Easy spray c18 column

Manufactured by Thermo Fisher Scientific

Sourced in United States

The EASY-Spray C18 column is a chromatographic column designed for liquid chromatography applications. It features a C18 stationary phase, which is a common reverse-phase material used for the separation and analysis of a wide range of compounds.

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Lab products found in correlation

43 protocols using easy spray c18 column

Liquid Chromatography-Orbitrap MS Proteomics Analysis

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The sample (5 μL)
was injected for analysis using a Thermo Fisher Easy nLC 1200 nanoflow
liquid chromatography system coupled to a Thermo Fisher Q-Exactive
high-resolution Orbitrap mass spectrometer. The sample was separated
on a Thermo Scientific Easy-Spray C18 Column (2 μm pore size,
150 mm long, 0.050 mm internal diameter) equipped with a trap column
(Acclaim PepMap 100 C18 LC column with a 3 μm particle size,
150 mm long, 0.075 mm internal diameter). Samples were separated using
water with 0.1% formic acid (solvent A) and 80% acetonitrile with
0.1% formic acid (solvent B), using a gradient of solvents flowing
at 300 nL/min during which the concentration of B increased from 2%
to 24% over 60 min, 24% to 36% over 10 min, and 36% to 98% over 1
min and then held at 98% for 15 min. Samples were ionized at 300 °C
and 200 V with an S-lens RF level of 80. A full scan (resolution of
70 000, automatic gain control target of 3 × 10⁶, maximum injection time of 40 ms, and scan range of 400 to 1600 m/z) coupled to a data-dependent MS-2 top
10 scan (resolution of 17 500, automatic gain control target
of 5 × 10⁴, maximum injection time of 150 ms, 0.8 m/z isolation window, normalized collision
energy of 27, and dynamic exclusion period of 10 s) was performed
to generate high-resolution parent and fragment masses of ionized
peptides.

Heck C.J., Seneviratne H.K, & Bumpus N.N. (2020). Twelfth-Position Deuteration of Nevirapine Reduces 12-Hydroxy-Nevirapine Formation and Nevirapine-Induced Hepatocyte Death. Journal of Medicinal Chemistry, 63(12), 6561-6574.

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Quantitative Proteomics Using iTRAQ

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The iTRAQ-labeled samples were analyzed using a Q Exactive^TM HF mass spectrometer (Thermo Fisher Scientific) coupled with an UltiMate™ 3000 RSLCnano HPLC System (Thermo Fisher Scientific). The iTRAQ-labeled peptides were pooled and desalted using Sep-Pak C18 cartridges (Waters, Milford, MA, USA). The desalted peptides were dried using SpeedVac and resuspended in 0.5% trifluoroacetic acid. The peptide mixtures were loaded onto an EASY-Spray™ C18 column (Thermo Fisher Scientific) and separated using a 0.1% formic acid solution with varying amounts of acetonitrile (5–80%). The top 15 most abundant precursor ions within the 375–1400 m/z scan range were dynamically selected for further fragmentation in high collision dissociation (HCD) mode, with the normalized collision energy set to 33 ± 1%. In the full MS scan, the resolution was set to 60,000 at m/z 200, AGC target to 3 × 10^-6, and maximum injection time to 50 ms. For the MS/MS scan, the resolution was set to 15,000, AGC target to 5 × 10^-4, and the maximum injection time was set to 100 ms. The release of the dynamic exclusion of selected precursor ions was set to 20 s.

Kuo P.J., Rau C.S., Wu S.C., Lin C.W., Huang L.H., Lu T.H., Wu Y.C., Wu C.J., Tsai C.W, & Hsieh C.H. (2021). Exosomes Secreted by Adipose-Derived Stem Cells Following FK506 Stimulation Reduce Autophagy of Macrophages in Spine after Nerve Crush Injury. International Journal of Molecular Sciences, 22(17), 9628.

