Ultimate 3000 rapid separation lc system

Manufactured by Thermo Fisher Scientific

Sourced in United States

The Ultimate 3000 Rapid Separation LC system is a high-performance liquid chromatography instrument designed for a wide range of analytical applications. It features a rapid separation capability and is capable of achieving high resolution and sensitivity across a variety of sample types.

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Ultimate 3000 (1640 citations) Ultimate 3000 system (297 citations) Ultimate 3000 LC system (140 citations) UltiMate® 3000 Rapid Separation LC (20 citations) Dionex Ultimate 3000 Rapid Separation LC system (8 citations) Ultimate 3000 Rapid Separation system (4 citations) Dionex UltiMate 3000 Rapid Separation (RS)LC system (2 citations)

20 protocols using ultimate 3000 rapid separation lc system

Proteome Profiling of Cell Lysates

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SDS-lysed patient and cell line samples were processed and digested according to the filter-aided sample preparation (FASP) method [23 (link),24 (link)]. All of the filter-processed samples used 20 μg of protein material. Peptides from both patient and cell line samples were cleaned up with the Oasis HLB μElution (Waters, Milford, MA, USA) protocol.4.4. Liquid Chromatography (LC)-MS Analysis.
Dried peptides were dissolved in 20 μL of 2% acetonitrile (ACN) and 0.5% formic acid (FA). Differently preserved THP-1 and Molm-13 samples were analyzed on an Orbitrap Elite mass spectrometer equipped with a nanospray Flex ion source coupled to an Ultimate 3000 Rapid Separation LC system (both from Thermo Scientific, Waltham, MA, USA). Approximately 0.5 μg peptides were pre-concentrated and separated, as previously described [5 (link)]. Patient samples without or with PBS wash(es) were analyzed on a Q Exactive HF Orbitrap mass spectrometer equipped with an Easy-Spray (Thermo Scientific) coupled to an Ultimate 3000 Rapid Separation LC system. Approximately 0.6 μg peptides were pre-concentrated on a 2 cm × 75 µm ID Acclaim PepMap 100 trapping column and separated on a 50 cm × 75 µm ID Easy-Spray PepMap RSLC analytical column (both from Thermo Scientific). Bound peptides were eluted within a 195 min run using a binary gradient with buffer A (0.1% FA in water) and buffer B (0.1% FA in ACN).

Wangen R., Aasebø E., Trentani A., Døskeland S.O., Bruserud Ø., Selheim F, & Hernandez-Valladares M. (2018). Preservation Method and Phosphate Buffered Saline Washing Affect the Acute Myeloid Leukemia Proteome. International Journal of Molecular Sciences, 19(1), 296.

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Proteomic Analysis of Whole Lung Tissue

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Samples from slides containing whole lung tissue sections were scraped excluding major blood vessels and processed as previously described (Herrera et al, 2020 (link)). Peptides were evaluated by liquid chromatography coupled tandem mass spectrometry using an UltiMate 3000 Rapid Separation LC system (Dionex Corporation) coupled to a Q Exactive HF mass spectrometer (Thermo Fisher Scientific). Raw spectra were aligned using MAXQuant software v1.6.17.0 (Cox & Mann, 2008 (link)) with the variable modifications of proline and methionine oxidation in addition to “matched between runs” being enabled. Raw data were then imported into R for differential analysis with MSqRob (Goeminne et al, 2018 (link)) using the default pipeline. Heat maps were plotted using scaled log₁₀-transformed LFQ counts.

Chenery A.L., Rosini S., Parkinson J.E., Ajendra J., Herrera J.A., Lawless C., Chan B.H., Loke P., MacDonald A.S., Kadler K.E., Sutherland T.E, & Allen J.E. (2021). IL-13 deficiency exacerbates lung damage and impairs epithelial-derived type 2 molecules during nematode infection. Life Science Alliance, 4(8), e202001000.

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Peptide Characterization by LC-MS/MS

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As previously described, peptides were resuspended in 10 μL of 3% acetonitrile, with 0.1% formic acid, and 1 μL was used for evaluation by liquid chromatography-coupled tandem MS using an UltiMate 3000 Rapid Separation LC system (Dionex Corporation) coupled to a Q Exactive HF mass spectrometer (Thermo Fisher Scientific).

Herrera J.A., Dingle L., Montero M.A., Venkateswaran R.V., Blaikley J.F., Lawless C, & Schwartz M.A. (2022). The UIP/IPF fibroblastic focus is a collagen biosynthesis factory embedded in a distinct extracellular matrix. JCI Insight, 7(16), e156115.

