Nano-scale Reversed-Phase Peptide Separation

The system was equipped with a 20 cm column of 75 μm I.D. fused silica capillary pulled to a 5 μm I.D. tip using a Sutter Instruments P-2000 CO₂ laser puller. The analytical column was packed with C-12 reversed phase chromatography material (Phenomenex, Jupiter 4 μm, Proteo 90 $\acute{\mathrm{Å}}$ ) using an in-house constructed pressure bomb and compressed helium gas. The LC system was also configured with a trap column of 100 μm × 3 cm (C-12, Phenomenex, Jupiter 4 μ, Proteo 90 $\acute{\mathrm{Å}}$ ). Peptides were loaded into the trap column for 5 min using a flow rate of 2 μl/min and were eluted using a 60 min gradient presented in Supplementary Table 1.