LC-MS/MS analyses were performed on a custom-built automated LC system coupled on-line to an LTQ-Orbitrap mass spectrometer (Thermo Scientific, San Jose, CA) via a nanoelectrospray ionization interface as previously described14 (link). Briefly, 0.75 μg of peptides were loaded onto a home-made 65-cm-long reversed-phase capillary column with 75-μm-inner diameter packed using 3 μm Jupiter C18 particles (Phenomenex, Torrance, CA). The mobile phase was held at 100% A (0.1% formic acid) for 20 min, followed by a linear gradient from 0 to 60% buffer B (0.1% formic acid in 90% acetonitrile) over 85 min. The instrument was operated in data-dependent mode with an m/z range of 400–2000, in which a full MS scan with a resolution of 100K was followed by 6 MS/MS scans. The 6 most intensive precursor ions were dynamically selected in the order of highest intensity to lowest intensity and subjected to collision-induced dissociation using a normalized collision energy setting of 35% and a dynamic exclusion duration of 1 min. The heated capillary was maintained at 200 °C, while the ESI voltage was kept at 2.2 kV.
Jupiter c18 particles
Manufactured by Phenomenex
Jupiter C18 particles are a type of chromatography packing material used for the separation and purification of various compounds. They are made of silica-based particles with a C18 (octadecyl) bonded phase. These particles are designed to provide efficient and reproducible separation performance for a wide range of analytes.
Automatically generated - may contain errors
Lab products found in correlation
Orbitrap fusion lumos mass spectrometer, by Thermo Fisher Scientific (4 mentions)
Nanoacquity uplc system, by Waters Corporation (3 mentions)
Ltq orbitrap mass spectrometer, by Thermo Fisher Scientific (1 mentions)
Orbitrap velos mass spectrometer, by Thermo Fisher Scientific (1 mentions)
Ultimate 3000 nano rslc system, by Thermo Fisher Scientific (1 mentions)
Nanoacquity, by Waters Corporation (1 mentions)
Q exactive mass spectrometer, by Thermo Fisher Scientific (1 mentions)
7 protocols using jupiter c18 particles
1
LC-MS/MS Proteomic Analysis Protocol
2
Orbitrap Velos LC-MS Peptide Separation
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Samples were analyzed on an Orbitrap Velos mass spectrometer (Thermo Fisher, Waltham, MA) that was interfaced with a 75 μm i.d.×65 cm long LC column packed with 3 μm Jupiter C18 particles (Phenomenex) and a 5 μL injection loop. The mobile phase solvents consisted of (A) 0.2% acetic acid and 0.05% TFA in water and (B) 0.1% TFA in 90% acetonitrile. An exponential gradient was used for the separation, which started with 100% A and gradually increased to 60% B over 100 min.
3
Orbitrap Mass Spectrometry Proteomics Analysis
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The enriched samples were analyzed with an Orbitrap Fusion Lumos mass spectrometer (Thermo Scientific) coupled with a NanoAcquity UPLC system (Waters, Milford) in sensitive acquisition settings. Precursor ions were acquired at a range of m/z 400–1600 with 120 K resolving power and the isolation of precursor for MS/MS analysis was performed with a 1.4 Th. Higher-energy collisional dissociation (HCD) with 30% collision energy was used for sequencing with a target value of 1e5 ions determined by automatic gain control. Resolving power for acquired MS2 spectra was set to 30k at m/z 200 with 150 ms maximum injection time. The peptide samples were loaded onto the trap column (3 cm × 150 µm i.d) via the back-flushing technique and separated with a 100 cm long analytical capillary column (75 µm i.d.) packed in-house with 3 µm Jupiter C18 particles (Phenomenex, Torrance). The long analytical column was placed in a dedicated 95 cm long column heater (Analytical Sales and Services) regulated to a temperature of 45 °C. NanoAcquity UPLC system was operated at a flow rate of 300 nL/min over 2 h with a linear gradient ranging from 95% solvent A (H2O with 0.1% formic acid) to 40% of solvent B (acetonitrile with 0.1% formic acid).
