Nanoflow Dual-Trap Single-Column Proteomics

Dual trap single column (DTSC)^{17 (link)} was adapted for nanoflow to enable single cell analysis^{18 (link)}. The trapping columns were 0.17 μL media bed (EXP2 from Optimize Technologies) packed with 10 μm diameter, 100 Å pore PLRP-S (Agilent) beads, and the analytical column was a PepSep 15 cm x 75 μm packed with 1.9 μm C18 stationary phase (Bruker). The configuration was installed on a Thermo Ultimate 3000 nanoRSLC equipped with one 10-port valve and one 6-port valve and a nano flow selector. Mobile phase A as 0.1% formic acid in water. Mobile phase B was 0.1% formic acid in acetonitrile. Peptides were separated using the following gradient all using linear transitions between conditions: starting conditions of 9% B at 500 nL/min; 22% B over 8 minutes; 37% B over 4.7 minutes; 1000 nL/min flowrate and 98% B over 0.2 minutes; hold at 98% B for 1 minute; reduce to 9% B at 1,000 nL/min over 0.1 minutes; hold at 9% B at 1,000 nL/min for 0.9 min; return to 500 nL/min flowrate in 0.1 min (15 min total). Valves and trapping columns were heated to 55° C and the analytical column was heated to 60° C. See Kreimer et al. for additional details about the dual trap chromatography setup.^{18 (link)}