min, and 27–95% buffer solution B in 10 min. Finally, The column was equilibrated with 95% buffer solution B 13C-nicotinic acid (Toronto Research Chemicals) was added as the internal standard. MultiQuant 3.0.2 software (AB Sciex) was used to integrate the extracted MRM peaks.

Lv L., Jiang H., Song D., Zhou X., Chen F., Ren L., Xie Y, & Zeng M. (2023). Sirt3 improves monosodium urate crystal-induced inflammation by suppressing Acod1 expression. Arthritis Research & Therapy, 25, 121.

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Targeted Metabolomics of Testes

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Targeted metabolomics by LC MS/MS was performed as reported before (70 (link)). Briefly, metabolites from testes were extracted in 80% cold methanol and analyzed by a Shimadzu LC Nexera X2 UHPLC coupled with a QTRAP 5500 LC MS/MS (AB Sciex) in multiple reaction monitoring mode. Each metabolite was tuned with standards for optimal transitions (see details in Table S3). The extracted multiple reaction monitoring peaks were integrated using MultiQuant 3.0.2 software (AB Sciex).

Zhu S., Huang J., Xu R., Wang Y., Wan Y., McNeel R., Parker E., Kolson D., Yam M., Webb B., Zhao C., Sigado J, & Du J. (2022). Isocitrate dehydrogenase 3b is required for spermiogenesis but dispensable for retinal viability. The Journal of Biological Chemistry, 298(9), 102387.

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RPE Cell Metabolomics Sample Prep

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To collect cells for metabolomics, RPE cells were rinsed once with cold 154mM NaCl solution and scraped in cold 80% methanol on dry ice. The metabolites were separated with an ACQUITY UPLC BEH Amide analytic column (2.1 × 50 mm, 1.7μm, Waters) by a Shimadzu LC Nexera X2 UHPLC coupled with a QTRAP 5500 LC MS/MS (AB Sciex). Each metabolite was tuned with standards for optimal transitions (Supplementary Table 4). The extracted multiple reaction monitoring peaks were integrated using MultiQuant 3.0.2 software.

Engel A.L., Wang Y., Khuu T.H., Worrall E., Manson M.A., Lim R.R., Knight K., Yanagida A., Qi J.H., Ramakrishnan A., Weleber R.G., Klein M.L., Wilson D.J., Anand-Apte B., Hurley J.B., Du J, & Chao J.R. (2021). Extracellular Matrix Dysfunction in Sorsby Patient-Derived Retinal Pigment Epithelium. Experimental eye research, 215, 108899.

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Metabolite Profiling by LC-MS and GC-MS

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Metabolite preparation and analysis with LC-MS and GC-MS were performed as previously reported.^{14 (link),23 (link)} Medium samples were centrifuged to remove debris, and 10 μL of the supernatant was mixed with 40 μL of cold methanol to extract the metabolites. Supernatants containing aqueous metabolites from both media and cells were dried at 4 °C and analyzed by LC-MS or GC MS. LC-MS used a Shimadzu LC Nexera X2 UHPLC coupled with a QTRAP 5500 LC MS/MS (AB Sciex). An ACQUITY UPLC BEH Amide analytic column (2.1 × 50 mm, 1.7 μm, Waters) was used for chromatographic separation. Each metabolite was tuned with standards for optimal transitions. The extracted multiple reaction monitoring (MRM) peaks were integrated using MultiQuant 3.0.2 software (AB Sciex). Table S6 lists the detailed parameters for the measured metabolites. For GC-MS, the samples were derivatized by methoxyamine and N-tertbutyldimethylsilyl-N-methyltrifluoroacetamide and analyzed by the Agilent 7890B/5977B GC-MS system with a DB-5MS column (30 m × 0.25 mm × 0.25 μm film). Mass spectra were collected from 80–600 m/z under selective ion monitoring mode. The detailed parameters are listed in Table S6, SI. The data was analyzed by Agilent MassHunter Quantitative Analysis Software and natural abundance was corrected by ISOCOR software.^{24 (link)}

Zhang R., Engel A.L., Wang Y., Li B., Shen W., Gillies M.C., Chao J.R, & Du J. (2020). Inhibition of Mitochondrial Respiration Impairs Nutrient Consumption and Metabolite Transport in Human Retinal Pigment Epithelium. Journal of proteome research, 20(1), 909-922.

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