Peptides were eluted from C18 Stage Tips with 2 × 20 µl solvent B (80% acetonitrile and 0.5% acetic acid). Acetonitrile was evaporated, and thereby, the volume reduced to 5 µl in a speed vacuum centrifuge. 10 µl solvent containing 2% acetonitrile and 0.1% trifluoroacetic acid was added.
Peptides were separated on line to the mass spectrometer by using an easy nano-LC system (Proxeon Biosystems). 5 µl samples were loaded with a constant flow of 700 nl/min onto a 15-cm fused silica emitter with an inner diameter of 75 µm (IntelliFlow; Proxeon Biosystems) packed in house with RP ReproSil-Pur C18-AQ 3 µm resin (Dr. Maisch). Peptides were eluted with a segmented gradient of 2–60% (for trypsin digest) and 5–60% (for EndoLysC digest) solvent B over 105 min with a constant flow of 250 nl/min. The nano-LC system was coupled to a mass spectrometer (LTQ-Orbitrap; Thermo Fisher Scientific) via a nanoscale LC interface (Proxeon Biosystems). The spray voltage was set to 2.1 kV, and the temperature of the heated capillary was set to 180°C.
Survey full-scan MS spectra (m/z = 300–1,650) were acquired in the Orbitrap with a resolution of 60,000 at the theoretical m/z = 400 after accumulation of 1,000,000 ions in the Orbitrap. The most intense ions (up to 10) from the preview survey scan delivered by the Orbitrap were sequenced by centromere identifier (collision energy 35%) in the LTQ after accumulation of 5,000 ions concurrently to full scan acquisition in the Orbitrap (TOP10 peptide sequencing). Maximal filling times were 1,000 ms for the full scans and 150 ms for the MS/MS. Precursor ion charge state screening was enabled, and all unassigned charge states as well as singly charged peptides were rejected. The dynamic exclusion list was restricted to a maximum of 500 entries with a maximum retention period of 90 s and a relative mass window of 5 ppm. Orbitrap measurements were performed with the lock mass option enabled for survey scans to improve mass accuracy (Olsen et al., 2005 (link)).
Peptides were separated on line to the mass spectrometer by using an easy nano-LC system (Proxeon Biosystems). 5 µl samples were loaded with a constant flow of 700 nl/min onto a 15-cm fused silica emitter with an inner diameter of 75 µm (IntelliFlow; Proxeon Biosystems) packed in house with RP ReproSil-Pur C18-AQ 3 µm resin (Dr. Maisch). Peptides were eluted with a segmented gradient of 2–60% (for trypsin digest) and 5–60% (for EndoLysC digest) solvent B over 105 min with a constant flow of 250 nl/min. The nano-LC system was coupled to a mass spectrometer (LTQ-Orbitrap; Thermo Fisher Scientific) via a nanoscale LC interface (Proxeon Biosystems). The spray voltage was set to 2.1 kV, and the temperature of the heated capillary was set to 180°C.
Survey full-scan MS spectra (m/z = 300–1,650) were acquired in the Orbitrap with a resolution of 60,000 at the theoretical m/z = 400 after accumulation of 1,000,000 ions in the Orbitrap. The most intense ions (up to 10) from the preview survey scan delivered by the Orbitrap were sequenced by centromere identifier (collision energy 35%) in the LTQ after accumulation of 5,000 ions concurrently to full scan acquisition in the Orbitrap (TOP10 peptide sequencing). Maximal filling times were 1,000 ms for the full scans and 150 ms for the MS/MS. Precursor ion charge state screening was enabled, and all unassigned charge states as well as singly charged peptides were rejected. The dynamic exclusion list was restricted to a maximum of 500 entries with a maximum retention period of 90 s and a relative mass window of 5 ppm. Orbitrap measurements were performed with the lock mass option enabled for survey scans to improve mass accuracy (Olsen et al., 2005 (link)).
