Proteomic Analysis of Microbial Samples by 1D-LC-MS/MS

Samples were analyzed by 1D-LC–MS/MS as described in Hinzke et al. (2019) [43 (link)]. Replicate samples (e.g., replicate 1 at 1%, 5%, and 10%) were run consecutively followed by two wash runs and a blank run to reduce carryover. For 1D-LC–MS/MS, 0.4 μg (pure culture samples) or 2 μg of peptide (mock community-spike in samples) were loaded onto a 5 mm, 300 μm i.d. C18 Acclaim PepMap 100 precolumn (Thermo Fisher Scientific) using an UltiMate 3000 RSLCnano Liquid Chromatograph (Thermo Fisher Scientific). After loading, the precolumn was switched in line with either a 50 cm × 75 μm (pure culture samples) or a 75 cm × 75 μm (mock community–spike in samples) analytical EASY-Spray column packed with PepMap RSLC C₁₈, 2 μm material. The analytical column was connected via an Easy-Spray source to a Q Exactive Plus hybrid quadrupole-Orbitrap mass spectrometer (Thermo Fisher Scientific). Peptides were separated on the analytical column using 140 (pure culture samples) or 260 (mock community-spike in) min gradients and mass spectra were acquired in the Orbitrap. The resolution used on the Q Exactive Plus for MS¹ scans, which provide the isotope pattern information used by Calis-p, was 70,000.