Probot fraction collector

Each high pH reversed-phase fraction was dissolved in 40 μL of 5% ACN, 0.1% TFA, following nano-LC peptide separation using the UltiMate3000 HPLC system (Dionex, Sunnyvale, CA). The peptides were trapped on a 0.3 × 5 mm μ precolumn filled with C18 PepMap, 5 μm, 100 Å particles (Dionex) and subsequently separated in a 0.1 × 150 mm analytical NanoEase column filled with Atlantis C18, 3 μm, 100 Å particles (Waters) using a linear gradient ranging from 5% ACN, 0.1% TFA to 50% of 80% ACN, 0.1% TFA over 85 min at a flow rate of 360 nL/min. The eluate was mixed at a 1 : 4 ratio postcolumn in a Probot fraction collector (Dionex) with 3 mg/mL CHCA (LaserBio Labs, Sophia-Antipolis, France) in 70% ACN, 0.1% TFA. Fractions were collected every 8 s for 60 min on an OptiTOF LC-MALDI plate. MALDI analysis was performed in a 4800 MALDI-TOF/TOF (AB Sciex). MS spectra were acquired across the m/z range of 800–4 000 using 625 laser shots per spectrum. A maximum of 12 precursors was chosen for fragmentation in each MS spectrum, starting with the weakest precursor. CID MS/MS spectra were acquired with using 3000 laser shots.

C18 trap column (C18 pepmap100, 5 µm particles, 100 Å pores, 300 µm i.d., 5 mm length), and C18 analytical column (C18 pepmap100, 3 µm particles, 15 cm length, 75 µm inner diameter., 100 Å pores) were from Acclaim (Vandoeuvre, France). The Ultimate3000 series HPLC and the Probot fraction collector were purchased from Dionex (Voisins le Bretonneux, France).