Peptides were analyzed in an Dionex UltiMate 3000 RSLC nano System coupled on-line to Q Exactive Orbitrap High Field Hybrid Quadrupole Mass Spectrometer (Thermo Fisher Scientific, Waltham, MA, United States) as described previously (Aryal et al., 2018 (link); Mohallem and Aryal, 2020 (link)). Briefly, reverse phase peptide separation was accomplished using a trap column (300 μm ID × 5 mm) packed with 5 mm 100 Å PepMap C18 medium coupled to a 50-cm long × 75 μm inner diameter analytical column packed with 2 μm 100 Å PepMap C18 silica (Thermo Fisher Scientific). The column temperature was maintained at 50°C. Sample was loaded to the trap column at a flow rate of 5 μL/min and eluted from the analytical column at a flow rate of 300 nL/min using a 120-min LC gradient. The column was washed and equilibrated by using three 30 min LC gradient before injecting next sample. Precursor ion (MS1) scans were collected at a resolution of 120,000 and MS/MS scans at a resolution of 15,000 at 200 m/z in data dependent acquisition mode.
Pepmap c18 medium
Manufactured by Thermo Fisher Scientific
The PepMap C18 medium is a high-performance reversed-phase chromatography material designed for the separation and purification of peptides. It is composed of spherical silica particles with a chemically bonded C18 alkyl ligand. The PepMap C18 medium offers excellent peak resolution, high loading capacity, and broad pH compatibility, making it suitable for a wide range of peptide analysis and purification applications.
Automatically generated - may contain errors
Lab products found in correlation
Dionex ultimate 3000 rslcnano system, by Thermo Fisher Scientific (2 mentions)
Pepmap c18 silica, by Thermo Fisher Scientific (2 mentions)
Q exactive hf orbitrap, by Thermo Fisher Scientific (2 mentions)
Acclaim pepmap 100 c18 analytical column, by Thermo Fisher Scientific (2 mentions)
Orbitrap fusion lumos mass spectrometer, by Thermo Fisher Scientific (1 mentions)
4 protocols using pepmap c18 medium
1
Peptide Analysis by Nano LC-MS/MS
2
Shotgun Proteomics Analysis by LC-MS/MS
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Samples were analyzed by reverse-phase LC-ESI-MS/MS system using the Dionex UltiMate 3000 RSLC nano System coupled to the Orbitrap Fusion Lumos Mass Spectrometer (Thermo Fisher Scientific). Peptides were loaded onto a trap column (300 mm ID ´ 5 mm) packed with 5 mm 100 Å PepMap C18 medium, then separated on a reverse phase column (50-cm long × 75 µm ID) packed with 2 µm 100 Å PepMap C18 silica (Thermo Fisher Scientific). The column temperature was maintained at 50°C. All MS measurements were performed in positive ion mode using a 120 minute LC gradient and standard data-dependent mode. MS data were acquired with a Top20 data-dependent MS/MS scan method.
3
Reverse-Phase HPLC-MS/MS Peptide Separation
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Samples were resuspended in 3% ACN, 0.1% Formic Acid (FA) and separated by reverse-phase HPLC with an Acclaim PepMap 100 C18 analytical column (75 μm ID × 50 cm) packed with 2 μm 100 Å PepMap C18 medium (Thermo Fisher Scientific), coupled with the Q-Exactive Orbitrap HF (Thermo Fisher Scientific) mass spectrometer. Peptides for global analysis were separated in a 160-min gradient. Peptides were loaded into the column with 2% mobile phase solution B (80%ACN with 0.1% FA in water). Solution B was linearly increased to 27% B until 110 min, followed by an increase to 40% B at 125 min. Mobile phase solution B was subsequentially increased 100% at 135 min, and held constant for an additional 10 min. The gradient was then returned to 2% B until the end of the run. Phosphopetideas and peptides for secretome analysis were separated in a 120-min gradient. Peptides were loaded into the column with 2% mobile phase B. Mobile phase solution B was increased linearly to 30% B until 80 min, followed by an increase to 45% B at 91 min. Mobile phase solution B was subsequentially increased 100% at 93 min, and held constant for an additional 5 min. The gradient was then returned to 2% B until the end of the run. Data-dependent acquisition MS/MS was performed for the top 20 precursors, with MS/MS spectra recorded from 400 to 1600 m/z.
4
Optimized Phosphoproteome and Secretome Analysis
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Samples were resuspended in 3% ACN, 0.1% Formic Acid (FA) and separated by reverse-phase HPLC with an Acclaim PepMap 100 C18 analytical column (75 μm ID × 50 cm) packed with 2 μm 100 Å PepMap C18 medium (Thermo Fisher Scientific), coupled with the Q-Exactive Orbitrap HF (Thermo Fisher Scientific) mass spectrometer. Peptides for global analysis were separated in a 160 minute gradient. Peptides were loaded into the column with 2% mobile phase solution B (80%ACN with 0.1% FA in water). Solution B was linearly increased to 27% B until 110 minutes, followed by an increase to 40% B at 125 minutes. Mobile phase solution B was subsequentially increased 100% at 135 minutes, and held constant for an additional 10 minutes. The gradient was then returned to 2% B until the end of the run. Phosphopetideas and peptides for secretome analysis were separated in a 120 minute gradient. Peptides were loaded into the column with 2% mobile phase B. Mobile phase solution B was increased linearly to 30% B until 80 minutes, followed by an increase to 45% B at 91 minutes. Mobile phase solution B was subsequentially increased 100% at 93 minutes, and held constant for an additional 5 minutes. The gradient was then returned to 2% B until the end of the run. Data-dependent acquisition MS/MS was performed for the top 20 precursors, with MS/MS spectra recorded from 400 to 1600 m/z.
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