Easy nlc 1000 hplc

Manufactured by Thermo Fisher Scientific

Sourced in United States

The EASY-nLC 1000 is a high-performance liquid chromatography (HPLC) system designed for the separation and analysis of complex samples. It features a nano-flow capability, making it suitable for applications requiring high sensitivity, such as proteomics and metabolomics research. The system is equipped with a solvent delivery system, a sample loading device, and a column compartment, allowing for efficient and reproducible chromatographic separations.

Automatically generated - may contain errors

Lab products found in correlation

21 protocols using easy nlc 1000 hplc

Orbitrap Elite Peptide Analysis

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

An aliquot of each sample was loaded onto an Acclaim PepMap trap column (2 cm × 75 µm) in line with an EASY-Spray analytical column (50 cm × 75 µm ID PepMap C18, 2-μm bead size) using the auto sampler of an EASY-nLC 1000 HPLC (Thermo Fisher Scientific) with solvent A consisting of 2% acetonitrile in 0.5% acetic acid and solvent B consisting of 80% acetonitrile in 0.5% acetic acid. The peptides were gradient eluted into an Orbitrap Elite mass spectrometer (Thermo Fisher Scientific) using the following gradient: 5%–35% in 60 min, 35%–45% in 15 min, followed by 45%–100% in 5 min. The gradient was held at 100% for another 10 min. MS1 spectra were recorded with a resolution of 60,000, an AGC target of 1e6, with a maximum ion time of 200 msec, and a scan range from 400 to 1500 m/z. Following each full MS scan, 15 data-dependent MS/MS spectra were acquired. The MS/MS spectra were collected in the Ion Trap with an AGC target of 3e4, maximum ion time of 150 msec, one microscan, 2 m/z isolation window, dynamic exclusion of 30 sec, and normalized collision energy (NCE) of 35.

Pinzaru A.M., Kareh M., Lamm N., Lazzerini-Denchi E., Cesare A.J, & Sfeir A. (2020). Replication stress conferred by POT1 dysfunction promotes telomere relocalization to the nuclear pore. Genes & Development, 34(23-24), 1619-1636.

+ Open protocol

+ Expand

Mesothelin Protein Identification by LC-MS/MS

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

The samples were injected on to Easy nLC 1000 HPLC (Thermo Scientific) coupled to an Orbitrap Fusion mass spectrometer (Thermo Scientific). The Easy nLC was configured with Acclaim Pepmap100 C18 2 cm trap column followed by 25 cm analytical column (Thermo Scientific) and the samples ran on a 90 min gradient at 250 nl/min. The precursor scans were performed in the Orbitrap at a resolution of 60,000 with a mass range of 375–1500m/z, followed by MS/MS analysis at a resolution of 7500 in the ion trap. Normalized collison energy was 29, and charge state 1 and unassigned charge states were excluded. Acquired MS/MS spectra were searched against human Uniprot protein database or Mesothelin (Uniprot number Q13421 iso #1) using the SEQUEST algorithm in the Proteome Discoverer 2.2 (Thermo Scientific, CA). Enzyme was set to Trypsin (Semi), precursor ion tolerance was set at 10 ppm, and the fragment ions tolerance was set at 0.6 Da along with methionine oxidation included as dynamic modification. Peptide validation was done by either the Percolator or Target Decoy PSM Validator with FDR set at 0.1%.

Liu X., Chan A., Tai C.H., Andresson T, & Pastan I. (2020). Multiple proteases are involved in mesothelin shedding by cancer cells. Communications Biology, 3, 728.

