Ultimate 3000 rslcnano lc system

Tryptic peptide mixtures were submitted to LC-MS/MS analysis using a Q-Exactive^TM mass spectrometer (Thermo Fisher Scientific) interfaced with an UltiMate 3000 RSLCnano LC system (Thermo Fisher Scientific).
Two biological replicates were analyzed for each bacterial strain and three technical replicates were acquired for each biological replicate.
Protein identification and quantification were achieved by using the MaxQuant software (version 1.6.3.4) (developed by the Computational Systems Biochemistry, The Max-Planck-Institute for Biochemistry, Martinsried, Germany) [34 (link)]. The Perseus software (version 1.6.0.7)) (developed by the Computational Systems Biochemistry, The Max-Planck-Institute for Biochemistry, Martinsried, Germany) was used for further processing the label free quantification data obtained by MaxQuant analysis and to build up the heat maps reporting the LFQ values for each protein in the six replicates of each strain [35 (link)].
The putative relative abundance level of each Slp in a single strain was calculated using a spectral counting approach [36 (link)].
Detailed protocols for LC-MS/MS data acquisition and parameters used for data processing are reported in Table S3.

The LC-MS analysis of crosslinked samples were conducted by Orbitrap Fusion Lumos Tribrid Mass Spectrometer (Thermo Fisher Scientific) coupled with UltiMate 3000 RSLC nano LC system (Thermo Fisher Scientific). The separation of samples was performed on a 50 cm reverse-phase column (in-house packed with Poroshell 120 EC-C18, 2.7 µm, Agilent Technologies) with 180 min gradient. High field asymmetric waveform ion mobility spectrometry (FAIMS) was enabled with internal stepping −40/–50/–60 V. Cross-linked samples were acquired with 120,000 resolution at MS1 levels, 30,000 resolution at MS2 levels, charge state 4–8 enabled for MS2, higher-energy collisional dissociation (HCD) at 30% for MS2 fragmentation.

LC–MS/MS analyses were performed on an UltiMate 3000 RSLC nano-LC system coupled with an Orbitrap Fusion Lumos Mass Spectrometer (Thermo Fisher Scientific, Waltham, Mass, USA). Dried peptide sample fractions were reconstituted in 0.1% formic acid and 1% acetonitrile, and separated on an Acclaim™ PepMap™100 C18 analytical column (130 Å, 2 µm, 75 µm × 250 mm). The mass spectrometer was operated in the data-dependent acquisition mode. All MS spectra detection was performed in positive-ion mode with charge states of 2–6 included, using orbitrap detector with a full scan MS spectra range of 300–1500 m/z at 60,000 resolutions. The top 20 most abundant precursors were subject to high-energy collision-induced dissociation.
The mass spectrometry raw data were analyzed by Proteome Discoverer (PD) version 2.4. The PD search parameters were set as follows: the precursor mass tolerance was set to 15 ppm and fragment mass tolerance to 0.02 Da. Carbamidomethyl/+ 57.021 Da (C) and Oxidation/+ 15.995 Da (M) were set as static modification, Deamidated/+ 0.984 Da (N, Q) was set as peptide dynamic modification and Acetyl/+ 42.011 Da was set as protein N-terminal dynamic modification. The minimum peptide length was set to 6. Peptide spectra matches were filtered with false-discovery rates of 1% (Strict) and 5% (Relaxed) on the peptide spectrum match and subsequently on the protein level.