Easy nlc 1000 uhplc

Manufactured by Thermo Fisher Scientific

Sourced in United States

The EASY-nLC 1000 UHPLC is a high-performance liquid chromatography system designed for ultra-high pressure liquid chromatography (UHPLC) applications. It is capable of generating pressures up to 1000 bar and flow rates up to 2 mL/min. The system is optimized for nano-flow and capillary-flow applications, making it suitable for use in proteomics and other analytical workflows that require high sensitivity and separation efficiency.

Automatically generated - may contain errors

Lab products found in correlation

28 protocols using easy nlc 1000 uhplc

Peptide Separation and Mass Spectrometry Analysis

Check if the same lab product or an alternative is used in the 5 most similar protocols

Peptides were dissolved in 25 μl 0.1% FA, and 10 μl of the sample was injected for analysis. Peptides were separated on reverse phase Thermo Scientific Acclaim PepMap 100: 75 μm × 2 cm (C18, 3 μm bead, 100 Å pore size) trap column and Thermo Scientific Acclaim PepMap C18, 2 μm particle size, RSLC 75 μm × 25 cm column. Data were acquired with Orbitrap QExactive coupled to Thermo Scientific Easy nLC-1000 UHPLC. Samples were run using a 90-min gradient from 5% “B” to 35% “B.” Buffer “A” was 0.1% FA in water, and buffer “B” was 0.1% FA in acetonitrile. All data were acquired in positive mode with a lock mass of 445.12002 using data-dependent acquisition with a 15-s dynamic exclusion. MS1 data were acquired in profile mode at a resolution of 70,000 with a maximum time of 75 msec and a range of 350 to 1500 m/z. MS2 spectra were acquired in profile mode at a resolution of 15,000 and maximum integration time of 75 msec. The isolation window was 1.6 m/z, and the collision energy was 20.

Baek J., Sas K., He C., Nair V., Giblin W., Inoki A., Zhang H., Yingbao Y., Hodgin J., Nelson R.G., Brosius FC I.I.I., Kretzler M., Stemmer P.M., Lombard D.B, & Pennathur S. (2023). The deacylase sirtuin 5 reduces malonylation in nonmitochondrial metabolic pathways in diabetic kidney disease. The Journal of Biological Chemistry, 299(3), 102960.

+ Open protocol

+ Expand

Cross-linking and Mass Spectrometry Analysis of Dna2-Cdc24-Pxd1 Complex

Check if the same lab product or an alternative is used in the 5 most similar protocols

Dna2-Cdc24-Pxd1(227–351) complex was prepared by incubating anti-FLAG beads bound by Cdc24-YFH and Dna2 from fission yeast with Pxd1(227–351) from E. coli, washing the beads, and eluting with 3× FLAG peptide. About 12 µg of purified complex in a volume of 20 µl was cross-linked by BS3 or DSS at a final concentration of 0.5 mM for 1 h at room temperature. The reactions were quenched with 20 mM NH₄HCO₃. Proteins were precipitated with ice-cold acetone, resuspended in 8 M urea, 100 mM Tris, pH 8.5. After trypsin digestion, the LC-MS/MS analysis was performed on an Easy-nLC 1000 UHPLC (Thermo Fisher Scientific) coupled to a Q Exactive-Orbitrap mass spectrometer (Thermo Fisher Scientific). Peptides were loaded on a pre-column (75 µm ID, 8 cm long, packed with ODS-AQ 12 nm–10 µm beads from YMC Co., Ltd.) and separated on an analytical column (75 µm ID, 11 cm long, packed with Luna C18 3 µm 100 Å resin from Phenomenex) using an acetonitrile gradient from 0–25% in 55 min at a flow rate of 200 nl/min. The top 10 most intense precursor ions from each full scan (resolution 70,000) were isolated for HCD MS2 (resolution 17,500; NCE 27) with a dynamic exclusion time of 60 s. Precursors with 1+, 2+, or unassigned charge states were excluded. pLink was used to identified cross-linked peptides with the cutoffs of FDR<5% and E_value<0.001 [42] (link).