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Proteomics Analysis of acSRSF1 HKO Mice

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Samples were processed for proteomics according to the FASP protocol and desalted on in-house prepared C18 tips^{82 (link)}. The peptides were resuspended to 0.5 µg/µL, and 1 µg was separated on an EASY Spray C18 column (50 cm × 75 cm, 2 µm particle size) (ThermoFisher Scientific, Toronto) using an EASY nLC-1200. The mobile phase was composed of 0.1% formic acid in water (A) and 80% acetonitrile with 0.1% formic acid (B). The gradient was as follows: 5−40% B (0–120 min), 40%−100% B (120–125 min), 100% B (125–135 min). The peptides were analyzed on a Thermo QExactive HF mass spectrometer in a Top 20 data-dependent acquisition mode. Proteins were identified using MaxQuant to search the mouse (UP000000589) proteomes from UniProtKB (February 2019). Peptide spectral matches and protein False Discovery Rates were set to 1% and required a minimum of 1 unique peptide for identification. To increase the number of identified matches, a match between runs was enabled with a match time window of 0.7 minutes. Protein abundances were calculated using the iBAQ algorithm in MaxQuant. Differential protein abundance was performed using student’s t-test with Bonferroni correction on iBAQ values between control and acSRSF1 HKO.

Arif W., Mathur B., Saikali M.F., Chembazhi U.V., Toohill K., Song Y.J., Hao Q., Karimi S., Blue S.M., Yee B.A., Van Nostrand E.L., Bangru S., Guzman G., Yeo G.W., Prasanth K.V., Anakk S., Cummins C.L, & Kalsotra A. (2023). Splicing factor SRSF1 deficiency in the liver triggers NASH-like pathology and cell death. Nature Communications, 14, 551.

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Tryptic Peptide Analysis by LC-MS/MS

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The TMT-labelled tryptic peptides were dissolved in 20 μL of 2% AcN/0.1% formic acid. Five μL samples were injected into a EASY-nLC™1000 liquid chromatography system (ThermoFisher Scientific) on-line coupled to a Q Exactive™ Plus Hybrid Quadrupole-Orbitrap™ mass spectrometer (ThermoFisher Scientific). The chromatographic separation of the peptides was achieved using a 50 cm long EASY-Spray™ C18 column (ThermoFisher Scientific), with an organic gradient: 4–26% B (solvent B = 98% AcN/0.1% formic acid) in 180 min, 26–95% B in 5 min, and 95% B for 8 min at a flow rate of 300 nL/min. The mass spectrometric (MS) acquisition method was comprised of one survey full spectrum ranging from m/z 350 to 1600, acquired with a resolution of R = 140,000 (at m/z 200), followed by data-dependent higher energy collision dissociation (HCD) fragmentations of maximum 16 most intense precursor ions with a charge state 2+ and 3+, applying 60 s dynamic exclusion. The tandem mass scans were acquired with a resolution of R = 70,000, targeting 2x10⁵ ions, setting isolation width to 2.0 Th and normalized collision energy to 33%.

Akusjärvi S.S., Ambikan A.T., Krishnan S., Gupta S., Sperk M., Végvári Á., Mikaeloff F., Healy K., Vesterbacka J., Nowak P., Sönnerborg A, & Neogi U. (2021). Integrative proteo-transcriptomic and immunophenotyping signatures of HIV-1 elite control phenotype: A cross-talk between glycolysis and HIF signaling. iScience, 25(1), 103607.

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Quantitative Proteomic Analysis by PRM

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Samples were analyzed on a Q-Exactive HF mass spectrometer equipped with an EASY-Spray ion source (Thermo Fisher Scientific). Peptides were resolved for nLC-MS analysis with a Dionex UltiMate 3000 nRSLC (Thermo Fisher Scientific) equipped with an EASY-Spray C18 column (2 μm particle size, 75 μm diameter, 250 mm length; Thermo Fisher Scientific, ES902). Peptides were separated with 60-min gradient using solvent A and solvent B (2 to 22% solvent B over 45 min, 22 to 38% solvent B over 15 min, both at a flow rate of 250 nl/min). One full duty cycle of the instrument consisted of a single MS-SIM MS1 scan followed by 30 PRM scans. For the full scan MS1, the instrument was set to 400 to 2000 m/z full scan range with a 15,000 resolution, 15 ms MIT, and 3 × 10⁶ AGC target. For the PRM scans, the instrument was set to 30,000 resolution, 60 ms MIT, 1 × 10⁵ AGC target, 0.8 m/z isolation window, NCE of 27, and 125 m/z fixed first mass. Spectrum data for both the MS1 and PRM scans were recorded in profile.

Justice J.L., Kennedy M.A., Hutton J.E., Liu D., Song B., Phelan B, & Cristea I.M. (2021). Systematic profiling of protein complex dynamics reveals DNA-PK phosphorylation of IFI16 en route to herpesvirus immunity. Science Advances, 7(25), eabg6680.

