Easy nlc 1000

Manufactured by Thermo Fisher Scientific

Sourced in United States, Germany, Denmark, Japan

The EASY-nLC 1000 is a high-performance liquid chromatography (nanoLC) system designed for sensitive and reproducible separation of complex peptide mixtures. It offers precise solvent delivery, robust performance, and compatibility with a range of detectors, making it a versatile tool for proteomics research and analysis.

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Easy nLC (237 citations) EASY-nLC 1000 HPLC system (62 citations) EASY-nLC 1000 UHPLC system (26 citations) EASY-nLC 1000 HPLC (21 citations) EASY-nLC 1000 liquid chromatography system (19 citations) EASY-nLC 1000 pump (6 citations) Thermo Easy nLC 1000 LC system (5 citations) EASY-nLC 1000 nanosystem (3 citations) Easy-nLC 1000 nano-LC system (3 citations) Thermo Scientific EASY-nLC 1000 System (2 citations)

591 protocols using easy nlc 1000

Proteomic Analysis of Antibody Digests

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Aliquots of IgG fractions (5–10 μg) were carboxymethylated and digested by trypsin. Digested samples from RA patients and mouse model were analysed by an LC-ESI-MS system consisting of a Paradigm MS4 (Michrome Bioresources, Inc.) or an EASY-nLC 1000 (Thermo Scientific) HPLC system and Orbitrap Elite hybrid (Thermo Scientific, Inc.) or Q Exactive (Thermo Scientific, Inc.) MS system equipped with a nanoESI ion source, and digested samples from ACC4, M2139 and 1E11 hybridoma IgGs were analysed by an LC-ESI-MS system consisting of an EASY-nLC 1000 (Thermo Scientific) and Orbitrap Fusion Tribrid mass spectrometer (Thermo Scientific, Inc.). Details of the method used for LC-ESI-MS analysis are described in Supplementary Methods.

Ohmi Y., Ise W., Harazono A., Takakura D., Fukuyama H., Baba Y., Narazaki M., Shoda H., Takahashi N., Ohkawa Y., Ji S., Sugiyama F., Fujio K., Kumanogoh A., Yamamoto K., Kawasaki N., Kurosaki T., Takahashi Y, & Furukawa K. (2016). Sialylation converts arthritogenic IgG into inhibitors of collagen-induced arthritis. Nature Communications, 7, 11205.

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Phosphopeptide Analysis by LC-MS/MS

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The phosphopeptides were dissolved in 5 μL of 0.25% formic acid (FA) and injected into an Easy-nLC 1000 (Thermo Fisher Scientific). Peptides were separated on a 45 cm in-house packed column (360 μm OD × 75 μm ID) containing C18 resin (2.2 μm, 100Å, Michrom Bioresources). The mobile phase buffer consisted of 0.1% FA in ultra-pure water (Buffer A) with an eluting buffer of 0.1% FA in 80% ACN (Buffer B) run over a linear 60 min gradient of 6%–30% buffer B at flow rate of 250 nL/min. The Easy-nLC 1000 was coupled online with a Velos LTQ-Orbitrap mass spectrometer (Thermo Fisher Scientific). The mass spectrometer was operated in the data-dependent mode in which a full-scan MS (from m/z 350–1500 with the resolution of 60,000 at m/z 400) was followed by top 10 collision-induced dissociation (CID) MS/MS scans of the most abundant ions with dynamic exclusion for 60 s and exclusion list of 500. The normalized collision energy applied for CID was 35% for 10 ms activation time.

Wang P., Zhao Y., Li Z., Hsu C.C., Liu X., Fu L., Hou Y.J., Du Y., Xie S., Zhang C., Gao J., Cao M., Huang X., Zhu Y., Tang K., Wang X., Tao W.A., Xiong Y, & Zhu J.K. (2017). Reciprocal regulation of the TOR kinase and ABA receptor balances plant growth and stress response. Molecular cell, 69(1), 100-112.e6.