Zhang J.M., Liu X.M., Ding Y.H., Xiong L.Y., Ren J.Y., Zhou Z.X., Wang H.T., Zhang M.J., Yu Y., Dong M.Q, & Du L.L. (2014). Fission Yeast Pxd1 Promotes Proper DNA Repair by Activating Rad16XPF and Inhibiting Dna2. PLoS Biology, 12(9), e1001946.

+ Open protocol

+ Expand

Proteomics Analysis: Peptide, Glycopeptide, and Phosphopeptide Identification

Check if the same lab product or an alternative is used in the 5 most similar protocols

EASY‐nLC 1000 UHPLC (Thermo Scientific) interfaced via a Nanospray Flex ion source to an LTQ‐Orbitrap Velos Pro or Orbitrap Fusion spectrometer (Thermo Scientific) was used for peptide, glycopeptide, and phosphopeptide analysis as described previously 27. Data were processed using Proteome Discoverer 1.4 software (Thermo Scientific).

Bagdonaite I., Pallesen E.M., Ye Z., Vakhrushev S.Y., Marinova I.N., Nielsen M.I., Kramer S.H., Pedersen S.F., Joshi H.J., Bennett E.P., Dabelsteen S, & Wandall H.H. (2020). O‐glycan initiation directs distinct biological pathways and controls epithelial differentiation. EMBO Reports, 21(6), e48885.

+ Open protocol

+ Expand

Shotgun Proteomics by Q-Exactive Plus

Check if the same lab product or an alternative is used in the 5 most similar protocols

Samples were run on a Q-Exactive Plus Orbitrap (Thermo Scientific, Waltham, MA, USA) equipped with EASY-nLC 1000 UHPLC. Separation of peptides was performed on an Acclaim PepMap RSLC 150 mm C18 column, 50 µm of internal diameter (2 µm bead diameter, 100 A) with a flow rate of 200 nL/min and in a gradient of 0.1% formic acid in water (buffer A) and 0.1% formic acid in acetonitril (buffer B). Content of buffer B was increased from 2% to 30% during 240 min and from 40% to 100% in 5 min. The sample load was 5 µL. The mass spectrometer was operated in the data dependent mode with automatic switching between full scan MS and MS/MS acquisition. Survey full scan MS spectra (m/z 300−1800) were acquired in the Orbitrap with a resolution of 70,000 (m/z 200) after accumulation of ions to a target value of 3 × 10⁶, based on predictive AGC from the previous full scan. Dynamic exclusion was set to 20 s. The 12 most intense multiply charged ions (z ≥ 2) were sequentially isolated and fragmented in the octopole collision cell, by higher-energy collisional dissociation (HCD) with a maximum injection time of 120 ms. A range from 50 to M + 50 Da was covered for MS2 (resolution of 17,500). A 2.5 Da isolation width was chosen.

Wiatr M., Staubach S., Figueiredo R., Stump-Guthier C., Ishikawa H., Schwerk C., Schroten H., Hanisch F.G., Rudolph H, & Tenenbaum T. (2020). Echovirus-30 Infection Alters Host Proteins in Lipid Rafts at the Cerebrospinal Fluid Barrier In Vitro. Microorganisms, 8(12), 1958.

+ Open protocol

+ Expand

Proteomic analysis of Arabidopsis

Check if the same lab product or an alternative is used in the 5 most similar protocols

Dried peptides were solubilized in a loading buffer (3% ACN, 0.1% FA) and measured on a Q Exactive HF coupled to an Easy nLC1000 UHPLC reverse-phase nano-liquid chromatography (Thermo Fisher Scientific, Hennigsdorf, Germany). The gradient ramped from 4 to 24% ACN over 20 min and then to 45% ACN over the next 15 min, followed by a 5-min washout with 80% ACN. The MS was run using a data-dependent MS/MS method with the following settings: full scans were acquired at a resolution of 120,000, AGC target of 3e6, maximum injection time of 50 ms, and an m/z ranging from 200 to 2,000. dd-MS2 scan was recorded at the 15,000 resolution with an AGC target of 1e5, maximum injection time of 150 ms, isolation window of 1.2 m/z, normalized collision energy 30, and dynamic exclusion of 20 s. Raw chromatograms were processed using the MaxQuant (Version 1.6.0.16, MPI of Biochemistry, Germany) software using the Arabidopsis TAIR database (Version 10, The Arabidopsis Information Resource, www.Arabidopsis.org).

