C18 t3 hss nano column

ULC/MS grade solvents were used for all chromatographic steps. Each sample was loaded using split-less nano-Ultra Performance Liquid Chromatography (10 kpsi nanoAcquity; Waters, Milford, MA, USA) in high-pH/low-pH reversed phase (RP) 2 dimensional liquid chromatography mode. 20 μg of digested protein from each sample was loaded onto a C18 column (XBridge, 0.3 × 50 mm, 5 μm particles, Waters). The following two buffers were combined: (A) 20 mM Ammonium formate, pH 10 and (B) Acetonitrile (ACN). Peptides were released from the column using a step gradient: 10.8%B, 13.8%B, 15.8%B, 17.8%B, 20.1%B, 23.4%B, 65%B. Each fraction flowed directly to the second dimension of chromatography. The buffers used in the low pH RP were: (A) H₂O + 0.1% Formic acid and (B) ACN + 0.1% Formic acid. Desalting of samples was performed online using a reverse-phase C18 trapping column (180 μm i.d., 20 mm length, 5 μm particle size, Waters). Then the peptides were separated using a C18 T3 HSS nano-column (75 μm i.d., 200 mm length, 1.8 μm particle size, Waters) run at 0.4 μL/minute. Finally, peptides were eluted from the column and loaded onto the mass spectrometer using the following protocol: 3% to 30%B over 60 min, 30% to 95%B over 5 min, 95% maintained for 7 min (followed by return to initial conditions).