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Tryptic Peptide LC-MS/MS Analysis

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The tryptic peptides were dissolved in 5 μL of 0.25% formic acid (FA) with 3% ACN and injected into an Easy-nLC 1000 (Thermo Fisher Scientific, MA, USA). Peptides were separated on a 45 cm in-house packed column (360 μm OD × 75 μm ID) containing C18 resin (2.2 μm, 100Å) (Bischoff Chromatography, Leonberg, Germany) with a column heater (Analytical Sales and Services, Flanders, NJ, USA) set at 50°C. The mobile phase buffer consisted of 0.1% FA in ultra-pure water (buffer A) with an eluting buffer of 0.1% FA in 80% ACN (buffer B) run over a linear 60 min (method comparisons) gradient of 5%-30% buffer B at a flow rate of 250 nL/min. The Easy-nLC 1000 was coupled online with an LTQ-Orbitrap Velos Pro mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA). The mass spectrometer was operated in the data-dependent mode in which a full MS scan (from m/z 3501500 with the resolution of 30,000 at m/z 400) was followed by the 10 most intense ions being subjected to collision-induced dissociation (CID) fragmentation. CID fragmentation was performed and acquired in the linear ion trap (normalized collision energy (NCE) 30%, the automatic gain control (AGC) 3e4, max injection time 100 ms, isolation window 3 m/z, and dynamic exclusion 60 s).

Suntravat M., Cromer W.E., Marquez J., Galan J.A., Zawieja D.C., Davies P., Castillo E.S, & Sánchez E.E. (2019). The Isolation and Characterization of a new Snake Venom Cysteine-Rich Secretory Protein (svCRiSP) from the Venom of the Southern Pacific Rattlesnake and its Effect on Vascular Permeability. Toxicon : official journal of the International Society on Toxinology, 165, 22-30.

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LC-MS/MS Analysis of Peptides and Phosphopeptides

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Dried peptide and phosphopeptide samples were dissolved in 4.8 μL of 0.25% formic acid with 3% (vol/vol) acetonitrile and 4 μL of each were injected into an EasynLC 1000 (Thermo Fisher Scientific). Peptides were separated on a 45-cm in-house packed column (360 μm OD × 75 μm ID) containing C18 resin (2.2 μm, 100 Å; Michrom Bioresources). The mobile phase buffer consisted of 0.1% formic acid in ultrapure water (buffer A) with an eluting buffer of 0.1% formic acid in 80% (vol/vol) acetonitrile (buffer B) run with a linear 60- or 90-min gradient of 6–30% buffer B at a flow rate of 250 nL/min. The Easy-nLC 1000 was coupled online with a hybrid high-resolution LTQ-Orbitrap Velos Pro mass spectrometer (Thermo Fisher Scientific). The mass spectrometer was operated in the data-dependent mode, in which a full-scan MS (from m/z 300 to 1,500 with the resolution of 30,000 at m/z 400), followed by MS/MS of the 10 most intense ions [normalized collision energy—30%; automatic gain control (AGC)—3E4, maximum injection time—100 ms; 90 s exclusion].

Charles Jacob H.K., Signorelli R., Charles Richard J.L., Kashuv T., Lavania S., Middleton A., Gomez B.A., Ferrantella A., Amirian H., Tao J., Ergonul A.B., Boone M.M., Hadisurya M., Tao W.A., Iliuk A., Kashyap M.K., Garcia-Buitrago M., Dawra R, & Saluja A.K. (2022). Identification of novel early pancreatic cancer biomarkers KIF5B and SFRP2 from “first contact” interactions in the tumor microenvironment. Journal of Experimental & Clinical Cancer Research : CR, 41, 258.

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Dimethyl-labeled Peptide Fractionation and MS

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Twenty-five µg of dimethyl-labeled peptides used in fractionations were dissolved in 5 µL of 0.3% formic acid (FA) with 3% ACN and 4 µL of each fraction injected into an EasynLC 1000 (Thermo Fisher Scientific). Peptides were separated on a 45 cm in-house packed column (360 µm OD × 75 µm ID) containing C18 resin (2.2 µm, 100Å, Michrom Bioresources, Auburn, CA, USA) with a 30 cm column heater (Analytical Sales and Services, Flanders, NJ, USA) set at 60°C. The mobile phase buffer for 25 µg peptide fractions consisted of 0.1% FA in ultrapure water (buffer A) with an eluting buffer of 0.1% FA in 80% ACN (buffer B) run with a 150 min gradient of 5–30% buffer B at a flow rate of 250 nL/min. The Easy-nLC 1000 was coupled online with a Velos Pro LTQ-Orbitrap mass spectrometer (Thermo Fisher Scientific). The mass spectrometer was operated in the data-dependent mode in which a full MS scan (from m/z 350–1500 with a resolution of 30,000 at m/z 400) was followed by MS/MS of the 10 most intense ions being subjected to collision-induced dissociation (CID) fragmentation (normalized collision energy–30%, automatic gain control–3E4, max injection time–100 ms).

