Peptide mixtures were analyzed by online nanoflow LC-MS/MS (nLC-MS/MS) using an EASY-nLC 1200 (Thermo Fisher Scientific) connected to a Q Exactive instrument (Thermo Fisher Scientific) through a nanoelectrospray ion source. The nano-LC system was operated in one column setup with a 50-cm analytical column (75-μm inner diameter and 350-μm outer diameter) packed with C18 resin (EASY-Spray PepMap RSLC C18 75 μm inner diameter, 2 μm particles; Thermo Fisher Scientific) configuration. Solvent A was 0.1% formic acid (FA), and solvent B was 0.1% FA in 80% ACN. Samples were injected in an aqueous 0.1% TFA solution at a flow rate of 500 nl/min. SILAC and hmSILAC immunoenriched methyl peptides were separated with a gradient of 5 to 30% solvent B over 120 min, followed by a gradient of 30 to 60% for 10 min and 60 to 95% over 5 min at a flow rate of 250 nl/min in the EASY-nLC 1200 system. The Q Exactive was operated in the data-dependent mode to automatically switch between full-scan MS and MS/MS acquisition. Survey full-scan MS spectra (from m/z 300 to 1650) were analyzed in the Orbitrap detector with resolution R = 60,000 at m/z 200. The 10 most intense peptide ions with charge states ≥2 were sequentially isolated and fragmented by higher-energy collision dissociation with a normalized collision energy setting of 28%. The maximum allowed ion accumulation times were 20 ms for full scans and 50 ms for MS/MS, and the target value for MSMS was set to 1 × 106. The dynamic exclusion time was set to 20 s.

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