All fractions for DDA library generation were injected on a Thermo Scientific Q Exactive HF mass spectrometer connected to an Easy nLC 1200 chromatography system (Thermo Scientific). Peptides (2 μg) were first loaded onto an EASY-SprayTM C18 Trap column (Thermo Scientific, P/N 164946, 3 μm, 75 μm * 2 cm), and then separated on an EASYSprayTM C18 LC Analytical Column (Thermo Scientific, ES803, 2 μm, 75 μm * 50 cm) with a linear gradient of buffer B (80% acetonitrile and 0.1% formic acid) at a flow rate of 250 nL/min over 120 min. MS detection method was positive ion, the scan range was 300–1650 m/z, and resolution for MS1 scan was 60000 at 200 m/z, target of automatic gain control (AGC) was 3e6, maximum IT was 25 ms, and dynamic exclusion was 30.0 s. Each full MS–SIM scan followed 20 ddMS2 scans. Resolution for MS2 scan was 15000, AGC target was 5e4, maximum IT was 25 ms, and normalized collision energy was 27 eV.
Easy spray c18 lc analytical column
Manufactured by Thermo Fisher Scientific
Sourced in United States
The EASY-SprayTM C18 LC Analytical Column is a reversed-phase high-performance liquid chromatography (HPLC) column designed for the separation and analysis of a wide range of compounds. It features a C18 stationary phase and is intended for use in analytical applications.
Automatically generated - may contain errors
Lab products found in correlation
Easy spray c18 trap column, by Thermo Fisher Scientific (9 mentions)
Easy nlc 1200 chromatography system, by Thermo Fisher Scientific (5 mentions)
Q exactive hf x mass spectrometer, by Thermo Fisher Scientific (2 mentions)
Easy nlc 1200, by Thermo Fisher Scientific (2 mentions)
Q exactive hf x, by Thermo Fisher Scientific (2 mentions)
Q exactive hf, by Thermo Fisher Scientific (1 mentions)
Orbitrap q exactive hf x mass spectrometer, by Thermo Fisher Scientific (1 mentions)
Acetonitrile, by Merck Group (1 mentions)
9 protocols using easy spray c18 lc analytical column
1
DDA Proteomics Analysis on Q Exactive HF
2
Detailed DDA Proteomic Workflow
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All fractions for DDA library generation were analysed by a Thermo Scientific QExactive HF X mass spectrometer connected to an Easy nLC 1200 chromatography system (Thermo Scientific). The peptide (1.5 μg) was first loaded onto an EASY-SprayTMC18 Trapcolumn (Thermo Scientific, P/N 164946, 3 µm, 75 um*2 cm), then separated on an EASY-SprayTM C18 LC Analytical Column (Thermo Scientific, ES802, 2 µm, 75 um*25 cm) with a linear gradient of buffer B (84% acetonitrile and 0.1% Formic acid) at a flow rate of 250 nl/min over 120 min. MS detection method was positive ion, the scan range was 300–1800 m/z, the resolution for the MS1 scan was 60,000 at 200 m/z, the target automatic gain control (AGC) was 3e6, the maximum IT was 25 ms, and the dynamic exclusion parameter was 30.0 s. Each full MS–SIM scan followed 20 ddMS2 scans. The resolution for the MS2 scan was 15,000, the AGC target was 5e4, the maximum IT was 25 ms, and the normalized collision energy was 30 eV.
3
Mass Spectrometric Analysis of Peptides
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For the DDA library construction, all fractions were analyzed using an interfaced Thermo Scientific Q Exactive HF X mass spectrometer and Easy nLC 1200 chromatography system (Thermo Scientific, Madison, WI, USA). The peptide (1.5 μg) was loaded onto an EASY-SprayTM C18 Trap column (P/N 164946, 3 μm, 75 μm × 2 cm, Thermo Scientific, Madison, WI, USA), and paired with the EASY-SprayTM C18 LC Analytical Column (ES802, 2 μm, 75 μm × 25 cm, Thermo Scientific, Madison, WI, USA) for chromatographic separation. A linear gradient of buffer B (80% acetonitrile and 0.1% Formic acid) at a flow rate of 250 nL/min maintained for 90 min ensured effective separation. Positive ionization, with scanning ranging from 300 to 1800 m/z, enabled detection of molecular weight. MS1 resolution was 60,000 at 200 m/z, with a target of AGC (automatic gain control) set at 3e6; the maximum IT was 25 ms, and dynamic exclusion was 30.0 s. Each full MS–SIM scan was preceded by 20 dd MS2 scans, with MS2 resolution at 15,000; the AGC target was 5e4, maximum IT was 25 ms and normalized collision energy was 30 eV.