Chodasiewicz M., Sokolowska E.M., Nelson-Dittrich A.C., Masiuk A., Beltran J.C., Nelson A.D, & Skirycz A. (2020). Identification and Characterization of the Heat-Induced Plastidial Stress Granules Reveal New Insight Into Arabidopsis Stress Response. Frontiers in Plant Science, 11, 595792.

+ Open protocol

+ Expand

Phosphoproteomic Analysis of Kinase Substrates

Check if the same lab product or an alternative is used in the 5 most similar protocols

The 2D-DIGE gel was silver-stained (Thermo Scientific) and protein spots were excised and digested with trypsin (Promega). The obtained peptides were analysed by a capillary liquid chromatography system (Waters/Micromass) connected to a Q-TOF Ultima mass spectrometer (Waters/Micromass). Raw data were acquired and processed using MassLynx version 4.0. (Waters/Micromass) to generate a peak list file for MS/MS ion search. The peak list files were searched against the NCBI non-redundant protein database restricted Mus musculus using the MS/MS ion search on Mascot search engine (Matrix Science). To identify in vitro phosphorylation sites, GST fusion proteins phosphorylated by active PKD2 or PKD3 were subjected to SDS–PAGE. The gel was stained with Coomassie Brilliant Blue and protein bands were excised and digested with trypsin. The resultant peptides were analysed by an ADVANCE UHPLC (Michrom Bioresources) connected to an Orbitrap Velos Pro mass spectrometer (for GST-Gads and GST-NCK1) or by an EASY-nLC1000 UHPLC (Thermo Fisher Scientific) connected to an Orbitrap Fusion Tribrid mass spectrometer (for GST-SHP-1).

Ishikawa E., Kosako H., Yasuda T., Ohmuraya M., Araki K., Kurosaki T., Saito T, & Yamasaki S. (2016). Protein kinase D regulates positive selection of CD4+ thymocytes through phosphorylation of SHP-1. Nature Communications, 7, 12756.

+ Open protocol

+ Expand

Glycoproteomic Analysis using EASY-nLC 1000 UHPLC and Orbitrap Fusion

Check if the same lab product or an alternative is used in the 5 most similar protocols

EASY-nLC 1000 UHPLC (Thermo Scientific) interfaced via nanoSpray Flex ion source to an -Orbitrap Fusion mass spectrometer (Thermo Scientific) was used for the glycoproteomic study. A precursor MS1 scan (m/z 350–1,700) of intact peptides was acquired in the Orbitrap at a nominal resolution setting of 120,000. The five most abundant multiply charged precursor ions in the MS1 spectrum at a minimum MS1 signal threshold of 50,000 were triggered for sequential Orbitrap HCD-MS2 and ETD-MS2 (m/z of 100–2,000). MS2 spectra were acquired at a resolution of 50,000. Activation times were 30 and 200 ms for HCD and ETD fragmentation, respectively; isolation width was four mass units, and one microscan was collected for each spectrum. Automatic gain control targets were 1,000,000 ions for Orbitrap MS1 and 100,000 for MS2 scans. Supplemental activation (20%) of the charge-reduced species was used in the ETD analysis to improve fragmentation. Dynamic exclusion for 60 s was used to prevent repeated analysis of the same components. Polysiloxane ions at m/z 445.12003 were used as a lock mass in all runs. The mass spectrometry glycoproteomics data have been deposited to the ProteomeXchange Consortium (Vizcaíno et al., 2014 (link)) via the PRIDE partner repository with the dataset identifier PXD011045.

Valoskova K., Biebl J., Roblek M., Emtenani S., Gyoergy A., Misova M., Ratheesh A., Reis-Rodrigues P., Shkarina K., Larsen I.S., Vakhrushev S.Y., Clausen H, & Siekhaus D.E. (2019). A conserved major facilitator superfamily member orchestrates a subset of O-glycosylation to aid macrophage tissue invasion. eLife, 8, e41801.

