Dionex nano ultimate 3000

Manufactured by Thermo Fisher Scientific

Sourced in Austria, United States

The Dionex nano Ultimate 3000 is a high-performance liquid chromatography (HPLC) system designed for nano-scale separations. It features a low-flow nanoflow pump and provides precise flow control for applications requiring low flow rates, such as proteomics and metabolomics studies.

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Lab products found in correlation

13 protocols using dionex nano ultimate 3000

Nano-LC-MS/MS Peptide Separation Protocol

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LC separation was done on a Dionex nano Ultimate 3000 (Thermo Scientific) with a Thermo Easy-Spray source. The digested peptides were reconstituted in 2% acetonitrile/0.1% trifluoroacetic acid, and 5 µL of each sample was loaded onto a PepMap 100 Å 3U 75 µm × 20 mm reverse phase trap where they were desalted online before being separated on a 100 Å 2U 50 µm × 150 mm PepMap EasySpray reverse phase column. Peptides were eluted using a 120 min gradient of 0.1% formic acid (A) and 80% acetonitrile (B) with a flow rate of 200 nL/min. The separation gradient was run with 0%–3% B over 3 min, 6%–10% B over 3 min, 10%–23% B over 74 min, 23%–50% B over 15 min, 50%–99% B over 5 min, a 4 min hold at 99% B, and finally 99%–2% B held at 2% B for 16 min.

Potapova A., Garvey W., Dahl P., Guo S., Chang Y., Schwechheimer C., Trebino M.A., Floyd K.A., Phinney B.S., Liu J., Malvankar N.S, & Yildiz F.H. (2024). Outer membrane vesicles and the outer membrane protein OmpU govern Vibrio cholerae biofilm matrix assembly. mBio, 15(2), e03304-23.

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DIA-based Proteome Profiling Workflow

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LC separation was carried out on a Dionex Nano Ultimate 3000 (Thermo Scientific) with a Thermo Easy-Spray source fitted with a PepSep emitter. The digested peptides were reconstituted in 2% acetonitrile/0.1% trifluoroacetic acid, and 5 µL of each sample was loaded onto a Thermo Scientific PepMap 100 C18 5 μm 0.3 mm × 5 mm reverse phase trap, where they were desalted online before being separated on a PepSep 8 cm ID 150 1.5 μm reverse phase column. Peptides were eluted using a 90 min gradient with a flow rate of 0.500 μL/min. The samples were run on an Orbitrap Exploris 480 (Thermo Scientific) in data-independent acquisition (DIA) mode; mass spectra were acquired using a collision energy of 30, resolution of 30 K, maximum inject time mode on auto, and an AGC target of 1000%, using an isolation window of 45.7 Da in the m/z range 350–1200 m/z. Raw spectrometry data and analysis are available from the Massive and Proteome Exchange repositories using the respective ID numbers (MSV000092680, PXD044608).

Zafarullah M., Li J., Salemi M.R., Phinney B.S., Durbin-Johnson B.P., Hagerman R., Hessl D., Rivera S.M, & Tassone F. (2023). Blood Proteome Profiling Reveals Biomarkers and Pathway Alterations in Fragile X PM at Risk for Developing FXTAS. International Journal of Molecular Sciences, 24(17), 13477.

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Peptide Separation and Mass Spectrometry Analysis

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LC separation was done on a Dionex Nano Ultimate 3000 (Thermo Scientific) with a Thermo EASY-Spray source. Digested peptides were reconstituted in 2% acetonitrile/0.1% TFA and 5 μl of each sample was loaded onto a PepMap 100 Å 3U 75 μm × 20 mm reverse phase trap where they were desalted online before being separated on a 100 Å 2U 50 μm × 150 mm PepMap EASY-Spray reverse phase column. Peptides were eluted using a 70 min gradient of 0.1% formic acid (A) and 80% acetonitrile (B) with a flow rate of 200 nL/min. The separation gradient was run with 2 to 5% B over 1 min, 5 to 10% B over 9 min, 10 to 20% B over for 27 min, 20 to 35% B over 10 min, 35 to 99% B over 10 min, a 2 min hold at 99% B, and finally 99 to 2% B held at 2% B for 5 min.

Klotz C., O'Flaherty S., Goh Y.J, & Barrangou R. (2017). Investigating the Effect of Growth Phase on the Surface-Layer Associated Proteome of Lactobacillus acidophilus Using Quantitative Proteomics. Frontiers in Microbiology, 8, 2174.

