Dionex ultimate 3000 nanolc

Manufactured by Thermo Fisher Scientific

Sourced in Germany, United States

The Dionex Ultimate 3000 NanoLC is a liquid chromatography system designed for high-performance nano-scale separations. It features precise flow control, low flow rates, and advanced detection capabilities suitable for a range of analytical applications.

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Lab products found in correlation

Orbitrap elite, by Thermo Fisher Scientific (2 mentions) Acclaim pepmap 100 c18 5 mm 300 μm trap column, by Thermo Fisher Scientific (1 mentions) Nanoease mzpeptide beh c18 column, by Waters Corporation (1 mentions) Q exactive instrument, by Thermo Fisher Scientific (1 mentions) Itraq labeling, by AB Sciex (1 mentions) Tripletof 5600, by AB Sciex (1 mentions) Ltq orbitrap xl mass spectrometer, by Thermo Fisher Scientific (1 mentions) Reprosil pur c18 aq 3 m resin, by Dr. Maisch (1 mentions) Ltq orbitrap velos mass spectrometer, by Thermo Fisher Scientific (1 mentions)

Close spelling variants of this product

Dionex Ultimate 3000 RSLC nanoLC Dionex Ultimate 3000 nanoLC system Dionex UltiMate 3000 RSLC nanoLC System Dionex UltiMate 3000 Rapid Separation nanoLC Ultimate 3000 nanoLC Dionex Ultimate 3000 Ultimate 3000

9 protocols using dionex ultimate 3000 nanolc

Nano-LC-ESI-Q-TOF Mass Spectrometry Workflow

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Each fraction was analysed by an LC system (Dionex‐ultimate 3000 Nano LC; Thermo Scientific) coupled to an ESI‐Q‐TOF mass spectrometer (maxis, impact; Bruker Daltonik, Bremen, Germany) in data‐dependent acquisition mode (m/z 350–1500). The peptides were loaded onto a C 18 capillary column (75 μm × cm) and eluted at a constant flow rate of 400 nl min^‐1 by a solvent mix consisting solvent A (0.1% formic acid in water) and solvent B (0.1% formic acid in acetonitrile) in a multi‐step gradient scheme: 2–30% B in 87 min, 30–50% B in 10 min, 50–80% B in 10 min and 80%B hold for 10 min. The mass spectrometer was set as one full MS scan followed by 10 MS/MS scans on the 10 most intense ions from the MS spectrum. The Source Capillary was set at 1900v, and the flow and temperature of dry gas was 2.0 l min^‐1 and 120°C respectively.

Ma G., Wang P., Yang Y., Wang W., Ma J., Zhou L., Ouyang J., Li R, & Zhang S. (2021). emPAI‐assisted strategy enhances screening and assessment of Mycobacterium tuberculosis infection serological markers. Microbial Biotechnology, 14(4), 1827-1838.

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Histone Tail Separation Using nanoLC

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Histone tails were separated as previously described (23 (link)) using a Dionex Ultimate 3000 nanoLC (Thermo Scientific) controlled by Chromeleon software. For each of the two biological replicates, two technical replicates were run. The nanoLC was equipped with a two column setup, a 5 cm pre-column (100 μm ID) packed with C₁₈ bulk material (ReproSil, Pur C18AQ 5 μm; Dr Maisch) and a 22 cm analytical column (75 μm ID) with picofrit packed with Polycat A resin (PolyLC, Columbia, MD, 3 μm particles, 1500 Å). Loading buffer was 0.1% formic acid in water. Buffer A and B were prepared as described previously (30 (link)). Briefly, solvent A was 75% acetonitrile, 20 mM propionic acid, adjusted to pH 6.0 using ammonium hydroxide, and solvent B was 25% acetonitrile adjusted to pH 2.5 with formic acid. The column oven of the nanoLC was set to 30°, in order to decrease trap pressure. Sample was loaded onto the pre-column for 10 min with loading buffer at 3 μl/min. Samples were run with a gradient of 5 min 100% solvent A, followed by 55–85% solvent B in 135 min and 85–100% in 10 min for column washing. Flowrate for the analysis was set to 250 nl/min.

Vandamme J., Sidoli S., Mariani L., Friis C., Christensen J., Helin K., Jensen O.N, & Salcini A.E. (2015). H3K23me2 is a new heterochromatic mark in Caenorhabditis elegans. Nucleic Acids Research, 43(20), 9694-9710.