4
Peptide Analysis by Orbitrap Mass Spectrometry
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The peptides of each sample were analyzed by a Thermo-Fisher Orbitrap Q Exactive-HFx mass spectrometer (Waltham, MA) connected to an Easy-nLC1200 chromatography system. Dried peptide samples re-dissolved in Solvent A (0.1% formic acid in water) were first loaded onto an EASY-SprayTM C18 Trap column (Thermo Scientific, P/N 164946, 3 μm, 75 μm*2 cm) and then separated on an EASY-SprayTM C18 LC Analytical Column (Thermo Scientific, ES802, 1.9 μm, 75 μm*8 cm) with a linear gradient of buffer B (84% acetonitrile and 0.1% formic acid) at a flow rate of 600 nl/min over 120 min. The MS positive ion detection method was used, with the scan range at 300–1400 m/z, resolution for MS1 scan of 60,000 at 200 m/z, target of AGC (automatic gain control) at 3e6, maximum IT at 20 ms, and dynamic exclusion at 30.0 s. Each full MS–SIM scan was performed following 20 ddMS2 scans. The resolution for the MS2 scan was 15,000, the AGC target was 5 e4, the maximum IT was 25 ms, and the normalized collision energy was 30 eV.
5
DDA Library Generation via LC-MS/MS
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For DDA library generation, all fractions were analyzed on a Thermo Scientific Q Exactive HF X mass spectrometer connected to an Easy nLC 1200 chromatography system (Thermo Scientific). Briefly, 1.5 μg of peptide was loaded onto an EASY-SprayTM C18 Trap column (Thermo Scientific) and separated on an EASY-SprayTM C18 LC Analytical Column (Thermo Scientific) with a linear gradient of buffer B (84% acetonitrile and 0.1% Formic acid) at a flow rate of 250 nl/min over 120 min. The Mass Spectrometry (MS) detection method was positive ion, and the scan range was 300-1800 m/z with a resolution of 60000 at 200 m/z for MS1 scan. The Automatic Gain Control (AGC) target was 3e6, and the maximum IT was 25ms, with dynamic exclusion set at 30.0s. Each full Mass Spectrometry Selected Ion Monitoring (MS-SIM) scan was followed by 20 ddMS2 scans, and the resolution for MS2 scan was 15000, with an AGC target of 5e4, maximum Injection Time (IT) of 25 ms, and normalized collision energy of 30 eV.
6
DDA Mass Spectrometry Peptide Analysis
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All fractions for DDA library generation were injected on a Thermo Scientific Q Exactive HF X mass spectrometer connected to an Easy nLC 1200 chromatography system (Thermo Scientific). The peptide was first loaded onto an EASY-Spray TM C18 Trap column (Thermo Scientific, P/N 164946, 3 μm, 75 μm × 2 cm), then separated on an EASY-SprayTM C18 LC Analytical Column (Thermo Scientific, ES802, 2 μm, 75 μm × 25 cm) with a linear gradient of buffer B (84% acetonitrile and 0.1% Formic acid) at a flow rate of 250 nl/min over 90 min. MS detection method was positive ion, the scan range was 300–1800 m/z, resolution for MS1 scan was 60,000 at 200 m/z, target of AGC (Automatic gain control) was 3 e6, maximum IT was 25 ms, dynamic exclusion was 30.0 s. Each full MS–SIM scan followed 20 ddMS2 scans. Resolution for MS2 scan was 15,000, AGC target was 5 e4, maximum IT was 25 ms and normalized collision energy was 30 eV.
7
Comprehensive DDA Profiling of Protein Fractions
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In the process of generating
a DDA library, all fractions, including those containing low- and
high-abundance proteins, were subjected to DDA runs. These were performed
by using a Thermo Scientific Q Exactive HF/HF-X mass spectrometer
coupled with an Easy nLC 1200 chromatography system (Thermo Scientific).
