Dionex ultimate 3000 nrslc

Manufactured by Thermo Fisher Scientific

The Dionex Ultimate 3000 nRSLC is a nano-scale reversed-phase liquid chromatography (nRPLC) system designed for high-performance liquid chromatography (HPLC) applications. The system is capable of handling flow rates from 20 nL/min to 2.5 μL/min, making it suitable for a variety of analytical tasks, including proteomics, metabolomics, and other applications that require sensitive and precise separation of complex samples.

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Lab products found in correlation

Q exactive hf mass spectrometer, by Thermo Fisher Scientific (3 mentions) Nanospray flex ion source, by Thermo Fisher Scientific (2 mentions) Easy spray c18 column, by Thermo Fisher Scientific (1 mentions) Easy spray ion source, by Thermo Fisher Scientific (1 mentions) Stainless steel emitter, by Thermo Fisher Scientific (1 mentions) Nupage sample buffer, by Thermo Fisher Scientific (1 mentions) Ltq orbitrap xl mass spectrometer, by Thermo Fisher Scientific (1 mentions) Mitochondria isolation kit, by Merck Group (1 mentions)

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Ultimate 3000 (1640 citations) Dionex Ultimate 3000 (733 citations)

5 protocols using dionex ultimate 3000 nrslc

Quantitative Proteomic Analysis by PRM

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Samples were analyzed on a Q-Exactive HF mass spectrometer equipped with an EASY-Spray ion source (Thermo Fisher Scientific). Peptides were resolved for nLC-MS analysis with a Dionex UltiMate 3000 nRSLC (Thermo Fisher Scientific) equipped with an EASY-Spray C18 column (2 μm particle size, 75 μm diameter, 250 mm length; Thermo Fisher Scientific, ES902). Peptides were separated with 60-min gradient using solvent A and solvent B (2 to 22% solvent B over 45 min, 22 to 38% solvent B over 15 min, both at a flow rate of 250 nl/min). One full duty cycle of the instrument consisted of a single MS-SIM MS1 scan followed by 30 PRM scans. For the full scan MS1, the instrument was set to 400 to 2000 m/z full scan range with a 15,000 resolution, 15 ms MIT, and 3 × 10⁶ AGC target. For the PRM scans, the instrument was set to 30,000 resolution, 60 ms MIT, 1 × 10⁵ AGC target, 0.8 m/z isolation window, NCE of 27, and 125 m/z fixed first mass. Spectrum data for both the MS1 and PRM scans were recorded in profile.

Justice J.L., Kennedy M.A., Hutton J.E., Liu D., Song B., Phelan B, & Cristea I.M. (2021). Systematic profiling of protein complex dynamics reveals DNA-PK phosphorylation of IFI16 en route to herpesvirus immunity. Science Advances, 7(25), eabg6680.

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Optimized Nanoscale Proteomics Workflow

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Samples were analyzed on a Q-Exactive HF mass spectrometer equipped with a Nanospray Flex Ion Source (Thermo Fisher Scientific). Peptides were resolved for nLC-MS analysis with a Dionex UltiMate 3000 nRSLC (Thermo Fisher Scientific) equipped with an in-house packed 50-cm column (360 μm outer diameter, 75 μm inner diameter; Thermo Fisher Scientific) packed with ReproSil-Pur C18 material (120 Å pore size, 1.9 μm particle size; ESI Source Solutions) equipped with a stainless steel emitter (Thermo Fisher Scientific). Peptides were resolved with a linear 150-min gradient using solvent A and solvent B (3 to 35% B over 150 min at a 250 nl/min flow rate). MS and MS/MS spectra were automatically obtained in a full MS/data-dependent MS2 method. The MS1 method operated with a full scan range of 350 to 1800 m/z with a resolution of 120,000 and a target AGC of 3 × 10⁶ with a MIT of 30 ms, and spectra were recorded in profile. For the data-dependent MS2 scans, HCD was performed on the top 10 most intense precursor ions, with a 30-s dynamic exclusion duration. MS2 isolation windows were set to 1.6 m/z, with a target AGC of 1 × 10⁵ and a MIT of 150 ms, a fixed first mass of 100 m/z, a resolution of 30,000, and an NCE of 28, and centroided spectral data were recorded.

