Ultimate 3000 xrs system

To reduce the complexity of tryptic peptides and improve the proteomic coverage, peptide fractionation was performed using high-pH reversed-phase liquid chromatography (LC) [30 (link)]. Each TMT10plex-labeled peptide mixture sample was redissolved with 45 μL 10 mM ammonium formate, pH 10. Twenty microliters of peptide solution were injected and separated on a 20-cm Hypersil GOLD C₁₈ column (1.9 μm particle size, 2.1 mm inner diameter, 175 Å pore size) heated to 35 °C on an Ultimate 3000 XRS system (Thermo Scientific), with a flow rate of 0.5 mL/min. Mobile phase A and B consisted of 10 mM ammonium formate in water (pH 10) and 10 mM ammonium formate in 95% acetonitrile (pH 10), respectively. The 13-min LC gradient was 0% B over 3 min, 0–28% B over 7 min, 28–90% B over 1 min, 90% B over 1 min, and 90–0% B over 1 min. For each TMT10plex set, a total of 72 fractions were collected after 3.5 min, with a collection rate of one fraction per 6 s. The 72 fractions were then concatenated into 24 fractions by combining fractions 1, 25, 49; 2, 26, 50; and so on. It was shown that the concatenation strategy allows more uniform peptide distributions on subsequent low-pH RPLC and thus improves protein identifications [30 (link)]. The concatenated fractions were concentrated in a SpeedVac and stored at -80 °C until LC-SPS-MS3 analysis.