Li X., Zhu P., Chen Y.J., Huang L., Wang D., Newton D.T., Hsu C.C., Lin G., Tao W.A., Staiger C.J, & Zhang C. (2023). The EXO70 inhibitor Endosidin2 alters plasma membrane protein composition in Arabidopsis roots. Frontiers in Plant Science, 14, 1171957.

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Peptide Separation and Mass Spectrometry

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The enriched peptides were injected into an Easy-nLC1000 (Thermo Scientific, USA). The eluted peptides were first separated on an in-house packed C₁₈ column (100 μm ID × 20 mm, RP-C18, 5 μm), and then on another in-house packed C₁₈ column (75μm ID × 100 mm, RP-C18, 3 μm) with a flow rate of 250 nL/min. The mobile phase buffer with 0.1% FA in water (buffer A) and an eluting buffer with 0.1% FA in ACN (buffer B) were run over a liner 60 min gradient. The Easy-nLC1000 was coupled online to a Q-Exactive HF-X mass spectrometer (Thermo Scientific, USA). The MS data of each sample were acquired at 300–1800 m/z at the resolution of 70 k for over 120 min. The top 10 most abundant ions from each MS scan were subsequently dissociated by high-energy collisional dissociation (HCD) in alternating data-dependent mode. The HCD-generated MS/MS spectra were acquired with a resolution no less than 17,500 at m/z 200.

Li Q., Li M., Ma H., Xue M., Chen T., Ding X., Zhang S, & Xiao J. (2023). Quantitative Phosphoproteomic Analysis Provides Insights into the Sodium Bicarbonate Responsiveness of Glycine max. Biomolecules, 13(10), 1520.

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Phosphopeptide LC-MS/MS Analysis

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Dried peptide and phosphopeptide samples were dissolved in 4.8 μL of 0.25% formic acid with 3% (v/v) acetonitrile (Sigma Aldrich, St. Louis, MI, USA), and 4 μL of each were injected into an EasynLC 1000 (Thermo Fisher, Waltham, MA, USA). Peptides were separated on a 45-cm in-house packed column (360 μm OD × 75 μm ID) containing C18 resin (2.2 μm, 100 Å) (Michrom Bioresources, Auburn, CA, USA). The mobile phase buffer consisted of 0.1% formic acid in ultrapure water (buffer A) with an eluting buffer of 0.1% formic acid in 80% (v/v) acetonitrile (buffer B) run with a linear 60- or 90-min gradient of 6–30% buffer B at flow rate of 250 nL/min. The Easy-nLC 1000 was coupled online with a hybrid high-resolution LTQ-Orbitrap Velos Pro mass spectrometer (Thermo Fisher, Waltham, MA, USA). The mass spectrometer was operated in the data-dependent mode, in which a full-scan MS (from m/z 300 to 1500 with the resolution of 30,000 at m/z 400), followed by MS/MS of the 10 most intense ions (normalized collision energy −30%; automatic gain control (AGC)—3E4, maximum injection time—100 ms; 90 s exclusion).

Charles Jacob H.K., Charles Richard J.L., Signorelli R., Kashuv T., Lavania S., Vaish U., Boopathy R., Middleton A., Boone M.M., Sundaram R., Dudeja V, & Saluja A.K. (2021). Modulation of Early Neutrophil Granulation: The Circulating Tumor Cell-Extravesicular Connection in Pancreatic Ductal Adenocarcinoma. Cancers, 13(11), 2727.

