Ultimate 3000 nanoflow lc system

Manufactured by Thermo Fisher Scientific

Sourced in United States

The Ultimate 3000 nanoflow LC system is a liquid chromatography instrument designed for high-performance separation of complex samples. It features advanced pump and autosampler technologies to deliver precise and consistent flow rates and sample injections. The system is capable of operating at nanoflow rates, making it suitable for applications that require high sensitivity and small sample volumes.

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Lab products found in correlation

Easy spray pepmap rslc c18, by Thermo Fisher Scientific (3 mentions) Reprosil pur c18 aq, by Dr. Maisch (2 mentions) Q exactive mass spectrometer, by Thermo Fisher Scientific (2 mentions) Q exactive hf orbitrap mass spectrometer, by Thermo Fisher Scientific (2 mentions) Oasis hlb, by Waters Corporation (1 mentions) Bca assay, by Thermo Fisher Scientific (1 mentions) C18 pepmap100 μ precolumn, by Thermo Fisher Scientific (1 mentions) Q exactive hf mass spectrometer, by Thermo Fisher Scientific (1 mentions) Proteasemax surfactant, by Promega (1 mentions) Trypsin, by Promega (1 mentions)

Close spelling variants of this product

Ultimate 3000 (1640 citations) Ultimate 3000 system (297 citations) Ultimate 3000 LC system (140 citations) Dionex Ultimate 3000 nanoflow LC system (2 citations)

5 protocols using ultimate 3000 nanoflow lc system

Peptide Mixture Analysis by LC-MS/MS

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Peptide mixture was injected into an Ultimate 3000 nanoflow LC system (Thermo Scientific, USA) in-line coupled to a Q Exactive mass spectrometer (Thermo Scientific). The chromatographic separation of the peptides was achieved using a 25 cm long in-house packed column (C18-AQ ReproSil-Pur®, Dr. Maisch GmbH, Germany) at 55°C with the following gradient: 4–30% ACN in 89 minutes, 26–95% ACN for 5 minutes, and 95% ACN for 8 minutes all at a flow rate of 250 nL/minutes.
The MS acquisition method comprised one full scan survey ranging from m/z 300 to m/z 1650 acquired with a resolution of R = 140,000 at m/z 200 and AGC target value of 5 × 10⁶, followed by data-dependent higher-energy collisional dissociation fragmentation scans from a maximum of 16 most intense precursor ions with a charge state ≥ 2. For dependent scans, the following parameters were used: precursor isolation width 4 Da, AGC target value of 2 × 10⁵, and normalized collision energy of 26. Scans were acquired in profile mode with a resolution of R = 17,500.

González F.E., Chernobrovkin A., Pereda C., García-Salum T., Tittarelli A., López M.N., Salazar-Onfray F, & Zubarev R.A. (2018). Proteomic Identification of Heat Shock-Induced Danger Signals in a Melanoma Cell Lysate Used in Dendritic Cell-Based Cancer Immunotherapy. Journal of Immunology Research, 2018, 3982942.

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Peptide Mixture Profiling by Nanoflow LC-MS/MS

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Peptide mixture was injected into an Ultimate 3000 nanoflow LC system (Thermo Fisher Scientific Inc., Waltham, MA, USA) in-line coupled to a QExactive mass spectrometer (Thermo Fisher Scientific Inc., Waltham, MA, USA). The chromatographic separation of the peptides was achieved using a 25 cm long in-house packed column (C18-AQ ReproSil-Pur^®, Dr. Maisch GmbH, Tübingen, Germany) at 55 °C with the following gradient: 4–30% ACN in 89 min, 26–95% ACN for 5 min and 95% ACN for 8 min all at a flow rate of 250 nL/min.
The MS acquisition method comprised one survey full scan ranging from m/z 300 to m/z 1650 acquired with a resolution of R = 140,000 at m/z 200 and AGC target value of 5 × 10⁶, followed by data-dependent higher-energy collisional dissociation fragmentation scans from maximum 16 most intense precursor ions with a charge state ≥2. For dependent scans, the following parameters were used: precursor isolation width 4 Da, AGC target value of 2*105, and normalized collision energy 26. Scans were acquired in profile mode with a resolution of R = 17,500.

Torres A., Vivanco S., Lavín F., Pereda C., Chernobrovkin A., Gleisner A., Alcota M., Larrondo M., López M.N., Salazar-Onfray F., Zubarev R.A, & González F.E. (2022). Haptoglobin Induces a Specific Proteomic Profile and a Mature-Associated Phenotype on Primary Human Monocyte-Derived Dendritic Cells. International Journal of Molecular Sciences, 23(13), 6882.