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Filter-Aided Mass Spectrometry Proteomics

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A previously published filter-aided sample preparation method was used for the digestion of the concentrated AWF proteins (Wisniewski et al., 2009 (link)). Briefly, proteins (200 μg) were washed three times with UA buffer (8 M urea and 150 mM Tris-HCl, pH 8.5) and then alkylated in 50 mM iodoacetic acid for 30 min at 25 °C in darkness. After washing three times with UA buffer, the proteins were digested with 2 μg trypsin (Promega, Madison, WI, USA) in 25 mM NH₄HCO₃ on a 30-kDa filter unit (Millipore, Burlington, MA, USA) for 18 h at 37 °C. The peptide concentration was determined based on the OD₂₈₀ value.
After the desalting step using a C18 cartridge, the peptides were analyzed using the Easy-nLC 1000 system coupled with the Orbitrap Fusion Lumos mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA). Total peptide solutions (1 μg from each sample) were separated using a peptide trap (Thermo Fisher Scientific, Waltham, MA, USA, EASY-Spray C18 column; 2 cm × 100 μm × 5 μm) and a 60 min linear solvent gradient (5–28% phase B (0.1% formic acid in 100% ACN), 5–40 min; 28–90% phase B, 40–42 min; 90% phase B, 42–60 min, buffer A (0.1% FA in H₂O)). The mass spectrometry (MS) operating parameters were as follows: data-dependent mode; MS1 resolution 60,000 at m/z 200; MS2 resolution 15,000 at m/z 120; m/z range 350–1,600 for the full scan.

Jiang S., Pan L., Zhou Q., Xu W., He F., Zhang L, & Gao H. (2023). Analysis of the apoplast fluid proteome during the induction of systemic acquired resistance in Arabidopsis thaliana. PeerJ, 11, e16324.

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Proteomics Analysis of Spike Pulldown

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Proteins from the spike pulldown experiments were subject to in-solution tryptic digestion and C18 clean-up as described above. Tryptic peptides were analyzed by LC–MS/MS. An UltiMate 3000 HPLC system (Thermo Scientific) with a nanoflow selector was coupled via an EASY-Spray Source (Thermo Scientific) to a Q Exactive HF mass spectrometer (Thermo Scientific). Peptides were injected onto a C18 trap cartridge (Thermo Scientific, 160454) at a flow rate of 25 µL/min for 1 min, using 0.1% FA (aq). Peptides were eluted from the trap cartridge and separated on an analytical column (EASY-Spray C18 column, 75 µm × 50 cm, Thermo Scientific, ES803A, at 45 °C) at a flow rate of 0.25 µL/min using the following gradient: 0–1 min, 1% B; 1–6 min, 1–6% B; 6–40 min, 6–18% B; 40–70 min, 18–35% B; 70–80 min, 35–45% B; 80–81 min, 45–99% B; 81–89.8 min, 99% B; 89.8–90 min, 99–1% B; 90–120 min, 1% B. Mobile phase A was 0.1% FA (aq) and mobile phase B was 80% ACN, 0.1% FA (aq). Precursor MS1 spectra were acquired using Orbitrap detection (resolution 60,000 at 200 m/z, scan range 350–1600). Data-dependent MS2 spectra of the most abundant precursor ions were obtained after higher-energy C-trap dissociation and Orbitrap detection (resolution 15,000 at 200 m/z) with TopN mode (loop count 15) and dynamic exclusion (duration 40 s) enabled.

Gutmann C., Takov K., Burnap S.A., Singh B., Ali H., Theofilatos K., Reed E., Hasman M., Nabeebaccus A., Fish M., McPhail M.J., O’Gallagher K., Schmidt L.E., Cassel C., Rienks M., Yin X., Auzinger G., Napoli S., Mujib S.F., Trovato F., Sanderson B., Merrick B., Niazi U., Saqi M., Dimitrakopoulou K., Fernández-Leiro R., Braun S., Kronstein-Wiedemann R., Doores K.J., Edgeworth J.D., Shah A.M., Bornstein S.R., Tonn T., Hayday A.C., Giacca M., Shankar-Hari M, & Mayr M. (2021). SARS-CoV-2 RNAemia and proteomic trajectories inform prognostication in COVID-19 patients admitted to intensive care. Nature Communications, 12, 3406.