+ Open protocol

+ Expand

Quantitative Proteome Profiling by MS

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

Proteins were digested and purified on carboxylated beads according to the SP3 protocol (Hughes et al., 2019 (link)). Peptides were analyzed by liquid-chromatography using the EASY-nLC1000 HPLC coupled to high-resolution mass spectrometric analysis on the Q-Exactive HF mass spectrometer (Thermo Fisher Scientific, Bremen, Germany). Peptides were separated on 50 cm EASY-spray columns (Thermo Fisher Scientific) with a single run of 140 min. MS acquisition was performed in a data-dependent mode with selection of the top 10 peptides from each MS spectrum for fragmentation and MS/MS analysis. Raw MS files were analyzed in the MaxQuant software and the Andromeda search engine (Cox et al., 2011 (link)). A database search was performed using the Uniprot database and included carbamidomethyl-cysteine as a fixed modification, as well as N-terminal acetylation and methionine oxidation as variable modifications. A reverse decoy database was used to determine false discovery rate of 1% at the peptide and protein levels. The label-free algorithm (LFQ) in MaxQuant was used to retrieve the quantitative information. Nuclear and cytosolic proteins were quantified separately.

Nataraj N.B., Noronha A., Lee J.S., Ghosh S., Mohan Raju H.R., Sekar A., Zuckerman B., Lindzen M., Tarcitano E., Srivastava S., Selitrennik M., Livneh I., Drago-Garcia D., Rueda O., Caldas C., Lev S., Geiger T., Ciechanover A., Ulitsky I., Seger R., Ruppin E, & Yarden Y. (2022). Nucleoporin-93 reveals a common feature of aggressive breast cancers: robust nucleocytoplasmic transport of transcription factors. Cell reports, 38(8), 110418.

+ Open protocol

+ Expand

Proteomic Analysis of Rif1 Interactome

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

Rif1 baits and co-purifying proteins were resolved by 4%–12% Bis-Tris gel and visualized by Coomassie blue staining. The gel was divided into upper and lower parts along the 39kDa molecular weight marker, with only the lower part fixed. The protein-containing upper and lower parts were cut into 5 and 1 regions respectively, and the gel samples were subjected to in-gel tryptic digestion. Peptides were extracted and purified, analyzed by LCMS using a Thermo Q Exactive Plus mass spectrometer, with a Thermo Easy-nLC 1000 HPLC and a Thermo Easy-Spray electrospray source. Identification and quantification of proteins was performed by searching against a mouse protein sequence database with the MaxQuant software (version 1.2.2.5) (Cox and Mann, 2008 (link)). Protein H/(H+L) ratios were derived using peptides’ H/L intensity values in MaxQuant output.

Delgado-Benito V., Rosen D.B., Wang Q., Gazumyan A., Pai J.A., Oliveira T.Y., Sundaravinayagam D., Zhang W., Andreani M., Keller L., Kieffer-Kwon K.R., Pękowska A., Jung S., Driesner M., Subbotin R.I., Casellas R., Chait B.T., Nussenzweig M.C, & Di Virgilio M. (2018). The Chromatin Reader ZMYND8 Regulates Igh Enhancers to Promote Immunoglobulin Class Switch Recombination. Molecular Cell, 72(4), 636-649.e8.

+ Open protocol

+ Expand

Characterization of Qconcat Proteins

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

The mass of purified intact stable isotopically labeled Qconcat A protein was analyzed by MALDI (Extended Data Fig. 2E) on a JEOL JMS-S3000 SpiralTOF mass spectrometer using the ultra-thin-layer sample preparation method^{60 (link),61 (link)} in which α-cyano-4-hydroxycinnamic acid (Sigma) was used as the matrix. The mass of Qconcat A was internally calibrated with horse myoglobin ([m+H]+ = 16,952.5 Da). Mass calibration and background subtraction were carried out with the JEOL msTornado control software, while additional analyses were carried out with the MoverZ software^{62 (link)}. The Qconcat A protein was also characterized by peptide mapping, wherein tryptic peptides from in-gel digestion were loaded onto a PicoFrit® column (New Objective, Woburn, MA) with an integrated emitter tip (360 mm O.D., 50 mm I.D., 10 mm tip) self-packed with 6 cm of reverse-phase C18 material (ReproSil-Pur C18-AQ, 3 mm beads from Dr. Maisch GmbH), and analyzed with a LTQ Orbitrap Velos mass spectrometer (Thermo Fisher Scientific), with a Agilent 1200 series HPLC system (Agilent) and a homebuilt micro electrospray source. The purified Qconcat B was characterized by peptide mapping on a Thermo Orbitrap Fusion mass spectrometer, with a Thermo Easy-nLC 1000 HPLC and a Thermo Easy-Spray electrospray source.