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Nano-LC-MS/MS Proteomics Workflow

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LC–MS/MS was performed on a Dionex Ultimate 3000 NanoLC connected to an Orbitrap Elite (Thermo Fisher, Waltham, MA, USA) equipped with an EasySpray ion source. The mobile phase A was composed of 0.1% aqueous formic acid, and mobile phase B was composed of 0.1% formic acid in acetonitrile. Peptides were loaded onto the analytical column (PepMap RSLC C18 2 μm, 100 Å, 50 μm i.d. × 15 cm) at a flow rate of 300 nL/min using a linear AB gradient composed of 2–25% A for 185 min, 25–90% B for 5 min, and then an isocratic hold at 90% for 5 min with re-equilibrating at 2% A for 10 min. The temperature was set to 40 °C for both columns. Nano-source capillary temperature was set to 275 °C, and spray voltage was set to 2 kV. MS1 scans were acquired in the Orbitrap Elite at a resolution of 60,000 full width at half maximum with an automated gain control target of 1 × 10⁶ ions over a maximum of 500 ms. MS2 spectra were acquired for the top 15 ions from each MS1 scan in normal scan mode in the ion trap with a target setting of 1 × 10⁴ ions, an accumulation time of 100 ms, and an isolation width of 2 Da. Normalized collision energy was set to 35%, and one microscan was acquired for each spectra.

Floris A., Mazarei M., Yang X., Robinson A.E., Zhou J., Barberis A., D’hallewin G., Azara E., Spissu Y., Iglesias-Ara A., Orrù S, & Tomasi M.L. (2020). SUMOylation Protects FASN Against Proteasomal Degradation in Breast Cancer Cells Treated with Grape Leaf Extract. Biomolecules, 10(4), 529.

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Nano-LC-MS Method for Tryptic Peptide Separation

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The LC-MS method was applied as previously
reported with slight modifications.^{14 (link),15 (link)} 200 nL of
the tryptic digests were separated by nano reverse phase (RP)-LC using
a Dionex UltiMate 3000 nanoLC system (Thermo Fisher Scientific, Breda,
Netherlands). After trapping on an Acclaim PepMap 100 C18 5 mm ×
300 μm trap column (Thermo Fisher Scientific), a nanoEase MZ
Peptide BEH C18 column of 75 μm × 100 mm, featuring 130
Å pores and 1.7 μm particles, was used for separation at
600 nL/min (Waters, Milford, USA). The binary gradient was from 3.0
to 21.7% B from 0.0 to 4.5 min, linear from 21.7 to 50% B from 4.5
to 5.5 min, isocratic at 50.0% B for 2.5 min, linear from 50 to 3%
from 8 to 9 min, and re-equilibration at 3% for 2.5 min. Solvents
A and B were an aqueous 0.1% TFA solution and 95% ACN, respectively.
Also, electrospray ionization mass spectrometry (ESI-MS) parameters
were as reported before.^{14 (link)}

Amez-Martín M., Wuhrer M, & Falck D. (2020). Immunoglobulin G Glycoprofiles are Unaffected by Common Bottom-Up Sample Processing. Journal of Proteome Research, 19(10), 4158-4162.

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Targeted Peptide Quantification by LC-MS

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Dry peptide samples were reconstituted in a water/acetonitrile (85:15) mixture containing 0.1% formic acid (100 μL) and 15 μL was injected on to an EASY-Spray C18 loading column (5 μm particle size, 100 μm × 2 cm; Fisher Scientific, DX164564) and resolved on a custom analytical column (2 μM particle size, 75 μm × 15 cm) using a Dionex Ultimate 3000 nano-LC (Thermo Fisher Scientific). Peptides were separated over a 60-min gradient of 15 to 33% acetonitrile (0.1% formic acid) and analyzed on a Q-Exactive instrument (Thermo Fisher Scientific) using a parallel reaction monitoring method targeting the SF3B1 peptide containing C1111 (amino acids 1110 – 1149, missed tryptic, +3 charge state). Selected precursor ions were isolated and fragmented by high-energy collision dissociation and fragments were detected in the Orbitrap at 17,500 resolution.
Raw data files were uploaded analyzed in Skyline (v.21.1.0.278) to determine the abundance of each peptide in inhibitor-treated samples relative to vehicle-treated samples. Peptide quantification was performed by calculating the sum of the peak areas corresponding to six fragment ions from each peptide. The peptides and fragment ions were preselected from in-house reference spectral libraries acquired in data-dependent acquisition mode to identify authentic spectra for each peptide and normalized to internal retention time standards.

Fekkes P, & Driessen A.J. (1999). Protein Targeting to the Bacterial Cytoplasmic Membrane. Microbiology and Molecular Biology Reviews, 63(1), 161-173.

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Nano-LC-MS/MS Analysis of Peptides

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LC-MS/MS was performed on a Dionex Ultimate 3000 NanoLC connected to an Orbitrap Elite (Thermo Fisher) equipped with an EasySpray ion source. The mobile phase A was composed of 0.1% aqueous formic acid, and mobile phase B was composed of 0.1% formic acid in acetonitrile. Peptides were loaded onto the analytical column (PepMap RSLC C18 2 μm, 100 Å, 50 μm i.d. × 15 cm) at a ow rate of 300 nL/min using a linear AB gradient composed of 2-25% A for 185 min, 25-90% B for 5 min, and then an isocratic hold at 90% for 5 min with re-equilibrating at 2% A for 10 min. The temperature was set to 40°C for both columns. Nano-source capillary temperature was set to 275°C, and spray voltage was set to 2 kV. MS1 scans were acquired in the Orbitrap Elite at a resolution of 60,000 full width at half maximum with an automated gain control target of 1 × 106 ions over a maximum of 500 ms. MS2 spectra were acquired for the top 15 ions from each MS1 scan in normal scan mode in the ion trap with a target setting of 1 × 104 ions, an accumulation time of 100ms, and an isolation width of 2 Da. Normalized collision energy was set to 35%, and one microscan was acquired for each spectra.

