For nLC-MS/MS analysis on the LTQ-Orbitrap Velos (Thermo-Fisher Scientific, San Jose, CA), the tryptic digest was reconstituted in 10 mL of 2% ACN with 0.5% formic acid (FA). The mass spectrometer was equipped with a ''Plug and Play'' nano ion source device (CorSolutions LLC, Ithaca, NY). The nanoLC was performed using a Dionex UltiMate3000 MDLC system (Dionex, Sunnyvale, CA). The gel extracted peptides (5-10 mL) were injected using a ''User Defined Program'' onto a PepMap C18 trap column (5 mm, 300 mm65 mm, Dionex) at a 20 mL/min flow rate for on-line desalting and then separated on a PepMap C18 reverse phase (RP) nano column (3 mm, 75 mm615 cm, Dionex) which was installed in the ''Plug and Play'' device with a 10-mm spray emitter (NewObjective, Woburn, MA) mounted in front of the Orbitrap ion transfer tube. The peptides were then eluted in a 60 min gradient of 10% to 40% ACN in 0.1% FA at 300 nL/min. The Orbitrap Velos was operated as described previously [9] . For repeat injections of the same samples, an exclusion list containing m/z values identified in the previous DDA run were generated using Proteome Discovery 1.1 software and applied to prevent resampling of the same ions. All data were acquired using Xcalibur 2.1 software (Thermo-Fisher Scientific).