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Nano LC-MS/MS Proteomics Workflow

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LC separation was done on a Dionex nano Ultimate 3000 (Thermo Scientific) with a Thermo Easy-Spray source. The digested peptides were reconstituted in 2% acetonitrile/0.1% trifluoroacetic acid, and 5 µl of each sample was loaded onto a PepMap 100 Å 3U 75 µm × 20 mm reverse-phase trap where they were desalted online before being separated on a 100 Å 2U 50 µm × 150 mm PepMap EasySpray reverse-phase column. Peptides were eluted using a 90-min gradient of 0.1% formic acid (A) and 80% acetonitrile (B) with a flow rate of 200 nl/min. The separation gradient was run with 2–5% B over 1 min, 5–10% B over 9 min, 10–20% B over 27 min, 20–35% B over 10 min, 35–99% B over 10 min, a 2-min hold at 99% B, and finally 99–2% B held at 2% B for 5 min.

Sharp K.A., Khoury M.J., Wirtz-Peitz F, & Bilder D. (2021). Evidence for a nuclear role for Drosophila Dlg as a regulator of the NURF complex. Molecular Biology of the Cell, 32(21), ar23.

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Liquid Chromatography Mass Spectrometry Peptide Separation

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LC separation was done on a Dionex Nano Ultimate 3000 (Thermo Scientific) with a Thermo Easy-Spray source^{17 (link)}. The digested peptides were reconstituted in 2% acetonitrile/0.1% TFA and 5 µl of each sample was loaded onto a PepMap 100 Å 3U 75 µm × 20 mm reverse-phase trap where they were desalted online before being separated on a 100 Å 2U 50-micron × 150 mm PepMap EasySpray reverse-phase column^{17 (link)}. Peptides were eluted using a 180-minute gradient of 0.1% formic acid (A) and 80% acetonitrile (B) with a flow rate of 200 nL/min. The separation gradient was run with 2% to 5% B over 1 minute, 5% to 10% B over 9 minutes, 10% to 20% B over for 27 minutes, 20% to 35% B over 10 minutes, 35% B to 99% B over 10 minutes, a 2 minute hold at 99% B, and finally 99% B to 2% B held at 2% B for 5 minutes^{17 (link)}.

Tiwari S., Mishra M., Salemi M.R., Phinney B.S., Newens J.L, & Gomes A.V. (2020). Gender-specific changes in energy metabolism and protein degradation as major pathways affected in livers of mice treated with ibuprofen. Scientific Reports, 10, 3386.

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Nano-LC-MS/MS Peptide Separation

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LC separation was done on a Dionex nano Ultimate 3000 (Thermo Scientific) with a Thermo Easy-Spray source. The digested peptides were reconstituted in 2% acetonitrile/0.1% trifluoroacetic acid and 1 μg in 5 μl of each sample was loaded onto a PepMap 100 Å 3U 75 um × 20 mm reverse phase trap where they were desalted online before being separated on a 100 Å 2U 50 micron × 150 mm PepMap EasySpray reverse phase column. Peptides were eluted using a 120 minutes gradient of 0.1% formic acid (A) and 80% acetonitrile (B) with a flow rate of 200 nl min⁻¹. The separation gradient was run with 2% to 5% B over 1minute, 5% to 50% B over 89 minutes, 50% to 99% B over for 2 minutes, a 4-minutes hold at 99% B, and finally 99% B to 2% B held at 2% B for 18 minutes.

He D., Feng H., Sundberg B., Yang J., Powers J., Christian A.H., Wilkinson J.E., Monnin C., Avizonis D., Thomas C.J., Friedman R.A., Kluger M.D., Hollingsworth M.A., Grandgenett P.M., Klute K.A., Toste F.D., Chang C.J, & Chio I.I. (2022). Methionine oxidation activates pyruvate kinase M2 to promote pancreatic cancer metastasis. Molecular cell, 82(16), 3045-3060.e11.

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Nano-HPLC Peptide Separation and Elution

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LC separation was done on a Dionex nano Ultimate 3000 (Thermo Scientific) with a Thermo Easy-Spray source. The digested peptides were reconstituted in 2% acetonitrile /0.1% trifluoroacetic acid, and 5µl of each sample was loaded onto a PepMap 100Å 3U 75 um x 20 mm reverse phase trap where they were desalted online before being separated on a 100 Å 2U 50 micron x 150 mm PepMap EasySpray reverse phase column. Peptides were eluted using a 90-minute gradient of 0.1% formic acid (A) and 80% acetonitrile (B) with a flow rate of 200nL/min. The separation gradient was run with 2% to 5% B over 1 minute, 5% to 10% B over 9 minutes, 10% to 20% B over 27 minutes, 20% to 35% B over 10 minutes, 35% to 99%B over 10 minutes, a 2 minute hold at 99%B, and finally 99% to 2%B held at 2% B for 5 minutes.

