*4.5. Proteomic Data Acquisition*

The proteomic data acquisition approach was modified from Haddad Momeni et al. [48]. Briefly, peptides were loaded onto a 2 cm C18 trap column (ThermoFisher 164705), connected in-line to a 15 cm C18 reverse-phase analytical column (Thermo EasySpray ES803) using 100% Buffer A (0.1% Formic acid in water) at 750 bar, using the Thermo EasyLC 1200 HPLC system (ThermoFisher, Roskilde, Denmark), and the column oven operating at 30 ◦C. Peptides were eluted over a 140 min gradient ranging from 10 to 60% of 80% acetonitrile, 0.1% formic acid at 250 nL/min, and a Q-Exactive instrument (ThermoFisher Scientific, Roskilde, Denmark) was run in a DD-MS2 top 10 method. Full MS spectra were collected at a resolution of 70,000, with an automatic gain control (AGC) target of 3 × <sup>10</sup><sup>6</sup> or maximum injection time of 20 ms and a scan range of 300 to 1750 *m*/*z*. The MS2 spectra were obtained at a resolution of 17,500, with an AGC target value of 1 × 106 or maximum injection time

of 60 ms, a normalized collision energy of 25, and an intensity threshold of 1.7 × 104. Dynamic exclusion was set to 60 s, and ions with a charge state <2 or unknown were excluded. MS performance was verified for consistency by running complex cell lysate quality control standards, and chromatography was monitored to check for reproducibility. The mass spectrometry data have been deposited to the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org (accessed on 21 December 2020)) via the PRIDE partner repository with the dataset identifier PXD 023249. The reviewer account details: Username: reviewer\_pxd023249@ebi.ac.uk; Password: xteOmcDd. The raw files were analyzed using Proteome Discoverer 2.4. Label-free quantitation (LFQ) was enabled in the processing and consensus steps, and spectra were matched against the *P. rubra* S4059 database obtained from Uniprot (UniProt ID: UP000305729). Dynamic modifications were set as Oxidation (M), Deamidation (N, Q), and Acetyl on the protein N-termini. Cysteine carbamidomethyl was set as a static modification.

The methods of proteomic analyses were modified from Beyene et al. [49]. Briefly, all the statistical analyses were performed using Perseus software (version 1.6.14.0, Max-Planck Institute of Biochemistry, Martinsried, Germany), https://maxquant.net/perseus/ (accessed on 20 September 2020)), and all results were filtered to a 1% false discovery rate (FDR). The normalized abundance values for each protein were log2 transformed, and at least two valid values were required in the bio-triplicates for quantitation. When the original signals were zero, they were imputed with random numbers from a normal distribution, in which the mean and standard deviation were chosen from low abundance values below the noise level (Width = 0.3; shift = 1.8) [49,50]. To identify proteins with significantly different abundances when S4059 were grown on chitin compared to on mannose, the FDR were estimated using Benjamini–Hochberg method, and a two-tailed unpaired *t*-test was used with a combination of *p*-value ≤ 0.05 and Log2 fold-change ≥1.5 [51]. The resulting significant proteins were exported from Perseus and visualized in GraphPad Prism 8 (Graphpad Software, San Diego, CA, USA, https://www.graphpad.com/scientific-software/prism/ (accessed 5 October 2020)) using volcano plots.
