Ultimate 3000 rs autosampler

Manufactured by Thermo Fisher Scientific

Sourced in United States, Switzerland, Germany

The UltiMate 3000 RS autosampler is a laboratory instrument designed for automated sample injection and handling. It is a core component of high-performance liquid chromatography (HPLC) and ultra-high-performance liquid chromatography (UHPLC) systems. The autosampler's primary function is to precisely and repeatedly introduce liquid samples into the analytical flow path for separation and detection.

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19 protocols using ultimate 3000 rs autosampler

RNA Analysis by LC-MS

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All RNAs were analyzed on Finnigan LCQ Advantage MAX ion trap instrumentation connected to a Thermo Scientific UHPLC (components: Ultimate 3000 RS Pump, Ultimate 3000 RS Autosampler, Ultimate 3000 RS Column Compartment, Ultimate 3000 Diode Array Detector). RNA mass spectra were acquired in the negative-ion mode with a potential of −4 kV applied to the spray needle (capillary temperature: 270°C, capillary voltage: −2−3 V). LC: 250 pmol RNA dissolved in 30 μl of 20 mM ethylenediaminetetraacetic acid (EDTA) solution; average injection volume: 30 μl; column: Waters xBridge C18 2.5 μm column (1.0 × 50 mm) at 30°C; flow rate: 100 μl/min; eluent A: 8.6 mM triethylamine (TEA), 100 mM 1,1,1,3,3,3-hexafluoroisopropanol in H₂O (pH 8.0); eluent B: methanol; gradient: 0–100% B in A within 30 min; UV detection at 260/280 nm. The correct assembly of all RNAs used in this study was confirmed by the mass data.

Plangger R., Juen M.A., Hoernes T.P., Nußbaumer F., Kremser J., Strebitzer E., Klingler D., Erharter K., Tollinger M., Erlacher M.D, & Kreutz C. (2019). Branch site bulge conformations in domain 6 determine functional sugar puckers in group II intron splicing. Nucleic Acids Research, 47(21), 11430-11440.

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Antibody Purification Peptide Analysis

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One third of the tryptic peptides of each antibody purification was analyzed by high‐resolution nanoflow MS/MS (LTQ Orbitrap Velos Pro coupled to Ultimate 3000 RSLC nano system equipped with an Ultimate3000 RS autosampler, ThermoFisher Scientific, Dreieich, Germany). Peptides were trapped on a trap column (C18, 75 μm × 2 cm, Acclaim PepMap100C18, 3 μm, ThermoFisher) and separated on a reversed phase column (nano viper Acclaim PepMap capillary column, C18; 2 μm; 75 μm × 50 cm, ThermoFisher) at a flow rate of 200 nl/min. The gradient was build with buffer A (water and 0.1% formic acid) and B (90% acetonitrile and 0.1% formic acid) using a gradient (4–55% buffer B in 56 min; 55–90% buffer B in 7 min). The effluent was directly sprayed into the mass spectrometer through a coated silica emitter (PicoTipEmitter, 30 μm, New Objective, Woburn, MA, USA) and ionized at 2.2 kV. MS spectra were acquired in a data‐dependent mode. For the collision‐induced dissociation (CID) MS/MS top 10 method, full‐scan MS spectra (m/z 300–1,700) were acquired in the Orbitrap analyzer using a target value of 10⁶. The 10 most intense peptide ions (charge states ≥ 2) were fragmented in the high‐pressure linear ion trap by low‐energy CID with normalized collision energy of 35%.

Dembla M., Kesharwani A., Natarajan S., Fecher‐Trost C., Fairless R., Williams S.K., Flockerzi V., Diem R., Schwarz K, & Schmitz F. (2018). Early auto‐immune targeting of photoreceptor ribbon synapses in mouse models of multiple sclerosis. EMBO Molecular Medicine, 10(11), e8926.

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UHPLC Analysis of Derivatized Methylglyoxal and Glyoxal

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Derivatized methylglyoxal and glyoxal were analyzed by the Thermo Scientific Dionex UltiMate 3000 UHPLC system (Thermo Fisher Scientific; Waltham, MA, USA) incorporated with a quaternary pump (LPG-3400D), UltiMate 3000 RS autosampler (WPS-3000), and fast separation photodiode array detector (DAD-3000). Derivatives were separated on a Kinetex C18 column (150 × 2.1 mm × 2.6 μm) (Phenomenex; Torrance, CA, USA) and a temperature-controlled column compartment (TCC–3000) was used to maintain its temperature at 40 °C. The binary mobile phase system consisted of 0.1% (v/v) formic acid in water (solvent C) and 0.1% (v/v) formic acid in acetonitrile (solvent D). The column eluted with a binary gradient system at a flow rate of 400 μL/min: 100% C from 0 to 4 min, 100–77% C in D from 4 to 25 min, and held at 77% C in D for 0.5 min, 77–100% C in D from 25.5 to 30 min, and then 100% C from 30 to 32 min. The injection volume was 10 μL. The peak areas of methylquinoxaline and quinoxaline were monitored at wavelengths of 316 and 314 nm, respectively. Data acquisition was carried out with the Chromeleon Chromatography Data System (Thermo Fisher Scientific; Waltham, MA, USA).