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Peptide Separation and Identification by LC-MS/MS

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Samples were desalted prior to LC-MS/MS analysis using Oasis cartridges for solid phase extraction (Oasis HLB, 1 cc, 10 mg, 30 µm particle size, Waters, Milfold, USA). Then, the peptide concentration for each sample was measured using the BCA assay (Thermo Fisher Scientific, Germering, Germany). Loaded sample quantity was 3 μg per injection. LC-MS/MS analysis was performed using an Orbitrap Q Exactive HF mass spectrometer (Thermo Fisher Scientific, San Jose, CA, USA) coupled with a UltiMate 3000 nanoflow LC system (Thermo Fisher Scientific, Germering, Germany). Mass spectrometry measurements were performed using datadependent acquisition (DDA) "top 15" mode. A trap column µ-Precolumn C18 PepMap100 (5 µm, 300 µm, i.d. 5 mm, 100 Å) (Thermo Fisher Scientific, USA) and an analytical column EASY-Spray PepMap RSLC C18 (2 µm, 75 µm, i.d. 500 mm, 100 Å) (Thermo Fisher Scientific, San Jose, CA, USA) were employed for separations. The column temperature was set to 50 o C. Mobile phases were as follows: (A) 0.1 % Formic acid (FA) in water; (B) 95 % ACN, 0.1 % FA in water. Samples were eluted using the gradient from 5 % B to 45 % B for 120 min at 270 nL/min flow rate.

Bubis J.A., Spasskaya D.S., Gorshkov V.A., Kjeldsen F., Kofanova A.M., Lekanov D.S., Gorshkov M.V., Karpov V.L., Tarasova I.A, & Karpov D.S. (2020). Rpn4 and proteasome-mediated yeast resistance to ethanol includes regulation of autophagy. Applied microbiology and biotechnology, 104(9).

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Peptide Analysis by Q-Exactive HF MS

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Samples were analyzed using a Q-Exactive HF mass spectrometer (Thermo Scientific, Bremen, Germany) coupled with an UltiMate 3000 Nanoflow LC system (Thermo Scientific, Germering, Germany). Peptides were concentrated at the trap column (μ-Precolumn C18 PepMap100, Thermo Scientific, 5 μm, 300 μm i.d. 5 mm, 100 Å) and eluted from an analytical column (EASY-Spray PepMap RSLC C18, Thermo Scientific, 2 μm, 75 μm i.d. 500 mm, 100 Å) with a gradient of acetonitrile.
Mass spectrometry measurements were performed using data-dependent acquisition mode (Top 12). MS1 spectra were acquired from 300 to 1400 Th, with a resolving power of 120 000 at m/z 200. Precursor ions were isolated with the m/z window of 1.4 Th followed by their fragmentation using higher-energy collision dissociation (HCD). Fragment ions were measured in an Orbitrap mass-analyzer with a resolving power of 15 000 at m/z 200. Each sample was analyzed in triplicate. The data were deposited in the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) via the PRIDE34 (link) partner repository with the dataset identifiers PXD007648 and 10.6019/PXD007648.

Gorshkov V., Bubis J.A., Solovyeva E.M., Gorshkov M.V, & Kjeldsen F. (2019). Protein corona formed on silver nanoparticles in blood plasma is highly selective and resistant to physicochemical changes of the solution †Electronic supplementary information (ESI) available: Supporting Information 1: Experimental details; figures and tables; protein abundance and corresponding sigmoid fit for all of the differentially abundant proteins detected in pH and temperature perturbation experiments. Supporting Information 2: Complete list of quantified proteins in all LC–MS runs in pH and temperature experiments, including protein metadata (Uniprot accession number, sequence coverage, GO annotation, number of identified peptides, etc.) Quantitative data provided as log-converted LFQ abundancies. Supporting Information 3: Results of lessening analysis. See DOI: 10.1039/c8en01054d. Environmental Science. Nano, 6(4), 1089-1098.

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Comprehensive Proteomics Analysis of Cell Lysates

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Proteomics data (available from the PRIDE [26 (link)] with the dataset identifier PXD014236) were collected earlier as described in [27 (link)]. Briefly, cells were lysed in ProteaseMAX Surfactant (Promega, Madison, WI, USA) lysis buffer using ultrasonic homogenization (Bandelin Sonopuls HD2070, Bandelin Electronic, Berlin, Germany). Protein extracts were reduced (10 mM dithiothreitol, 56 °C for 20 min), alkylated (10 mM iodoacetamide, at room temperature for 30 min in the dark), and digested overnight using trypsin (Promega, Madison, WI, USA). LC-MS/MS analysis was performed using an Orbitrap Q Exactive HF mass spectrometer (Thermo Fisher Scientific, San Jose, CA, USA) coupled with an UltiMate 3000 nanoflow LC system (Thermo Fisher Scientific, Germering, Germany) in data-dependent acquisition (DDA), “top 15” mode. A trap column µ-Precolumn C18 PepMap100 (5 µm, 100 Å, 300 µm i.d. × 5 mm) (Thermo Fisher Scientific, Waltham, MA, USA) and an analytical column EASY-Spray PepMap RSLC C18 (2 µm, 100 Å, 75 µm i.d. × 500 mm) (Thermo Fisher Scientific, San Jose, CA, USA) were employed for separations. Mobile phases were as follows: (A) 0.1% formic acid (FA) in water; (B) 95% acetonitrile, 0.1% FA in water. Samples were eluted using the gradient from 5% B to 45% B for 120 min at 270 nL/min flow rate.

Spasskaya D.S., Kulagin K.A., Grineva E.N., Osipova P.J., Poddubko S.V., Bubis J.A., Kazakova E.M., Kusainova T.T., Gorshkov V.A., Kjeldsen F., Karpov V.L., Tarasova I.A, & Karpov D.S. (2023). Yeast Ribonucleotide Reductase Is a Direct Target of the Proteasome and Provides Hyper Resistance to the Carcinogen 4-NQO. Journal of Fungi, 9(3), 351.

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