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Proteomic Analysis of DC and EV Samples

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DC samples were analyzed on an Easy nLC 1000 nano HPLC system coupled to a Q Exactive HF mass spectrometer (Thermo Scientific, San José, USA). C18-based reverse phase separation was used with a 50-cm analytical column (EASY-Spray, Thermo Fisher Scientific). Peptides were loaded in buffer A (0.1% formic acid (v/v)) and eluted with a 300-min linear gradient of 8-31% buffer B (90% AcN, 0.1% formic acid (v/v)) at 200 nL/min flow. Spectra were acquired using full ion-scan mode (120,000 FT-resolution) over the 400-1,500 mass-to-charge (m/z) range. Data acquisition was performed using a top 15 method (15,000 resolution). EV samples were analyzed on an Easy nLC 1000 nano-HPLC apparatus coupled to an Orbitrap Elite mass spectrometer (Thermo Scientific, San José, USA). Peptides were loaded onto a home-made C-18 reversed-phase nano-column (100 μm I.D., 45 cm) and separated in a continuous gradient consisting of 8-31% B for 180 min at 300 nL/min. Peptides were ionized using a Picotip emitter nanospray needle (New Objective, Woburn, MA, USA). Each MS run consisted of enhanced FT-resolution spectra (120,000 resolution) in the 390–1,200 m/z range followed by data-dependent MS/MS spectra of the 10 most intense parent ions (30,000 resolution) acquired along the chromatographic run.

Izquierdo-Serrano R., Fernández-Delgado I., Moreno-Gonzalo O., Martín-Gayo E., Calzada-Fraile D., Ramírez-Huesca M., Jorge I., Camafeita E., Abián J., Vicente-Manzanares M., Veiga E., Vázquez J, & Sánchez-Madrid F. (2022). Extracellular vesicles from Listeria monocytogenes-infected dendritic cells alert the innate immune response. Frontiers in Immunology, 13, 946358.

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Phosphopeptide Analysis by LC-MS/MS

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Dried phosphopeptide samples were dissolved in 4.8 μl of 0.25% formic acid with 3% (vol/vol) acetonitrile, and 4 μl of each was injected into an Easy-nLC 1000 (Thermo Fisher Scientific). Peptides were separated on a 45-cm in-house packed column (360 μm outside diameter [o.d.] × 75 μm inside diameter [i.d.]) containing C₁₈ resin (2.2 μm, 100 Å; Michrom Bioresources). The mobile phase buffer consisted of 0.1% formic acid in ultrapure water (buffer A) with an eluting buffer of 0.1% formic acid in 80% (vol/vol) acetonitrile (buffer B) run with a linear 60-min gradient of 6 to 30% buffer B at a flow rate of 250 nl/min. The Easy-nLC 1000 was coupled online with a hybrid high-resolution LTQ-Orbitrap Velos Pro mass spectrometer (Thermo Fisher Scientific). The mass spectrometer was operated in the data-dependent mode, in which a full-scan MS (from m/z 300 to 1,500 with the resolution of 30,000 at m/z 400) was followed by tandem mass spectrometry (MS/MS) of the 10 most intense ions (normalized collision energy, 30%; automatic gain control [AGC], 3E4; maximum injection time, 100 ms; 90 s exclusion).

Zhou X., Eckart M.R, & Shapiro L. (2021). A Bacterial Toxin Perturbs Intracellular Amino Acid Balance To Induce Persistence. mBio, 12(1), e03020-20.

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Mass Spectrometry Proteomics Analysis

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Each lane of the SDS PAGE gel containing proteins from the membrane fraction and the extracellular fraction were cut into 10 equidistant pieces and subjected to tryptic digestion as described previously (Bonn et al., 2014 (link)). The resulting tryptic peptide mixtures were subjected to LC-MS/MS analysis on a TripleTOF 5600 instrument (AB Sciex, Concord, Canada) coupled with an Easy nLC-1000 (Thermo Scientific, Waltham, MA, United States). Sample desalting and elution by a binary gradient of water/acetonitrile was performed on an in-house packed column. Overview scans with a resolution of R = 30,000 in the range of 350–1,250 m/z with a scan time of 250 ms were followed by 20 MS/MS fragment scans in the range of 300–1,600 m/z with a maximum scan time of 50 ms after collisionally induced dissociation (CID).
For analysis of the cytosolic proteins, proteins were reduced/alkylated and subjected to in solution tryptic digest (Junker et al., 2018 (link)). The resulting peptide mixture was subjected to LC-MS/MS analysis on a Velos Elite (Thermo Scientific) coupled with an Easy nLC-1000 (Thermo Scientific) as described in Otto et al. (2016) (link).

Hofmann J.D., Otto A., Berges M., Biedendieck R., Michel A.M., Becher D., Jahn D, & Neumann-Schaal M. (2018). Metabolic Reprogramming of Clostridioides difficile During the Stationary Phase With the Induction of Toxin Production. Frontiers in Microbiology, 9, 1970.

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