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Mass Spectrometry Analysis of Peptides

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Samples for PRM analysis were analyzed on a Q-Exactive HF mass spectrometer equipped with a Nanospray Flex Ion Source (ThermoFisher Scientific). Peptides were analyzed for nLC-MS analysis with a Dionex Ultimate 3000 nRSLC (ThermoFisher Scientific) equipped with a 50 cm fused silica capillary (75 μm x363 μm) column packed in-house with ReproSil-Pur C18 packing material (ReproSil-Pur 120 C18-AQ 1.9 μm, ESI Source Solutions r119.aq.0001). Peptides were resolved with a 71-minute gradient at a flow rate of 250 nL/min using Solvent A (99.9% LC-MS grade water with 0.1% formic acid) and Solvent B (99.7% LC-MS grade acetonitrile, 0.2% LC-MS grade water, 0.1% formic acid; 5 to 35% B over 60 minutes, 35 to 97% B over 1 minute, and held at 97% for 10 minutes). One full duty cycle of the instrument consisted of a single Full-MS1 scan followed by 4 PRM scans. For the Full-MS1 scan, the instrument was set to a 350–1,800 m/z full scan range with a 15,000 resolution, 15 ms MIT, and 1e6 AGC target. For the PRM scans, the instrument was set to a 30,000 resolution, 25 ms MIT, 5e5 AGC target, 1.2 m/z isolation window, NCE of 27, and a fixed first mass of 125 m/z. Spectrum data for both the Full-MS1 and PRM scans were recorded in profile.

Shi W., Sheng X., Dorr K.M., Hutton J.E., Emerson J.I., Davies H.A., Andrade T.D., Wasson L.K., Greco T.M., Hashimoto Y., Federspiel J.D., Robbe Z.L., Chen X., Arnold A.P., Cristea I.M, & Conlon F.L. (2021). Cardiac proteomics reveals sex chromosome-dependent differences between males and females that arise prior to gonad formation. Developmental cell, 56(21), 3019-3034.e7.

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Proteomic Analysis of Inclusion Bodies

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Cell-free inclusion bodies were reduced in 1× NuPage Sample Buffer (Invitrogen), incubated at 70 °C for 10 min, then alkylated with iodoacetamide (100 mM) at room temperature for 30 min before being heated to 95 °C for 2 min. Soluble protein was resolved by 1 dimensional gel-electrophoresis (4–12% Bis-Tris NuPAGE gel) and digested in-gel with trypsin, as previously described^{43 (link)}. Digested peptides were concentrated by vacuum centrifugation, desalted using StageTips^{44 (link)} constructed using Empore C₁₈ extraction discs (3M Analytical Biotechnologies). Desalted peptides were analyzed by nanoliquid chromatography–tandem mass spectrometry using a Dionex Ultimate 3000 nRSLC coupled to an LTQ-Orbitrap XL mass spectrometer (ThermoFisher Scientific, San Jose, CA), as previously described^{9 (link)}. MS/MS spectra were extracted by Proteome Discoverer and analyzed using SEQUEST by searching E. coli and contaminant protein databases. Probabilistic calculation of false-positive rates (<1% FDR) was performed by Scaffold/X! Tandem (Proteome Software) using the PeptideProphet and ProteinProphet algorithms⁴⁵.

Castellana M., Wilson M.Z., Xu Y., Joshi P., Cristea I.M., Rabinowitz J.D., Gitai Z, & Wingreen N.S. (2014). Enzyme clustering accelerates processing of intermediates through metabolic channeling. Nature biotechnology, 32(10), 1011-1018.

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Quantification of OPA1 Acetylation

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To acquire precise quantification of acetylation on OPA1 K834 in various contexts, targeted mass spectrometry was performed. 3 × 15 cm dishes of relevant cells were harvested, and mitochondria were enriched using mitochondria isolation kit (Sigma-Aldrich). The mitochondria samples were subject to DDA LC-MS/MS to determine the mitochondrial proteome as previously described, and the abundances of MCU, VDAC3, and CYC1 were used to calculate the normalization factors across samples. PRM assays were carried out by nLC-MS/MS using a Dionex Ultimate 3000 nRSLC coupled to a Q Exactive HF mass spectrometer (Thermo Fisher Scientific). Mitochondrial tryptic peptides were eluted on C18 column by a linear gradient from 0%–35% B over 90 min. The PRM method was set up with the following parameters: profile mode MS2 resolution of 30,000, with AGC target of 1e5, maximum inject time of 300 ms, isolation window of 0.8, and normalized collision energy of 27, and a peptide inclusion list containing the targeted OPA1 peptide GVEVDPSLIK(ac)DTWHQVYR. Summed fragment area normalized by the proteome-derived normalization factors was used for quantification.

Sheng X, & Cristea I.M. (2021). The antiviral sirtuin 3 bridges protein acetylation to mitochondrial integrity and metabolism during human cytomegalovirus infection. PLoS Pathogens, 17(4), e1009506.

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