Nanoesi emitter

The nano-Ultra Performance Liquid Chromatography was coupled online through a nanoESI emitter (10 μm tip; New Objective) to a quadrupole orbitrap mass spectrometer (Orbitrap Exploris 480; Thermo Scientific) using a FlexIon nanospray apparatus (Proxeon).
Data were acquired in PRM mode, while monitoring all Erk2 peptides of interest (heavy and light VADPDHDHTGFLTEYVATR with 0, 1, 2, or 3 phosphorylations, as well as light mutant peptides with or without phosphorylations and nonmodified Erk2 peptides GQVFDVGPR, FDMELDDLPK, and ICDFGLAR). Identification of the additional Erk2 peptides to whom no synthetic standards were used was done during method optimization, using a data-dependent acquisition analysis of the samples, while searching the data using the Byonic search engine (64 (link)) against the rat Erk2 protein sequence (WT and mutants) and common laboratory protein contaminants.
For the PRM analysis, MS1 resolution was set to 120,000 (at 200 m/z), mass range of 375 to 1500 m/z, standard automatic gain control target, and maximum injection time was set to 100 ms. MS2 resolution was set to 15,000, quadrupole isolation 2 m/z, higher-energy collision dissociation energy 30%, standard automatic gain control target, and maximum injection time mode set to auto.

The nanoLC was coupled online through a nanoESI emitter (7 cm length, 10 mm tip; New Objective; Woburn, MA, USA) to a quadrupole ion mobility time-of-flight mass spectrometer (Synapt G2 HDMS, Waters) tuned to 20,000 mass resolution (full width at half height). Data were acquired using Masslynx version 4.1 in data independent acquisition mode (DIA), HDMSE positive ion mode. The ions were separated in the T-Wave ion mobility chamber and transferred into the collision cell. Collision energy was alternated from low to high throughout the acquisition time. In low-energy (MS1) scans, the collision energy was set to 5 eV and this was ramped from 27 to 50 eV for high-energy scans. For both scans, the mass range was set to 50–2,000 Da with a scan time set to 1 second. A reference compound (Glu-Fibrinopeptide B; Sigma) was infused continuously for external calibration using a LockSpray and scanned every 30 seconds.