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Peptide Analysis by LC-MS/MS

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Sample peptides were analyzed by an UltiMate 3000 Rapid Separation LC system (Dionex Corporation, Sunnyvale, CA) coupled to a Q Exactive HF Hybrid Quadrupole‐Orbitrap MS (Thermo Fisher Scientific, Waltham, MA).

Vasilogianni A., Al‐Majdoub Z.M., Achour B., Annie Peters S., Barber J, & Rostami‐Hodjegan A. (2022). Quantitative Proteomics of Hepatic Drug‐Metabolizing Enzymes and Transporters in Patients With Colorectal Cancer Metastasis. Clinical Pharmacology and Therapeutics, 112(3), 699-710.

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Reverse Phase HPLC Analysis of Compounds

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The instrument consisted of a Dionex-Ultimate^® 3000 Rapid Separation LC system, which was equipped with an auto sampler, a quaternary pump, a degasser, a column oven, and a DAD detector.[22 (link)23 (link)] The chromatographic analysis was carried out using a reverse phase Acclaim Polar Advantage II C18 column. The column temperature was maintained at 40°C. The mobile phase was consisted of 0.1% formic acid solution (A) and acetonitrile (B) with gradient elution system as shown in Table 1 at a flow rate of 1 ml/min while the separation time was 18 min and the injection volume was 5 μl. The spectral data from the DAD-3000RS DAD was set at 210, 254 and 320 nm. For quantitative analysis, the wavelength was set at 320 nm. The peak identification was based on the retention time and the DAD spectrum against the standard presented in the chromatogram. The data acquisition was performed by Chromeleon software version 6.8 (Dionex, Thermo Fisher Scientific Inc.).

Saidan N.H., Aisha A.F., Hamil M.S., Majid A.M, & Ismail Z. (2015). A novel reverse phase high-performance liquid chromatography method for standardization of Orthosiphon stamineus leaf extracts. Pharmacognosy Research, 7(1), 23-31.

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Pineapple Protein Mass Spectrometry

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All MD2 pineapple protein extractions
and mass spectrometry were performed in the Bio-MS Research facility,
Faculty of Biology, Medicine, and Health, University of Manchester.
Digested samples were analyzed by liquid chromatography-mass spectrometry
(LC–MS)/MS using an UltiMate 3000 Rapid Separation LC system
(RSLC, Dionex Corporation, Sunnyvale, CA) coupled to an Orbitrap Elite
(Thermo Fisher Scientific, Waltham, MA) mass spectrometer. Peptide
mixtures were separated using a gradient from 92% A (0.1% FA in water)
and 8% B (0.1% FA in acetonitrile) to 33% B, in 104 min at 300 nL
min^–1, using a 75 mm × 250 μm i.d. 1.7
M CSH C18, analytical column (Waters). Peptides were selected for
fragmentation automatically by data-dependent analysis.

Ariffin N., Newman D.W., Nelson M.G., O’cualain R, & Hubbard S.J. (2024). Proteogenomic Gene Structure Validation in the Pineapple Genome. Journal of Proteome Research, 23(5), 1583-1592.

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Liquid Chromatography-Tandem Mass Spectrometry

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Digested samples were analysed by liquid chromatography (LC) coupled tandem MS (LC-MS/MS) using an UltiMate® 3000 Rapid Separation LC system (RSLC, Dionex Corporation) coupled to a Q Exactive HF (Thermo Fisher), peptide mixtures were separated using a multistep gradient from 95% A (0.1% formic acid, FA, Thermo Fisher, in water) and 5% B (0.1% FA in acetonitrile) to 7% B at 1 min, 18% B at 58 min, 27% B at 72 min and 60% B at 74 min at 300 nL/min, using a 75 mm x 250 µm, inner diameter 1.7 µm, CSH C18 analytical column (Waters). Peptides were selected for fragmentation automatically by data dependent analysis.

Mallikarjun V., Richardson S.M, & Swift J. (2020). BayesENproteomics: Bayesian Elastic Nets for Quantification of Peptidoforms in Complex Samples. Journal of proteome research, 19(6).

