C18 acclaim pepmap100 precolumn

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)).

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.