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Peptide Identification by Nano-UPLC-MS/MS

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Peptides were reconstituted in solvent A (2% ACN/0.1% FA) and approximately, two µg samples (2/12 µL) were injected on a 50 cm long EASY-Spray C18 column (Thermo Fisher Scientific) connected to an Ultimate 3000 nanoUPLC system (Thermo Fisher Scientific) using a 90 min long gradient: 4-26% of solvent B (98% ACN/0.1% FA) in 90 min, 26-95% in 5 min, and 95% of solvent B for 5 min at a flow rate of 300 nL/min. Mass spectra were acquired on a Q Exactive HF hybrid quadrupole orbitrap mass spectrometer (Thermo Fisher Scientific) ranging from m/z 375 to 1700 at a resolution of R=120,000 (at m/z 200) targeting 5x10⁶ ions for a maximum injection time of 80 ms, followed by data-dependent higher-energy collisional dissociation (HCD) fragmentations of precursor ions with a charge state 2+ to 8+, using 45 s dynamic exclusion. The tandem mass spectra of the top 18 precursor ions were acquired with a resolution of R=60,000, targeting 2x10⁵ ions for a maximum injection time of 54 ms, setting quadrupole isolation width to 1.4 Th and normalized collision energy to 33%.

Shafi A.M., Végvári Á., Zubarev R.A, & Penha-Gonçalves C. (2023). Brain endothelial cells exposure to malaria parasites links type I interferon signalling to antigen presentation, immunoproteasome activation, endothelium disruption, and cellular metabolism. Frontiers in Immunology, 14, 1149107.

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Proteomic Profiling via iTRAQ-LC-MS/MS

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Using a Q ExactiveTM HF mass spectrometer (Thermo Fisher Scientific) coupled with an UltiMate™ 3000 RSLCnano HPLC System (Thermo Fisher Scientific), and Sep‐Pak C18 cartridges (Waters) to analysed, pooled and desalted the iTRAQ‐labelled samples. The mixtures of desalted peptides were loaded onto an EASY‐Spray™ C18 column (Thermo Fisher Scientific) after drying using SpeedVac and resuspension in 0.5% trifluoroacetic acid. Then the samples were separated using a 0.1% formic acid solution with varying amounts of acetonitrile (5%–80%). For further fragmentation in high collision dissociation (HCD) mode, the top 15 abundant precursor ions with the normalized collision energy set to 33% ± 1% were dynamically selected (within the 375–1400 m/z scan range). The resolution in the full MS scan was set to 60,000 at 200 m/z. The AGC target is 3e6. The maximum injection time is about 50 ms. For the MS/MS scan, the resolution was set to 15,000 with the 5e4 AGC target and the 100 ms maximum injection time. The release of the dynamic exclusion of selected precursor ions is around 20 s.²⁰

Lee K., Chen H., Cheng K., Liu T., Lee K., Teng C., Huang C., Hsieh M, & Kuo H. (2023). Use of iTRAQ‐based quantitative proteomic identification of CHGA and UCHL1 correlated with lymph node metastasis in colorectal carcinoma. Journal of Cellular and Molecular Medicine, 27(14), 2004-2020.

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Quantitative Proteomics Using iTRAQ

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The iTRAQ-labeled samples were analyzed using the Q Exactive^TM HF mass spectrometer (Thermo Fisher Scientific), coupled with the UltiMate™ 3000 RSLCnano HPLC System (Thermo Fisher Scientific). The iTRAQ-labeled peptides were pooled and desalted in Sep-Pak C18 cartridges (Waters, Milford, MA, USA). Then, desalted peptides were dried using SpeedVac and resuspended in 0.5% trifluoroacetic acid. The peptide mixtures were loaded onto an EASY-Spray™ C18 column (Thermo Fisher Scientific)) and separated using 0.1% formic acid solution, with varying amounts of acetonitrile (5~80%). The top 15 abundant precursor ions, within the 375–1400 m/z scan range, were dynamically selected for further fragmentation in high collision dissociation (HCD) mode, with normalized collision energy set to 33 ± 1. In the full MS scan, the resolution was set to 60,000 at m/z 200, AGC target to 3e6, and maximum injection time to 50 ms. For the MS/MS scan, the resolution was set to 15,000, AGC target to 5e4, and the maximum injection time to 100 ms. The release of the dynamic exclusion of selected precursor ions was set to 20 s.

Rau C.S., Kuo P.J., Wu S.C., Huang L.H., Lu T.H., Wu Y.C., Wu C.J., Lin C.W., Tsai C.W, & Hsieh C.H. (2021). Enhanced Nerve Regeneration by Exosomes Secreted by Adipose-Derived Stem Cells with or without FK506 Stimulation. International Journal of Molecular Sciences, 22(16), 8545.

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