Sharp K.A., Khoury M.J., Wirtz-Peitz F., & Bilder D. (2021). Evidence for a Nuclear Role forDrosophilaDlg as a Regulator of the NURF Complex.

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Mass Spectrometry Proteomics of E. coli

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The co-precipitates were analyzed on a Q-exactive mass spectrometer equipped with a Dionex Ultimate 3000 Nano (Thermo Fisher Scientific). Peptides were trapped on a Acclaim PePmap 100 (75 μm × 2 cm, nanoviper, C₁₈, 3 μm, Thermo Fisher Scientific) and separated on a Venusil XBP C₁₈ (2.1 × 150 mm, 5 μm, Agela Technologies) using a gradient formed between solvent A (0.1% formic acid in water) and solvent B (0.1% formic acid in acetonitrile). The gradient started at 5% solvent B and the concentration of solvent B was increased to 80% within 60 min. Database search was performed using the MASCOT software against the E. coli K12 database.

Li H., Wang Y., Chen Q., Xia X., Shen J., Wang Y, & Shao B. (2021). Identification of Functional Interactome of Colistin Resistance Protein MCR-1 in Escherichia coli. Frontiers in Microbiology, 11, 583185.

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High-Resolution Peptide Separation and Analysis

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Peptides were resuspended in 0.2% formic acid, and the concentration of each sample was determined using a NanoDrop One spectrophotometer (Thermo Scientific). The samples were then prepared in autosampler vials and loaded onto a 75-μm i.d. × 360-μm o.d. capillary column (New Objective) that was packed in-house69 with 1.7-μm BEH C18 particles and held at 50 °C throughout the analysis. Separations were performed with a Dionex UltiMate 3000 nano HPLC system (Thermo Scientific). The mobile phases were 0.2% formic acid in water (A) and 0.2% formic acid in 80% ACN (B). The peptides were analysed by an Orbitrap Eclipse system (Thermo Scientific) with the following parameters: Orbitrap MS1 resolution of 240,000; MS1 automatic gain control target of 1 × 10⁶; MS1 maximum injection time of 50 ms; MS1 scan range of 300–1,500 m/z; dynamic exclusion of 20 ms; advanced peak determination⁷⁰ toggled on; MS2 isolation window of 0.7 m/z; MS2 collision energy of 25%; ion trap MS2 resolution setting of turbo; MS2 automatic gain control target of 3 × 10⁴; MS2 maximum injection time of 14 ms; MS2 scan range of 150–1,200 m/z.

Tai J., Guerra R.M., Rogers S.W., Fang Z., Muehlbauer L.K., Shishkova E., Overmyer K.A., Coon J.J, & Pagliarini D.J. (2023). Hem25p is required for mitochondrial IPP transport in fungi. Nature cell biology, 25(11), 1616-1624.

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Nano-LC-MS/MS Proteomic Analysis

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Dried samples were reconstituted in 5 µL 30% FA containing 10 fmol each of four synthetic standard peptides and diluted with 40 µL mobile phase A (98% H₂O, 2% ACN, 0.1% FA). 5 µL of this solution was then injected into a Dionex Ultimate 3000 nano LC-system coupled to a QExactive orbitrap mass spectrometer equipped with a nanospray ion source (Thermo Fisher Scientific, Austria). As a pre-concentration step, peptides were loaded on a 2 cm x 100 µm C18 Pepmap100 pre-column (Thermo Fisher Scientific, Austria) at a flow rate of 10 µL/min using mobile phase A. Elution from the pre-column to a 50 cm × 75 µm Pepmap100 analytical column (Thermo Fisher Scientific, Austria) and subsequent separation was achieved at a flow rate of 300 nL/min using a gradient of 8–40% mobile phase B (80% ACN, 2% H₂O, 0.1% FA) over 90 min. For mass spectrometric detection, MS scans were performed in the range from m/z 400–1400 at a resolution of 70,000 (at m/z = 200). MS/MS scans of the 8 most abundant ions were achieved through HCD fragmentation at 30% normalized collision energy and analyzed in the orbitrap at a resolution of 17,500 (at m/z = 200).

Del Favero G., Janker L., Neuditschko B., Hohenbichler J., Kiss E., Woelflingseder L., Gerner C, & Marko D. (2021). Exploring the dermotoxicity of the mycotoxin deoxynivalenol: combined morphologic and proteomic profiling of human epidermal cells reveals alteration of lipid biosynthesis machinery and membrane structural integrity relevant for skin barrier function. Archives of Toxicology, 95(6), 2201-2221.

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