Kim S.J., Fernandez-Martinez J., Nudelman I., Shi Y., Zhang W., Raveh B., Herricks T., Slaughter B.D., Hogan J., Upla P., Chemmama I.E., Pellarin R., Echeverria I., Shivaraju M., Chaudhury A.S., Wang J., Williams R., Unruh J.R., Greenberg C.H., Jacobs E.Y., Yu Z., de la Cruz M.J., Mironska R., Stokes D.L., Aitchison J.D., Jarrold M.F., Gerton J.L., Ludtke S.J., Akey C.W., Chait B.T., Sali A, & Rout M.P. (2018). Integrative Structure and Functional Anatomy of a Nuclear Pore Complex. Nature, 555(7697), 475-482.

+ Open protocol

+ Expand

Shotgun Proteomics using Q Exactive

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

An aliquot of the experimental sample and the control were loaded onto an Acclaim PepMap trap column (2 cm × 75 µm) in line with an EASY-Spray analytical column (50 cm × 75 µm ID PepMap C18, 2 μm bead size) using the auto sampler of an EASY-nLC 1000 HPLC (Thermo Fisher Scientific) with solvent A consisting of 2% acetonitrile in 0.5% acetic acid and solvent B consisting of 80% acetonitrile in 0.5% acetic acid. The peptides were gradient eluted into a Thermo Fisher Scientific Q Exactive mass spectrometer using the following gradient: 5–35% in 60 min, 35–45% in 10 min, followed by 45–100% in 10 min. High-resolution full MS spectra were recorded with a resolution of 70,000 at m/z 400, an AGC target of 1e6, with a maximum ion time of 120 ms, and a scan range from 400 to 1500 m/z. The top 20 MS/MS spectra were collected with an AGC target of 5e4, maximum ion time of 120 ms, one microscan, 2 m/z isolation window, and Normalized Collision Energy (NCE) of 27 and a dynamic exclusion of 30 s.

Zhang N., Mendieta-Esteban J., Magli A., Lilja K.C., Perlingeiro R.C., Marti-Renom M.A., Tsirigos A, & Dynlacht B.D. (2020). Muscle progenitor specification and myogenic differentiation are associated with changes in chromatin topology. Nature Communications, 11, 6222.

+ Open protocol

+ Expand

Optimized Phosphopeptide Analysis via Q Exactive HF-X

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

The phosphopeptides were analyzed using Q Exactive HF-X Hybrid Quadrupole-Orbitrap Mass Spectrometer (Thermo Fischer Scientific) coupled online to a nanoflow EASY-nLC1000 HPLC (Thermo Fisher Scientific). Briefly, the phosphopeptides were loaded onto an in house packed 50 cm C18 column with a 75 μM inner diameter (1.9 μM ReproSil particles, Dr. Maisch GmbH). The temperature of the column was maintained at 50°C by an in-house made column oven. The phosphopeptides were separated in a duration of 140-minute gradient with two mobile phase system buffer A (0.1% formic acid) and buffer B (60% ACN plus 0.1% formic acid) at a flow rate of 300 nl/min. The electro sprayed peptides were analyzed by the Q Exactive HF-X Hybrid Quadrupole-Orbitrap Mass Spectrometer (Thermo Fischer Scientific) in a data dependent mode, with one survey scan at a target of 3×10⁶ ions (300–1650 m/z, R=60,000 at 200 m/z), followed by Top10 MS/MS scans with HCD (high energy collisional dissociation) based fragmentation (target 1×10⁵ ions, maximum filling time 120ms, Isolation window 1.6 m/z, and normalized collision energy 27%), detected in the Orbitrap (R=15,000 at 200 m/z). Apex trigger 4 to 7s, charge exclusion (unassigned, 1, 5, −8 & >8), and dynamic exclusion 40s were enabled.