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Analytical and Preparative RP-HPLC Methods

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Analytical RP-HPLC was performed with an Ultimate 3000 Rapid Separation LC System (DAD-3000RS diode array detector) using an Acclaim RSLC 120 C18 column (2.2 μm, 120 Å, 3 × 50 mm, flow 1.2 mL min^–1) from Dionex (Sunnyvale, CA, USA). Data recording and processing was done with Dionex Chromeleon Management System (version 6.80). Preparative RP-HPLC was performed with a Waters Prep LC Controller System using a Dr. Maisch GmbH Reprospher column (C18-DE, 100 × 30 mm, particle size 5 μm, pore size 100 Å, flow rate 40 mL min^–1). Compounds were detected by UV absorption at 214 nm using a Waters 486 Tunable Absorbance Detector. The following eluents were used for all RP-HPLC measurements: “A” (Milli-Q deionized H₂O with 0.1% TFA); “D” (Milli-Q deionized H₂O–HPLC-grade acetonitrile (10 : 90) with 0.1% TFA). LC-MS data were collected after coupling the analytical system described above with a LCQ Fleet Ion Trap mass spectrometer (Thermo Scientific, San Jose, CA, USA). LC-MS data recording and processing was done with Xcalibur (version 2.2, Thermo Scientific). High resolution MS spectra, recorded on a LTQ OrbitrapXL Hybrid Ion Trap-Orbitrap mass spectrometer (Thermo Scientific), were provided by the analytical service of the Department of Chemistry and Biochemistry at the University of Bern (group P.D. Dr. Stefan Schürch).

Bartoloni M., Jin X., Marcaida M.J., Banha J., Dibonaventura I., Bongoni S., Bartho K., Gräbner O., Sefkow M., Darbre T, & Reymond J.L. (2015). Bridged bicyclic peptides as potential drug scaffolds: synthesis, structure, protein binding and stability †Electronic supplementary information (ESI) available: Crystallographic data; spectroscopic data with 1H NMR spectra, HPLC profiles and MS spectra. CCDC 1063793 and 1063796–1063799. For ESI and crystallographic data in CIF or other electronic format see DOI: 10.1039/c5sc01699a. Chemical Science, 6(10), 5473-5490.

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Proteomic Analysis via SDS-PAGE and LC-MS/MS

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Following SDS-PAGE, gel lanes were sliced and subjected to in-gel digestion with trypsin^{58 (link)} with modifications^{11 (link)}. Peptide samples were analysed by liquid chromatography (LC)-tandem MS using a nanoACQUITY UltraPerformance LC system (Waters) coupled online to an LTQ Velos mass spectrometer (Thermo Fisher Scientific) or using an UltiMate 3000 Rapid Separation LC system (Thermo Fisher Scientific) coupled online to an Orbitrap Elite mass spectrometer (Thermo Fisher Scientific). Peptides were concentrated and desalted on a Symmetry C₁₈ preparative column (20 mm × 180 μm, 5-μm particle size; Waters) and separated on a bridged ethyl hybrid C₁₈ analytical column (250 mm × 75 μm, 1.7-μm particle size; Waters) using a 45-min linear gradient from 1% to 25% or 8% to 33% (v/v) acetonitrile in 0.1% (v/v) formic acid at a flow rate of 200 nl/min. Peptides were selected for fragmentation automatically by data-dependent analysis.

Horton E.R., Byron A., Askari J.A., Ng D.H., Millon-Frémillon A., Robertson J., Koper E.J., Paul N.R., Warwood S., Knight D., Humphries J.D, & Humphries M.J. (2015). Definition of a consensus integrin adhesome and its dynamics during adhesion complex assembly and disassembly. Nature cell biology, 17(12), 1577-1587.

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Mass Spectrometry Workflow for Peptide Analysis

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For each sample, 0.5 µg tryptic peptides dissolved in 2% acetonitrile (ACN) and 0.5% formic acid (FA) were injected into an Ultimate 3000 Rapid Separation LC system (Thermo Scientific) coupled to a QExactive HF mass spectrometer (MS) (Thermo Scientific, Bremen, Germany) using settings as described in detail previously [43 (link)], except the LC gradient composition was shorter, i.e., 5% solvent B (i.e., 100% ACN) during trapping over 5 min followed by 5–8% B for 0.5 min, 8–24% B for the next 65.5 min, 24–35% B for 24 min, and 35–90% B for 5 min, hold 90% B for 8 min and ramp to 5% B for 5 min, before keeping 5% B for 20 min.

Aasebø E., Brenner A.K., Hernandez-Valladares M., Birkeland E., Berven F.S., Selheim F, & Bruserud Ø. (2021). Proteomic Comparison of Bone Marrow Derived Osteoblasts and Mesenchymal Stem Cells. International Journal of Molecular Sciences, 22(11), 5665.

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