Bednarska K, & Fecka I. (2021). Potential of Vasoprotectives to Inhibit Non-Enzymatic Protein Glycation, and Reactive Carbonyl and Oxygen Species Uptake. International Journal of Molecular Sciences, 22(18), 10026.

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Betanin Quantification in Beetroot Samples

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Betanin determination of diluted beetroot juice samples (2.5 mg/mL in 1:1 0.1 % formic acid in water: 2 % HCl in MeOH) and beetroot food extracts was carried out on a Dionex UltiMate 3000 RSLC HPLC System (Dionex, Camberley, UK) equipped with an UltiMate 3000 RS pump, an UltiMate 3000 RS autosampler and a QExactive Quadrupole-Orbitrap Mass Spectrometer (Thermo Fisher Scientific, Waltham, USA). Electrospray ionisation at negative ion mode was performed with a spray voltage of 2.00 kV and capillary temperature of 280 °C. The total ion current (TIC) with a range of 100–1000 m/z and 70,000 resolution was measured. The ion m/z 549 was used for quantification of betanin. Sample aliquots (3 µL) were injected on a Phenomenex Luna C18(2) (250 × 2.0 mm, 5um particle size) reverse-phase column thermostatically regulated at 40 °C. The mobile phase consisted of water with 1 % acetic acid (solvent A), and acetonitrile with 1 % acetic acid (solvent B). After a 6-min equilibration with 20 % B, the elution programme was as follows: 0–30 min, 10–100 % B, (0.2 mL/min) followed by a washing stage (100 % B, 30–36 min, 0.2 mL/min) and re-equilibration at the initial conditions for 3 min. Betanin with a retention time of 2.56 min was quantified by external standard determination.

Clifford T., Constantinou C.M., Keane K.M., West D.J., Howatson G, & Stevenson E.J. (2016). The plasma bioavailability of nitrate and betanin from Beta vulgaris rubra in humans. European Journal of Nutrition, 56(3), 1245-1254.

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HPLC Analysis of Phenolic Compounds

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Electronic balance (serial number: 1508, OHAUS, Germany) and vortex mixer (VM-2000, 220 V, Digisystem Laboratory Instruments Inc., Taiwan) were used for this study. Chromatographic analyses using high performance liquid chromatography (HPLC) of the extract were carried out on Thermo Scientific Dionex Ultimate 3000 Rapid Separation LC (RSLC) systems (Thermo Fisher Scientific Inc., MA, USA), coupled to a quaternary rapid separation pump (LPG-3400RS), Ultimate 3000RS autosampler (WPS-3000), and rapid separation diode array detector (DAD-3000RS). Phenolic compounds were separated on an Acclaim C18 (4.6 × 250 mm; 5 μm) column (Dionex, USA) which was controlled at 30°C using a temperature controlled column compartment (TCC-3000). Data acquisition, peak integration, and calibrations were performed with Dionex Chromeleon software (Version 6.80 RS 10).

Rahman M., Khatun A., Nesa M.L., Hossain H, & Jahan I.A. (2015). Bioactive Polyphenols from the Methanol Extract of Cnicus arvensis (L.) Roth Demonstrated Antinociceptive and Central Nervous System Depressant Activities in Mice. Evidence-based Complementary and Alternative Medicine : eCAM, 2015, 794729.

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Label-free Quantification and PTM Analysis of Interactome

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For LC/MS interactome studies, a Dionex UltiMate 3000 RSLCnano system equipped with a Dionex UltiMate 3000 RS autosampler, an Acclaim PepMap RSLC analytical column and an Acclaim PepMap 100 trap column (Thermo Scientific) were used.
For label-free quantification (LFQ) analysis and protein identification, raw data files were analyzed using MaxQuant software suite v1.6.5.0 [48 (link)] against Andromeda search engine [49 (link)] and in-house standard parameters. Results were analyzed and visualized using LFQ-Analyst [50 (link)].
For PTM analysis, raw files were searched with Byonic v3.0.0 (ProteinMetrics) using GlyGly at lysine as a variable modification. Only peptides and proteins falling within a false discovery rate (FDR) of 1% based on a decoy database were further analyzed.

Kuser-Abali G., Zhang Y., Szeto P., Zhao P., Masoumi-Moghaddam S., Fedele C.G., Leece I., Huang C., Cheung J.G., Ameratunga M., Noguchi F., Andrews M.C., Wong N.C., Schittenhelm R.B, & Shackleton M. (2023). UHRF1/UBE2L6/UBR4-mediated ubiquitination regulates EZH2 abundance and thereby melanocytic differentiation phenotypes in melanoma. Oncogene, 42(17), 1360-1373.