Floris A., Mazarei M., Yang X., Robinson A., Zhou J., Barberis A., D'Hallewin G., Azara E., Spissu Y., Iglesias-Ara A., Orrù S., & Tomasi M.L. (2020). SUMOylation protects FASN against proteasomal degradation in breast cancer cells treated with grape leaf extract.

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Quantitative Tear Protein Analysis

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Aliquots of lyophilized tear protein extracts (25 lg total protein) were used for isobaric tags for relative and absolute quantitation (iTRAQ) labeling (AB Sciex, Framingham, MA, USA) followed by LC-MS/MS analysis as previously described. 18 Liquid chromatography was performed using a commercial LC system (Dionex Ultimate 3000 Nano LC; Thermo Fisher Scientific, Inc., Sunnyvale, CA, USA) and mass spectrometry with a commercial device (TripleTOF 5600; AB Sciex, Framingham, MA, USA) according to procedures described elsewhere. 18

Fenner B.J., Liu Y.C., Koh S.K., Gao Y., Deng L., Beuerman R.W., Zhou L., Theng J.T.S, & Mehta J.S. (2019). Mediators of Corneal Haze Following Implantation of Presbyopic Corneal Inlays. Investigative ophthalmology & visual science, 60(4).

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Peptide Separation and Analysis by Nano-LC-MS/MS

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Peptides were separated by reverse‐phase chromatography using a Dionex Ultimate 3000 nanoLC (Dionex/Thermo Fisher Scientific) on a C18 capillary column (Acclaim PepMap100 C18, 2 μm, 100 Å, 75 μm i.d. × 20 cm/ThermoFisher) at an eluent flow rate of 300 nl/min. Mobile phase A contained 5% acetonitrile, 0.1% formic acid, and mobile phase B contained 80% acetonitrile and 0.1% formic acid. The column was preequilibrated with 5% mobile phase B followed by an increase to 65% mobile phase B in 75 min. The eluted peptides were ionized online by electrospray ionization (ESI) and transferred into an LTQ Orbitrap XL mass spectrometer (ThermoFisher) which was operated in the positive mode to measure full scan MS spectra (300–1700 m/z in the Orbitrap analyzer at resolution R = 30 000) followed by isolation and fragmentation (MS/MS) of the 10 most intense ions (in the LTQ iontrap) by collision‐induced dissociation (CID).

Hatakeyama D., Sunada H., Totani Y., Watanabe T., Felletár I., Fitchett A., Eravci M., Anagnostopoulou A., Miki R., Okada A., Abe N., Kuzuhara T., Kemenes I., Ito E, & Kemenes G. (2022). Molecular and functional characterization of an evolutionarily conserved CREB‐binding protein in the Lymnaea CNS. The FASEB Journal, 36(11), e22593.

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Trypsin Digestion and LC-MS/MS Analysis

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The resultant peptides of trypsin digestion were desalted by solid phase extraction using C₁₈ Empore disc cartridges (Supelco/Sigma-Aldrich, Taufkirchen, Germany) [22 (link)]. Desalted peptide mixtures were separated by reverse-phase chromatography using the Dionex Ultimate 3000 nanoLC (Dionex/Thermo Fisher Scientific, Idstein, Germany) on in-house manufactured 25 cm fritless silica micro-columns with an inner diameter of 100 μm. Columns were packed with the ReproSil-Pur C18-AQ 3 µm resin (Dr. Maisch GmbH, Entringen, Germany). Peptides were separated on a 5%–60% acetonitrile gradient (90 min) with 0.1% formic acid at a flow rate of 350 nL/min. The eluted peptides were ionized online by electrospray ionization and transferred into an LTQ Orbitrap Velos mass spectrometer (Thermo Fisher Scientific, Bremen, Germany) which was operated in the positive mode to measure full scan MS spectra (from m/z 300–1700 in the Orbitrap analyzer at resolution R = 60,000) followed by isolation and fragmentation of the twenty most intense ions (in the LTQ part) by collision-induced dissociation.

Murugaiyan J., Eravci M., Weise C, & Roesler U. (2016). Label-Free Quantitative Proteomic Analysis of Harmless and Pathogenic Strains of Infectious Microalgae, Prototheca spp.. International Journal of Molecular Sciences, 18(1), 59.

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