Initially, a peptide sample (1.5 μg) was loaded onto an EASY-Spray
C18 Trap column (Thermo Scientific, P/N 164946, 3 μm, 75 μm,
2 cm). Subsequently, the sample was separated on an EASY-Spray C18
LC Analytical Column (Thermo Scientific, ES803, 2 μm, 75 μm,
50 cm) using a linear gradient of buffer B (80% acetonitrile and 0.1%
formic acid) at a flow rate of 250 nL/min over 90 min. The MS detection
method employed positive ion mode with a scan range of 300–1650 m/z. The resolution for the MS1 scan was
60,000 at 200 m/z, and the target
automatic gain control (AGC) was 1e6 with a maximum IT of 50 ms. Dynamic
exclusion was observed for 30.0 s. For each full MS–SIM scan,
20 ddMS2 scans were performed. The resolution for the MS2 scan was
15,000, with an AGC target of 1e5 and a maximum IT of 25 ms. The normalized
collision energy was 27 eV.
a DDA library, all fractions, including those containing low- and
high-abundance proteins, were subjected to DDA runs. These were performed
by using a Thermo Scientific Q Exactive HF/HF-X mass spectrometer
coupled with an Easy nLC 1200 chromatography system (Thermo Scientific).
Initially, a peptide sample (1.5 μg) was loaded onto an EASY-Spray
C18 Trap column (Thermo Scientific, P/N 164946, 3 μm, 75 μm,
2 cm). Subsequently, the sample was separated on an EASY-Spray C18
LC Analytical Column (Thermo Scientific, ES803, 2 μm, 75 μm,
50 cm) using a linear gradient of buffer B (80% acetonitrile and 0.1%
formic acid) at a flow rate of 250 nL/min over 90 min. The MS detection
method employed positive ion mode with a scan range of 300–1650 m/z. The resolution for the MS1 scan was
60,000 at 200 m/z, and the target
automatic gain control (AGC) was 1e6 with a maximum IT of 50 ms. Dynamic
exclusion was observed for 30.0 s. For each full MS–SIM scan,
20 ddMS2 scans were performed. The resolution for the MS2 scan was
15,000, with an AGC target of 1e5 and a maximum IT of 25 ms. The normalized
collision energy was 27 eV.
8
Peptide Fractionation for DDA Library
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Peptide fractions for DDA library generation were analysed using a Q Exactive HF X mass spectrometer (Thermo Scientific Corp) coupled to an Easy nLC 1200 chromatography system (Thermo Scientific Corp). Peptide samples (1.5 μg) were first loaded onto an EASY-Spray C18 Trap column (P/N 164946, 3 μm, 75 μm × 2 cm; Thermo Scientific Corp), then separated on an EASY-Spray C18 LC Analytical Column (ES802, 2 μm, 75 μm × 25 cm; Thermo Scientific Corp) over a 120 min gradient from buffer B (0.1% formic acid and 84% acetonitrile). The column flow rate was maintained at 250 nL/min. MS detection was performed in positive ion mode, full scans were performed between 300 and 1800 m/z, the resolution for the MS1 scan was 60,000 at 200 m/z, the automatic gain control (AGC) target for the MS scan was set to 3e6, and the maximum injection time (IT) was 25 ms. The dynamic exclusion was set to 30.0s. Each full mass spectrometry (MS)-selected ion monitoring (SIM) scan followed 20 ddMS2 scans. The MS2 scan was performed at 15,000 resolution, the AGC target was 5e4, the maximum IT was 25 ms, and the collision energy was 30 eV.
9
Mass Spectrometry-Based Proteomics Protocol
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All fractions for library generation were injected on a Thermo Scientific Q Exactive HF X mass spectrometer connected to an Easy nLC 1200 chromatography system (Thermo Scientific). Each fraction (1.5 μg) was first loaded onto an EASY-Spray C18 trap column (Thermo Scientific, P/N 164946, 3 μm, 75 μm × 2 cm), then peptides were separated using an EASY-Spray C18 LC analytical column (Thermo Scientific, ES802, 2 μm, 75 μm × 25 cm) with a linear gradient of 84% acetonitrile (Merck, Hunterdon County, New Jersey, USA) and 0.1% formic acid at a flow rate of 250 nL/min for 90 minutes. The MS detection method: positive ion; the scan range: 300 to 1800 m/z; the resolution for MS1 scans: 60000 at 200 m/z; the target of automatic gain control: 3e6; maximum injection time (IT) 25 ms; and dynamic exclusion: 30.0 second. Each full MS-single ion monitoring (SIM) scan followed 20 ddMS2 scans. Resolution for MS2 scans: 15000; automatic gain control (AGC) target: 5e4; maximum IT25 ms; and normalized collision energy: 30 eV.
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