*4.4. Growing Bacteria and Sample Preparation for Proteomic Analyses*

The protein samples were prepared according to Chevallier et al. [47]. *P. rubra* S4059 wild type and GH19 mutant were grown in 20 mL MMM with crystalline chitin for 2 days at 25 ◦C, 200 rpm. All experiments were carried out in biological triplicates. A five milliliter culture was harvested (5000× *g*, 20 min), and then the culture supernatant was transferred into a new 15 mL Falcon tube, and ice-cold acetone (−20 ◦C) was added to the supernatant to a final concentration of 80%. Then the mixture was kept at −20 ◦C overnight and harvested at 2000× *g* for 20 min. Acetone was carefully removed. The harvested bacterial cells were washed twice with ice-cold phosphate-buffered saline (PBS), and the pellet was lysed using 20 μL of lysis buffer (consisting of 6 M Guanidinium Hydrochloride, 10 mM Tris (2-carboxyethyl) phosphine hydrochloride, 40 mM 2-chloroacetamide, 50 mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) pH 8.5). Samples were inactivated at 95 ◦C for 5 min and were then sonicated on high 3 times for 10 s in a 4 ◦C Bioruptor sonication water bath (Diagenode). A Bradford assay (Sigma) was used to determine protein concentration, and 50 μg of each sample was used for digestion. Samples were diluted 1:3 with 10% Acetonitrile, 50 mM HEPES pH 8.5, LysC (MS grade, Wako, Japan) added in a 1:50 (enzyme to protein) ratio, and samples were incubated at 37 ◦C for 4 h. Samples were further diluted to 1:10 with 10% Acetonitrile, 50 mM HEPES pH 8.5, trypsin (MS grade, Promega) added in a 1:100 (enzyme to protein) ratio, and samples were incubated overnight at 37 ◦C. Enzyme activity was quenched by adding 2% trifluoroacetic acid (TFA) to a final concentration of 1%. Before mass spectrometry analysis, the peptides were desalted on SOLAu C18 plates (ThermoFisher Scientific, Roskilde, Denmark). After each solvent application, the plate was centrifuged for 1 min at 350× *g*. For each sample, the C18 material was activated with 200 μL of 100% Methanol (HPLC grade, Sigma), then 200 μL of 80% Acetonitrile, 0.1% formic acid. The C18 material was subsequently equilibrated 2 x with 200 μL of 1% TFA, 3% Acetonitrile, after which the samples were loaded. After washing the tips twice with 200 μL of 0.1% formic acid, the peptides were eluted using 40% Acetonitrile, 0.1% formic acid, and transferred into clean 500 μL Eppendorf tubes. The eluted peptides were concentrated in an Eppendorf Speedvac and reconstituted in 1% TFA, 2% Acetonitrile for Mass Spectrometry (MS, Merck, Darmstadt, Germany) analysis.