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HPLC-DAD Analysis of Carbonyl Compounds and Green Tea Catechins

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HPLC-DAD was performed with a Thermo Scientific HPLC system consisting of an Ultimate 3000 RS pump, an Ultimate 3000 RS autosampler, and an Ultimate 3000 DAD. The column used was a 4.6 mm i.d.×150 mm, 5 μm, Gemini C18 column (Phenomenex, Torrance, CA) to analyze the OPD/DNPH derivatives with MGO/MDA and the concentrations of EC, EGC, ECG, and EGCG in green tea with a flow rate of 1.0 mL/min. For the OPD/DNPH derivatives with MGO/MDA, column elution started with 20% solvent B (A: 100% water with 0.1% formic acid; B: 100% acetonitrile with 0.1% formic acid) for 5 mins, followed by a linear increase to 100% solvent B from 5 to 20 min, and then equilibrated to 20% B from 21 to 25 min for the next run. For green tea catechins, column elution started with 5% solvent B for 5 mins, followed by a linear increase to 35% solvent B from 5 to 25 min, and then a linear increase to 95% solvent B from 25 to 30 min, keep 95% solvent B for 3 min, then equilibrated to 5% B for the next run. The detection wavelength was 315 nm for MGO-OPD derivatives and 305 nm for MDA-DNPH derivatives, and 280 nm for green tea catechins. The injection volume of each sample was 80 μL. The temperature of autosampler was set to 8 °C. For contents of catechins in green tea, four catechins, EC, EGC, ECG, and EGCG were used as standards.

Zhang S., Zhao Y., Ohland C., Jobin C, & Sang S. (2018). Microbiota facilitates the formation of the aminated metabolite of green tea polyphenol (−)-epigallocatechin-3-gallate which trap deleterious reactive endogenous metabolites. Free radical biology & medicine, 131, 332-344.

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LC-MS Metabolite Profiling of Urine and Plasma

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Urine and plasma samples were analysed separately in randomised batches. In brief, LC-MS analyses were carried out on a Dionex UltiMate 3000 RSLC HPLC System (Dionex), equipped with an UltiMate 3000 RS pump, an UltiMate 3000 RS autosampler and a QExactive Quadrupole-Orbitrap Mass Spectrometer (Thermo Fisher Scientific). Electrospray ionisation at both negative and positive ion modes was performed with a spray voltage of 2•00 kV and capillary temperature of 280°C. The total ion current with a range of 50-1000 m/z and 70 000 resolution was measured. Sample aliquots (2 µl) were injected on an Accucore (Thermo Fisher) C18 (150 × 2•5 mm, 5-μm particle size) reverse-phase column, thermostatically regulated at 40°C. The mobile phase consisted of water with 0•1 % formic acid (solvent A) and acetonitrile with 0•1 % formic acid (solvent B). Starting conditions were 95 % A, and compounds were separated with a gradient of 95 % A to 5 % A over 18 min, with a 5-min wash cycle before being re-equilibrated to 95 % A; flow rate was 0•35 ml/min. All samples were randomised in their running order, and a quality-control sample was analysed on ten occasions throughout the batch. The quality-control was a pool of all samples in a single aliquot.

Langer S., Kennel A, & Lodge J.K. (2018). The influence of juicing on the appearance of blueberry metabolites 2 h after consumption: a metabolite profiling approach. The British journal of nutrition, 119(11).

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Peptide Fractionation via HPLC

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Fractionation of the peptide mixture was performed on a Dionex Ultimate 3000 HPLC system equipped with an UltiMate 3000 RS pump, an UltiMate 3000 RS column compartment and an UltiMate 3000 RS autosampler (Dionex). The column was initially equilibrated for 70 min in buffer A (20 mM ammonium formate, pH 10) as described above before sample injection. The tryptic digest was injected onto a Phenomenex column (Gemini-NX 3u C18 110A; 1502.00 mm) using a linear gradient increasing at a rate of 1% B per minute from 2 ± 45% mobile phase B (A: 20 mM HCOONH4 at pH 10; B: 20 mM HCOONH4, 80% CAN at pH 10) at a flow rate of 200 μl/min while monitoring a UV absorbance at 214 nm/280 nm. Fractions were collected every minute during the course of the run. Subsequently, all 48 collected fractions were mixed into 14 components, lyophilized, and kept at −80 °C before Nano-LC-MS/MS analysis.

Zhang Q., Wu L., Bai B., Li D., Xiao P., Li Q., Zhang Z., Wang H., Li L, & Jiang Q. (2020). Quantitative Proteomics Reveals Association of Neuron Projection Development Genes ARF4, KIF5B, and RAB8A With Hirschsprung Disease. Molecular & Cellular Proteomics : MCP, 20, 100007.

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Degassed Solvent nanoLC Separation

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Degassed solvents were used for nanoLC (mobile phase (A): 0.1% (v/v) FA in water; organic phase (B) 0.1% (v/v) FA in ACN). The vials with the samples (15 µL) were placed into a rack and put onto the Dionex Ultimate 3000 RS Autosampler.

Kulyyassov A, & Ogryzko V. (2020). In Vivo Quantitative Estimation of DNA-Dependent Interaction of Sox2 and Oct4 Using BirA-Catalyzed Site-Specific Biotinylation. Biomolecules, 10(1), 142.

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