A Thermo Scientific Q Exactive with an Easy-nLC 1000 nano-LC was utilized (Thermo Fisher, San Jose, CA). 18 μL of sample was injected onto a Thermo Scientific EASY-spray PepMap C18, 3 μm, 75 μm × 150 mm column (Thermo Fisher, San Jose, CA) with the column temperature set to 35 °C. Mobile phases were A – H2O with 0.1% formic acid, and B – ACN with 0.1% formic acid. At a flow rate of 20 μL/min, the sample gradient was as follows: 40 minute linear gradient from 2% to 60% B; 20 minute linear gradient to 90% B and hold for 10 minutes; 5 minute linear gradient back to initial conditions and hold for 5 minutes. MS settings were: positive mode; m/z range 150-2000; MS resolution 70,000 and automatic gain control (AGC) 1E6; data dependent MS/MS top 5 with S/N 5, resolution of 140,000, AGC 1E5, normalized collision energy (NCE) 30, and isolation window 4.0 m/z; spray voltage 1.80 kV; and S lens 60 V. Samples were analyzed in a random order.
Easy nlc 1000 nano lc
Manufactured by Thermo Fisher Scientific
The Easy-nLC 1000 is a nano-liquid chromatography (nano-LC) system designed for high-performance separation of complex samples. It features a compact and robust design, capable of delivering consistent and reliable performance in nano-scale separations.
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Lab products found in correlation
Q exactive, by Thermo Fisher Scientific (2 mentions)
Thermo easy column sc200, by Thermo Fisher Scientific (1 mentions)
Thermo easy column sc001 traps, by Thermo Fisher Scientific (1 mentions)
Q exactive mass spectrometer, by Thermo Fisher Scientific (1 mentions)
Nanospray 3 source, by AB Sciex (1 mentions)
Easy spray source, by Thermo Fisher Scientific (1 mentions)
Qtrap 5500, by AB Sciex (1 mentions)
3 protocols using easy nlc 1000 nano lc
1
Nano-LC-MS/MS Proteomic Analysis Pipeline
2
Quantitative Mass Spectrometry Protocol
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The mass spectrometry (MS) data were collected by Shanghai Applied
Protein Technology, which was performed on a Q-Exactive mass spectrometer
(Thermo Scientific) coupled to an EASY-nLC1000 nano LC. The peptides
were loaded onto a reverse-phase trap column (RP-C18, 150 μm
× 20 mm, Thermo EASY column SC001 traps) connected to the C18
reversed-phase analytical column (RP-C18, 150 μm × 100
mm, Thermo EASY column SC200). Mobile phase A was 0.1% FA (v/v) in
acetonitrile–H2O (acetonitrile, 2%), and mobile
phase B was 0.1% FA in acetonitrile–H2O (acetonitrile,
84%). A linear gradient was performed at a flow rate of 300 nL/min:
0–220 min, 0–55% B; 220–228 min, 55–100%
B; 228–240 min, 100% B.
The mass spectrometer was operated
in the positive ion mode with the peptide recognition mode enabled.29 (link) A data-dependent top10 method was used to acquire
the MS data by survey scans (300–1800 m/z) for HCD fragmentation. The resolution for scans was set
to 70 000 at m/z 200 and
the resolution for HCD spectra was set to 17,500 at m/z 200, and the isolation window was 2 m/z. In each scan cycle, the number of precursors
selected for tandem MS was 20, and the charge state screening parameter
was set at 2. The underfill ratio was defined as 0.1%, and the normalized
collision energy was 30 eV.
Protein Technology, which was performed on a Q-Exactive mass spectrometer
(Thermo Scientific) coupled to an EASY-nLC1000 nano LC. The peptides
were loaded onto a reverse-phase trap column (RP-C18, 150 μm
× 20 mm, Thermo EASY column SC001 traps) connected to the C18
reversed-phase analytical column (RP-C18, 150 μm × 100
mm, Thermo EASY column SC200). Mobile phase A was 0.1% FA (v/v) in
acetonitrile–H2O (acetonitrile, 2%), and mobile
phase B was 0.1% FA in acetonitrile–H2O (acetonitrile,
84%). A linear gradient was performed at a flow rate of 300 nL/min:
0–220 min, 0–55% B; 220–228 min, 55–100%
B; 228–240 min, 100% B.
The mass spectrometer was operated
in the positive ion mode with the peptide recognition mode enabled.29 (link) A data-dependent top10 method was used to acquire
the MS data by survey scans (300–1800 m/z) for HCD fragmentation. The resolution for scans was set
to 70 000 at m/z 200 and
the resolution for HCD spectra was set to 17,500 at m/z 200, and the isolation window was 2 m/z. In each scan cycle, the number of precursors
selected for tandem MS was 20, and the charge state screening parameter
was set at 2. The underfill ratio was defined as 0.1%, and the normalized
collision energy was 30 eV.
3
Quantitative Protein Profiling with PRM and SRM
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Q Exactive coupled to EASY-Spray
source and EASY-nLC 1000 nanoLC (Thermo Scientific) were utilized
for PRM assays. QTRAP 5500, NanoSpray III source (SCIEX), and EASY-nLC
II were used for SRM assays. Peptides were loaded at 5 μL/min
onto pre-columns (2 cm × 100 μm, 5 μm C18) and separated
on analytical columns (15 cm × 75 μm, 3 μm C18) using
acetonitrile–water gradients at 400 nL/min.
source and EASY-nLC 1000 nanoLC (Thermo Scientific) were utilized
for PRM assays. QTRAP 5500, NanoSpray III source (SCIEX), and EASY-nLC
II were used for SRM assays. Peptides were loaded at 5 μL/min
onto pre-columns (2 cm × 100 μm, 5 μm C18) and separated
on analytical columns (15 cm × 75 μm, 3 μm C18) using
acetonitrile–water gradients at 400 nL/min.
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