Batista T.M., Jayavelu A.K., Albrechtsen N.J., Iovino S., Lebastchi J., Pan H., Dreyfuss J., Krook A., Zierath J.R., Mann M, & Kahn C.R. (2020). A Cell-Autonomous Signature of Dysregulated Protein Phosphorylation Underlies Muscle Insulin Resistance in Type 2 Diabetes. Cell metabolism, 32(5), 844-859.e5.

+ Open protocol

+ Expand

Characterization of Qconcat Proteins

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

+ Open protocol

+ Expand

HDL Proteome Quantification by Mass Spec

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

For this study, we used 2 mass spectrometers. The Q Exactive Quadrupole Orbitrap (Thermo Fisher Scientific) was used for absolute quantification of APOA1 and APOA4 pool sizes as well as the relative quantification of the HDL proteome across the 6 HDL sizes. We utilized the Orbitrap Fusion Lumos (Thermo Fisher Scientific) for tracer enrichment quantification of APOA4 and APOA1 across 6 HDL sizes. Both instruments were coupled to an EASY-nLC 1000 HPLC pump (Thermo Fisher Scientific). The Q Exactive was fronted with a Nanospray FLEX ion source and the Lumos with an EASY-Spray ion source (Thermo Fisher Scientific).

Andraski A.B., Singh S.A., Higashi H., Lee L.H., Aikawa M, & Sacks F.M. (2023). The distinct metabolism between large and small HDL indicates unique origins of human apolipoprotein A4. JCI Insight, 8(8), e162481.

+ Open protocol

+ Expand

LC-MS/MS Peptide Analysis Protocol

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

Peptide samples (900 ng for each) were reconstituted in 0.1% FA/H₂O and analyzed by LC‐MS/MS with an Easy n‐LC 1000 HPLC and a Q Exactive mass spectrometer (Thermo Fisher Scientific, USA). The peptides were separated with a C18 column (75 µm × 20 cm) packed with Reprosil‐Pur C18 AQ particles (3.0 μm, Dr. Maisch HPLC GmbH) with solvent A (0.1% FA) and solvent B (ACN/0.1% FA) at a flow rate of 280 nl/min with a gradient: 4% B (0 min), 8% B (5 min), 22% (58 min), 32% B (70 min), 90% B (71 min), and 90% B (78 min).
A Q Exactive mass spectrometer (Thermo Fisher Scientific, USA) was used for MS analysis in data‐dependent acquisition mode. Each MS1 spectrum was obtained at 70,000 high‐resolution (m/z 200) at 300–1600 m/z. The automatic gain control (AGC) target value was 3E6 for a maximum filling time of 60 ms. The top 20 most abundant precursor ions were selected with a 2.0‐m/z isolation window and fragmented with a normalized collision energy of 27. MS/MS spectra were acquired at 17,500 resolution (m/z 200) with a 50,000 target value over a maximum injection time of 80 ms by setting up an isolation window of 2.0 m/z and dynamic exclusion time of 40 s.

Zhang T., Chen X., Sun L., Guo X., Cai T., Wang J., Zeng Y., Ma J., Ding X., Xie Z., Niu L., Zhang M., Tao N, & Yang F. (2021). Proteomics reveals the function reverse of MPSSS‐treated prostate cancer‐associated fibroblasts to suppress PC‐3 cell viability via the FoxO pathway. Cancer Medicine, 10(7), 2509-2522.

+ 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!