LC/MS analysis

LC-ESI/MS/MS analysis was performed using a Q-Exactive™ Plus Hybrid Quadrupole-Orbitrap™ Plus mass spectrometer (ThermoFisher) coupled to an EASY-nanoLC 1000 system (ThermoFisher). The samples were loaded (2 μL) onto a 75 μm i.d. × 25 cm Acclaim^® PepMap 100 RP column (ThermoFisher). The peptides were eluted at a flow rate of 300 nL/min with an acetonitrile gradient in aqueous formic acid (0.1%) as mobile phase A. Peptide elution occurred in the following sequence: 0%–4% B (buffer B) for 1 min, 4%–12% B over 127 min, 12%–22% B over 112 min, 22%–30% B over 40 min, 30%–70% B over 6 min, hold at 70% B for 6 min, followed by increase in B to 95% B over 1 min, and an isocratic wash at 95% B for 6 min. Full-scan mass spectra were acquired using the Orbitrap™ mass analyzer in the mass-to-charge ratio (m/z) of 375–1,500 and with a mass resolving power set to 70,000. Ten data-dependent high-energy collisional dissociations were performed with a mass resolving power set to 35,000, a fixed first m/z 100, an isolation width of 2.0 m/z, and the normalized collision energy setting of 27.

The maximum injection time was 120 ms for parent ion analysis and 120 ms for product ion analysis. Target ions already selected for MS/MS were dynamically excluded for 30 s. An automatic gain control target value of 3 × 10⁶ ions was used for full MS scans and 5 × 10⁵ ions for MS/MS scans. Peptide ions with charge states of one or greater than seven were excluded from MS/MS acquisition. The tandem mass spectra were processed using Matrix Science Distiller version 2.5 without charge state deconvolution and deisotoping. The processed files were used for protein database searches using Mascot (version 2.5.1; Matrix Science, London, United Kingdom). The UniProt Mouse Reference database (downloaded May 3, 2014, 69,021 entries) was used. A parent ion tolerance and MS2 fragment tolerance were set to 10 ppm and 0.05 Da, respectively. Carbamidomethyl of cysteine was specified as a fixed modification and oxidation of methionine was set as a variable modification.

Protein identifications were performed using Scaffold, version 4.4.8 (Proteome Software, Inc., Portland, OR), implementing the Protein and Peptide Prophet algorithms [23,24]. Peptide identifications were accepted with >90.0% probability. Protein identifications were accepted if they could be established at >95.0% probability and contained at least two peptides with unique sequences. Protein probabilities were assigned using the Protein Prophet algorithm. Proteins that contained similar peptides, but could not be differentiated based on identification of unique peptide sequences, were grouped to satisfy the principles of parsimony.