The enriched peptides were analysed with the EASY‐nLC1000 system (Thermo Finnigan) connected to a Q Exactive mass spectrometer (Thermo Finnigan). Briefly, peptide samples were dissolved in 0.1% formic acid, directly loaded onto a reverse‐phase precolumn (Thermo EASY column SC200, 150 μm × 100 mm). A reverse‐phase analytical column (Thermo EASY column SC001 traps, 150 μm × 20 mm) was used for peptide separation, as described previously (Li, Sun, et al., 2016 (link)). The resulting peptides were evaluated with the Q Exactive mass spectrometer for 120 min. The mass spectrometer was operated in positive ion mode. MS data were acquired using a data‐dependent procedure, dynamically choosing the most abundant precursor ions from the survey scan (350–1800 m/z) for high‐energy collision dissociation fragmentation. Survey scans were acquired at a resolution of 70,000 at m/z 200 and the resolution for high‐energy collision dissociation spectra was set to 17,500 at m/z 200. Automatic gain control was used to prevent overfilling of the ion trap and 5 × 104 ions were accumulated for generating the MS/MS spectra. Each LC–MS/MS analysis was repeated three times to reduce technical variation.
Thermo easy column sc001 traps
Manufactured by Thermo Fisher Scientific
The Thermo EASY column SC001 traps are designed for use in liquid chromatography-mass spectrometry (LC-MS) applications. They serve as a sample concentration and cleanup device, facilitating the preparation of samples for analysis. The traps are made of inert materials and are compatible with a range of solvents and sample types.
Automatically generated - may contain errors
Lab products found in correlation
Q exactive mass spectrometer, by Thermo Fisher Scientific (3 mentions)
Thermo easy column sc200, by Thermo Fisher Scientific (3 mentions)
Easy nlc 1000 system, by Thermo Fisher Scientific (1 mentions)
Easy nlc 1000, by Thermo Fisher Scientific (1 mentions)
Agilent 1100 quaternary hplc, by Agilent Technologies (1 mentions)
Easy nlc 1000 nano lc, by Thermo Fisher Scientific (1 mentions)
3 protocols using thermo easy column sc001 traps
1
Peptide Analysis via Q Exactive MS
2
Peptide Mixture Analysis by LC-MS/MS
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Peptide mixtures were subjected to nano-liquid chromatography associated with MS for protein identification. All of the mass analyses were performed using a LC-MS/MS system, which consisted of an Agilent 1100 quaternary HPLC (Agilent, EASY-nLC1000, USA) and a Q-Exactive mass spectrometer (Thermo Finnigan, Germany) with the application of a distal 180°C source temperature. An RP trap column (Thermo EASY column SC200, 150 μm × 100 mm) and a C18 reverse-phase column (Thermo EASY column SC001 traps, 150 μm × 20 mm) was used for desalting and separating samples, respectively. Mobile phase A consisted of HPLC-grade water containing 0.1% formic acid (FA), and phase B consisted of 84% HPLC-grade acetonitrile (ACN) containing 0.1% FA. The analytical separation was run at a flow rate of 400 nl/min by using a linear gradient of phase B as follows: 0–45% for 100 min, 45–100% for 8 min, and 100% for 12 min. Each LC-MS/MS analysis was repeated three times to reduce technical variation (Lee et al., 2015 (link)).
3
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.
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