4
Optimized nanoLC-MS/MS for Proteomics
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Analytical capillary columns (100 cm × 75 μm i.d.) and trap columns (3 cm × 150 μm i.d) were packed in-house with 3 μm Jupiter C18 particles (Phenomenex, Torrance, CA). The long analytical column was placed in a column heater (Analytical Sales and Services, Pompton Plains, NJ) regulated to a temperature of 45°C. Ultimate 3000 nanoRSLC system (Thermo Scientific, Sunnyvale, CA) was operated at a flow rate of 350 nl/min over 2 h with linear gradient ranging from 95% solvent A (water with 0.1% formic acid) to 40% of solvent B (acetonitrile with 0.1% formic acid). The enriched samples were analyzed on an Orbitrap Fusion Lumos mass spectrometer (Thermo Scientific) equipped with an in-house customized nanoelectrospray ion source. Precursor ions were acquired (m/z 300–1500 at 120k resolving power and the isolation of precursor for MS/MS analysis was performed with a 1.4 Th. Higher-energy collisional dissociation (HCD) with 30% collision energy was used for sequencing with a target value of 5E4 ions determined by automatic gain control. Resolving power for acquired MS2 spectra was set to 30k at m/z 200 with 150 ms maximum injection time.
5
Optimized Nanoflow LC-MS/MS Protocol
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Long analytical capillary columns (100 cm × 75 µm internal diameter) and dual-fritted trap columns (2 cm × 150 µm internal diameter) were packed in house with 3-µm Jupiter C18 particles with a pore size of 300 Å (Phenomenex). The columns were placed in a column heater regulated to a temperature of 45 °C. A NanoAcquity ultrahigh-performance liquid chromatography system (Waters) run in back-flush mode for trapping of samples was operated at a flow rate of 300 nl min−1 over 2 h with a linear gradient ranging from 95% solvent A (H2O with 0.1% formic acid) to 40% of solvent B (acetonitrile with 0.1% formic acid). The enriched samples were analyzed on an Orbitrap Fusion Lumos mass spectrometer (Thermo Scientific) equipped with an in-house customized nanoelectrospray ion source with the following instrumental parameters: spray voltage, 2.2 kV; capillary temperature, 275 °C and radio frequency lens level, 30.0. The precursor ion scan was operated with a scan range of 300 to 1,500 m/z; AGC target of 5 × 105; maximum injection time of 50 ms and resolution of 120,000 at 200 m/z. The MS/MS scan was performed with an isolation width of 1.4 Th, HCD with 30% collision energy, AGC target of 1 × 105, maximum injection time of 200 ms and resolution of 30,000 at 200 m/z.
6
NanoLC-MS/MS Proteomic Analysis Protocol
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Analytical capillary columns (100 cm × 75 µm i.d.) and trap columns (2 cm × 150 µm i.d.) were packed in-house with 3 µm Jupiter C18 particles (Phenomenex, Torrance). The long analytical column was placed in a column heater (Analytical Sales and Services) regulated to a temperature of 45 °C. NanoAcquity UPLC system (Waters, Milford) was operated at a flow rate of 300 nL/min over 2 h with a linear gradient ranging from 95% solvent A (H2O with 0.1% formic acid) to 40% of solvent B (acetonitrile with 0.1% formic acid). The enriched samples were analyzed on an Orbitrap Fusion Lumos mass spectrometer (Thermo Scientific) equipped with an in-house customized nanoelectrospray ion source. Precursor ions were acquired (m/z 300–1500) at 120 K resolving power and the isolation of precursor for MS/MS analysis was performed with a 1.4 Th. Higher-energy collisional dissociation (HCD) with 30% collision energy was used for sequencing with an auto gain control (AGC) target of 1e5. Resolving power for acquired MS2 spectra was set to 30k at with 200 ms maximum injection time.
7
Peptide Separation and Analysis by LC-MS/MS
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