Easy nlc 1000 system nano hplc

Manufactured by Thermo Fisher Scientific

Sourced in United States

The EASY‐nLC 1000 system is a nano HPLC (High-Performance Liquid Chromatography) instrument. The core function of this system is to provide high-resolution separation of complex sample mixtures using nanoscale liquid chromatography techniques.

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4 protocols using easy nlc 1000 system nano hplc

Proteomic Analysis of Biological Samples

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Each sample was split into two volumes and subsequently analyzed on an EASY‐Spray C18 LC column (75 μm × 120 mm, 3 μm) (Thermo Fisher Scientific). A Q Exactive mass spectrometer (Thermo Fisher Scientific) combined with an EASY‐nLC 1000 system (Nano HPLC, Thermo Fisher Scientific) was used to analyze the samples. Ten microliters of each sample was separated using mobile phase A (99.9% ultrapure water, 0.1% formic acid) and mobile phase B (99.9% acetonitrile, 0.1% formic acid), with a gradient of 5% to 95% mobile phase B for 126 min, at a flow rate of 0.3 μl/min. Then, the eluate was analyzed using the Q Exactive mass spectrometer.
The MS data were acquired in high sensitivity mode, using the following analytical parameters: ion source EASY‐Spray, spray voltage 2.3 kV; capillary temperature, 320℃; full scan automatic gain control (AGC), target 3E6; resolution, 70,000 (full width at half maximum, fwhm); full scan maximum injection time, 20 ms; and scan range, 300 − 1,800 m/z. The MS/MS spectrum parameters were as follows: resolution, 17,500 (fwhm); AGC target, 1E5; maximum injection time, 120 ms; and intensity threshold, 8.30E3. A total of 30 data‐dependent MS/MS scans were acquired for each full scan. Precursor ions were further fragmented in a collision cell using normalized collision energy of 32%, and the fragments were quantitated using an Orbitrap.

Deng X., Du B., Zhu F., Gao Y, & Li J. (2020). Proteomic analysis of Aspergillus niger 3.316 under heat stress. MicrobiologyOpen, 9(5), e1012.

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High-pH Fractionation and LC-MS/MS Analysis

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The TMT-labelled peptides were dissolved in 100 μL buffer A (98% double-distilled water, 2% acetonitrile, pH 10) and fractionated via high pH reversed-phase fractionation chromatography with a RIGOL L-3000 HPLC System (Beijing RIGOL Technologies Inc., Beijing, China). Fractions were collected every 1.75 min in 45 tubes and then dried and combined into 10 tubes for further LC-MS/MS analysis.
LC-MS/MS analysis was performed with an EASY-nLC 1000 System (Nano HPLC, Thermo Fisher Scientific, USA) coupled online to a Q Exactive Mass Spectrometer with an EASY-Spray Ion Source (Thermo Fisher Scientific, USA). The samples were loaded onto an Acclaim PepMap 100 precolumn (2 cm × 100 μm, 5 μm, C18, Thermo Fisher Scientific, USA). Peptide separation was conducted using an EASY-Spray column (12 cm × 75 μm, 3 μm, C18, Thermo Fisher Scientific, USA) with buffer A (100% ultrapure water and 0.1% formic acid) and buffer B (100% acetonitrile and 0.1% formic acid) at a flow rate of 350 nL·min⁻¹. The eluted peptides were analysed using the Q Exactive online system. MS data acquisition was performed using a data-dependent top 20 method.

Tan W., Wang Y., Wang K., Wang S., Liu J., Qin X., Dai Y., Wang X, & Gao X. (2020). Improvement of Endothelial Dysfunction of Berberine in Atherosclerotic Mice and Mechanism Exploring through TMT-Based Proteomics. Oxidative Medicine and Cellular Longevity, 2020, 8683404.

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Nano-LC-MS/MS Proteomic Analysis Protocol

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The LC–MS/MS measurements were conducted with a Q-Exactive tandem mass spectrometer and an Easy-nLC 1000 nano HPLC system (Thermo Fisher Scientific). The trap column used for the nano HPLC was a 2 cm × 75 μm capillary column packed with 3 μm C18-silica particles (Thermo Fisher Scientific), and the separation column was a 12.5 cm × 75 μm capillary column packed with 3 μm C18-silica particles (Nikkyo Technos, Japan). The flow rate for the nano HPLC was 300 nL/min. The separation was conducted using a 10−40% linear acetonitrile gradient over 70 min, in the presence of 0.1% formic acid. The nanoLC–MS/MS data were acquired in the data-dependent acquisition (DDA) mode controlled by the Xcalibur 4.0 program (Thermo Fisher Scientific). The DDA settings were as follows: the resolution was 70 000 for a full MS scan and 17 500 for an MS2 scan; the AGC target was 3.0E6 for a full MS scan and 5.0E5 for an MS2 scan; the maximum IT was 60 ms for both the full MS and MS2 scans; the full MS scan range was 310–1500 m/z; and the top 10 signals in each full MS scan were selected for the MS2 scan. The DDA measurement was performed three times for each sample, and two biological replicates were measured in each condition except for the set of antibiotics experiment.

Yamakawa A., Niwa T., Chadani Y., Kobo A, & Taguchi H. (2023). A method to enrich polypeptidyl-tRNAs to capture snapshots of translation in the cell. Nucleic Acids Research, 51(5), e30.

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Peptide Identification by Nano-HPLC-MS/MS

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Peptides were detected using an EASY‐nLC 1000 Nano HPLC system and a Q‐Exactive mass spectrometer, both manufactured by Thermo Fisher Scientific. The basic RPLC fractionated peptide digests, which had been vacuum‐dried, were reconstituted with 0.1% formic acid and loaded onto a trap column (100 μm × 2 cm, Nanoviper) at a rate of 3 μL/min. Peptides were separated using a gradient of 5–30% solvent B, which consisted of 0.1% formic acid in 95% acetonitrile, on an analytical column (75 μm × 50 cm, RSLC C18) for 80 min at a flow rate of 300 nL/min. The run time is fixed to 90 min. The mass spectrometer operates in data‐dependent acquisition mode. A comprehensive full‐scan MS was conducted within the m/z range of 350–1500 at a resolution of 120,000 at 200 m/z. The automatic gain control (AGC) target value for the MS1 was set to 3,000,000 and the ion fill time was established at 50 ms. The strongest ions with charge states ≥2 were separated every 3 sec. HCD fragmentation with 38% normalized collision energy was then used to fragmented them. The AGC target for MS2 was established at 100,000 and the ion filling time was established at 100 ms. Dynamic exclusion was established at 30s with a 10 ppm mass window. For each sample, two analyses were conducted.

Ding Y., Chen Y., Sun J., Shi Y., Li G., Luan X., Wang S., Li X., Jiang W., Wang L, & Zhang G. (2024). Identification of potential biomarkers for neuromyelitis optica by quantitative proteomics. Annals of Clinical and Translational Neurology, 11(5), 1184-1196.

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