+ Open protocol

+ Expand

Proteomic analysis of cell samples

Check if the same lab product or an alternative is used in the 5 most similar protocols

Cell pellets were solubilized in a buffer containing 6 M urea and 2 M thiourea in 50 mM Tris-HCl pH 7.5. Following protein reduction (1 mM DTT) and alkylation (5 mM iodoacetamide), proteins were digested with trypsin overnight at room temperature. Resulting peptides were purified on C18 tips. Liquid-chromatography mass-spectrometric analysis was performed on the EASY-nLC1000 UHPLC coupled to the Q-Exactive mass spectrometer (Thermo Scientific). Cells from each donor and treatment were analyzed in two technical replicates. MS files were analyzed in MaxQuant with an FDR threshold of 1% on the peptide and protein levels.

Nathan G., Kredo-Russo S., Geiger T., Lenz A., Kaspi H., Hornstein E, & Efrat S. (2015). MiR-375 Promotes Redifferentiation of Adult Human β Cells Expanded In Vitro. PLoS ONE, 10(4), e0122108.

+ Open protocol

+ Expand

Glycopeptide Enrichment and Analysis

Check if the same lab product or an alternative is used in the 5 most similar protocols

LWAC fractions from total cell lysate digests were screened by preliminary LC-MS for glycopeptide content, and those most enriched in glycopeptides were pooled together and further fractionated by isoelectric focusing as previously described [92 (link)]. Mass spectrometry analysis was performed on an EASY-nLC 1000 UHPLC (Thermo Scientific) interfaced via nanoSpray Flex ion source to an LTQ-Orbitrap Velos Pro spectrometer (Thermo Scientific) as previously described [56 ] with minor changes and as described in detail in S1 Text.

Bagdonaite I., Nordén R., Joshi H.J., Dabelsteen S., Nyström K., Vakhrushev S.Y., Olofsson S, & Wandall H.H. (2015). A Strategy for O-Glycoproteomics of Enveloped Viruses—the O-Glycoproteome of Herpes Simplex Virus Type 1. PLoS Pathogens, 11(4), e1004784.

+ Open protocol

+ Expand

Liquid Chromatography-Mass Spectrometry Workflow

Cited 1 time

Check if the same lab product or an alternative is used in the 5 most similar protocols

LC-MS/MS runs were performed on the EASY-nLC1000 UHPLC (Thermo Scientific) coupled to the Q-Exactive Plus or Q-Exactive HF mass spectrometers (Thermo Scientific) (Scheltema et al., 2014 (link)). Peptides were separated with a 50 cm EASY-spray PepMap column (Thermo Scientific) using a water-acetonitrile gradient, with a flow rate of 300 nl/min at 40°c. Peptides were loaded to the column with buffer A (0.1% formic acid) and separated using a 105 min linear gradient of 7%–28% buffer B (80% acetonitrile, 0.1% formic). The resolutions of the MS and MS/MS spectra were 70,000 and 17,500 for Q-Exactive Plus, respectively. The resolutions of the MS and MS/MS spectra were 60,000 and 30,000 for the Q-Exactive HF, respectively. The m/z range was set to 300–1700 or 380– 1800 Th. MS data were acquired in a data-dependent mode, with target values of 3E+06 and 1E+05 or 5E+04 for MS and MS/MS scans, respectively, and a top-10 method.

Marmor-Kollet H., Siany A., Kedersha N., Knafo N., Rivkin N., Danino Y.M., Moens T.G., Olender T., Sheban D., Cohen N., Dadosh T., Addadi Y., Ravid R., Eitan C., Toth Cohen B., Hofmann S., Riggs C.L., Advani V.M., Higginbottom A., Cooper-Knock J., Hanna J.H., Merbl Y., Van Den Bosch L., Anderson P., Ivanov P., Geiger T, & Hornstein E. (2020). Spatiotemporal Proteomic Analysis of Stress Granule Disassembly Using APEX Reveals Regulation by SUMOylation and Links to ALS Pathogenesis. Molecular cell, 80(5), 876-891.e6.

+ Open protocol

+ Expand

About PubCompare

Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.

We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.

However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.

Ready to get started?

Revolutionizing how scientists
search and build protocols!