Ultra-sensitive Peptide Analysis by LC-MS/MS

All peptide samples were analyzed by 1D-LC-MS/MS as previously described (96 (link)), with the modification that a 75-cm analytical column was used. Briefly, an UltiMate 3000 RSLCnano liquid chromatograph (Thermo Fisher Scientific) was used to load peptides with loading solvent A (2% acetonitrile, 0.05% trifluoroacetic acid) onto a 5-mm, 300-μm-internal-diameter (i.d.) C₁₈ Acclaim PepMap100 precolumn (Thermo Fisher Scientific). Since peptide concentrations were very low, complete peptide samples (80 μl) were loaded onto the precolumn. Peptides were eluted from the precolumn onto a 75-cm by 75-μm analytical EASY-Spray column packed with PepMap RSLC C₁₈, 2-μm material (Thermo Fisher Scientific) heated to 60°C. Separation of peptides on the analytical column was achieved at a flow rate of 225 nl min⁻¹ using a 460-min gradient going from 98% buffer A (0.1% formic acid) to 31% buffer B (0.08% formic acid, 80% acetonitrile) in 363 min and then to 50% B in 70 min and to 99% B in 1 min and ending with 26 min 99% B. Eluting peptides were analyzed in a Q Exactive Plus hybrid quadrupole-Orbitrap mass spectrometer (Thermo Fisher Scientific). Carryover was reduced by running two wash runs (injection of 20 μl acetonitrile) between samples. Data acquisition in the Q Exactive Plus was done as previously described (5 (link)).