Peptides from pooled samples of each condition were separated with a linear gradient from 3 to 30% B (eluent A: 0.1% FA in water and eluent B: 95% ACN and 0.1% FA) on an in-house packed 40 cm × 0.75 μm C18 column (3-μm C18 beads, Dr. Maisch ProntoSIL) connected to Easy-nLC 1200 (Thermo Fisher Scientific). Measurements were acquired on a Orbitrap Exploris 480 mass spectrometer (Thermo Fisher Scientific) at a normalized AGC target of 200% for both MS1 and MS2. The scan range was set to 350 to 1150 m/z, RF lens was set to 50%, and the maximum injection time was 264 ms for MS1 and 54 ms for MS2. Twenty dependent scans were acquired, and dynamic exclusion was set to 20 s. Charge states between 2 and 6 were selected, and the Orbitrap resolution for MS1 was set to 120,000 and 30,000 for MS2. Peptides were fragmented with HCD at a collision energy of 30%. The total method length was 132 min.
3 μm c18 beads
Manufactured by Thermo Fisher Scientific
The 3-μm C18 beads are a type of chromatographic media used in liquid chromatography applications. They have a particle size of 3 micrometers and a C18 (octadecyl) stationary phase.
Automatically generated - may contain errors
Lab products found in correlation
Orbitrap exploris 480, by Thermo Fisher Scientific (1 mentions)
Easy nlc 1200, by Thermo Fisher Scientific (1 mentions)
Ltq orbitrap velos pro mass spectrometer, by Thermo Fisher Scientific (1 mentions)
Acclaim pepmap100 nanoviper column, by Thermo Fisher Scientific (1 mentions)
Acclaim pepmap100, by Thermo Fisher Scientific (1 mentions)
Ultimate 3000 rslc system, by Thermo Fisher Scientific (1 mentions)
2 protocols using 3 μm c18 beads
1
Peptide Separation and Identification
2
Liquid Chromatography-Mass Spectrometry of Tryptic Peptides
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Tryptic peptides were injected into an Ultimate 3000 RSLC system (Thermo Scientific, Sunnyvale, CA) connected online with positive electrospray ionization on a LTQ-Orbitrap Velos Pro mass spectrometer (Thermo Scientific, Bremen, Germany). The tryptic plasma peptides (2.5 μg) were eluted with a 180 min biphasic acetonitrile (I) gradient from a 50 cm analytical column (Dionex #164570, Acclaim PepMap100 nanoViper column, 75 μm i.d. × 50 cm, packed with 3 μm C18 beads (Thermo Scientific)), whereas the tryptic platelet-derived peptides were separated with a 90 min gradient on a 15 cm analytical column (Acclaim PepMap 100, 15 cm × 75 µm i.d. nanoViper column, packed with 2 µm C18 beads). The eluting peptides were ionized in the electrospray and analysed by the LTQ-Orbitrap Velos Pro. The mass spectrometer was operated in the DDA-mode (data-dependent-acquisition) to automatically switch between full scan MS and MS/MS acquisition. The LC-MS analysis is described in detail in the Supplementary methods section.
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