*3.1. Protein Extraction*

Specimens of *B. verrucosa* were sampled at Praia da Memória, Porto, Portugal (Lat/Long WGS84; 41◦1400.0 N 8◦4327.0 W). Then whole animal bodies (four specimens) were kept at −80 ◦C, freeze dried and subsequently homogenized in a blender until obtaining a dry powder. Lyophilized material of *B. verrucosa* (0.1 g) was mixed with 500 μL Tris-HCl (40 mM), MgCl2 (5 mM), Dithiothreitol (DTT) (1 mM), protease inhibitors (87,785, Thermo Scientific, Waltham, MA, USA), at pH 8.0, (buffer 1) in vortex (2 × 30 s). The mixture was centrifuged at 16,000× *g*, during 20 min at 4 ◦C. The supernatant (soluble protein fraction, SF) was stored at −20 ◦C and the pellet was homogenized with 500 μL urea (7 M), thiourea (2 M), CHAPS (4%, *w*/*v*), dithiothreitol (65 mM) and ampholytes (0.8%, *v*/*v*), at pH 4–7 in vortex (2 × 30 s) and incubated overnight, at 4 ◦C. The homogenate was centrifuged at 16,000× *g*, during 20 min at 4 ◦C, and the supernatant (insoluble protein fraction, IF) collected and stored at −20 ◦C. Total protein concentration was estimated according to the Bradford method [110].

### *3.2. Two-Dimensional Gel Electrophoresis*

Two-dimensional gel electrophoresis (2DE) was performed as described previously [49]. Duplicate IF and SF (~400 μg of protein) were diluted to 300 μL urea (7 M), thiourea (2 M), CHAPS (4%, *w*/*v*), dithiothreitol (65 mM) and ampholytes (0.8%, *v*/*v*), at pH 4–7 and loaded onto 17 cm, pH 4–7 immobiline dry strips (Bio-Rad, Hercules, CA, USA) with active hydration (50 Volt) for 12 h. Proteins were separated by isoelectric focusing (IEF) in a Protean IEF cell (Bio-Rad) with the following program: step 1, 15 min at 250 V; step 2, 3 h voltage gradient to 10,000 V (linear ramp); step 3, 10,000 V until achieving 60,000 V/h (linear ramp). Second-dimension sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed in a Hoefer SE 900 vertical slab electrophoresis system (Hoefer, Holliston, MA, USA), with 12% (*w*/*v*) acrylamide gels, at 480 mA and 20 ◦C. After electrophoresis run the gels were stained with colloidal Coomassie blue G-250 [111]. The 2DE protein profiles were analyzed by gel scanning with a GS-800 calibrated densitometer (Bio-Rad) and the D analysis software (Bio-Rad) as described previously [49]. Protein spots detected by this procedure were excised from the gels for subsequent identification.

### *3.3. MALDI-TOF MS Analysis*

Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF/TOF) mass spectrometry (MS) measurements were performed to identify protein spots from 2DE gels. Protein spots were washed, distained, reduced, alkylated, and digested with trypsin following the procedure described by Osório and Reis [112]. The solution containing the peptides was collected and stored at −20 ◦C until application to a MALDI plate. Peptides were acidified with trifluoroacetic acid (TFA) and concentrated using C18 micro-columns (C18 Tips, 10 μL, Thermo Scientific, 87782). Peptides were thereafter eluted from the micro-column directly onto the MALDI plate with 1.5 μL of α-CHCA matrix (8 mg/mL) prepared in acetonitrile (50%, *v*/*v*), TFA (0.1%, *v*/*v*) and 6 mM ammonium phosphate. MALDI mass spectra were externally calibrated following the manufacturer's instructions (TOF/TOF calibration mixture, AB SCIEX) and internal calibration was applied using trypsin autolysis peaks. Peptide mass spectra data was collected in positive ion reflector mode in the range of *m*/*z* 700–4000 (4800 Plus MALDI TOF/TOF Analyzer, AB SCIEX).

Proteins were identified by combining Peptide Mass Fingerprint and MS/MS information. Proteins were searched in a locally stored NCBI copy of protein sequences of the genomes of the sea anemones *Exaiptasia pallida* (26,042 protein count, GenBank accession: GCA\_001417965.1) and

*Nematostella vectensis* (24,780 protein count, GenBank accession: GCA\_000209225.1), using the Mascot search engine (Version 2.4). The search included peaks with a signal-to-noise ratio greater than 10 and allowed for up to two missed trypsin cleavage sites, mass tolerance of 50 ppm, cysteine carbamidomethylation (fixed modification), methionine oxidation (variable modification), and a charge state of +1. For a match to be considered significant, protein scores with a probability greater than 95% (*p* < 0.05), calculated by the Mascot software, were required [112]. The data generated from 2D-MALDI procedures were also searched against UniProtKB protein sequence database in the Metazoa section [113,114], using the same parameters mentioned before.

### *3.4. In Solution Protein Digestion and MS/MS Analysis*

For LC-MS/MS analysis, SF and IF protein samples were processed by filter aided sample preparation (FASP) method [115] with the following modifications. Protein samples (40 μg) were alkylated and digested with trypsin (recombinant, proteomics grade, Roche, Basel, Switzerland), at enzyme to protein ratio of 1:100 ( *w*/*w*), for 16 h at 37 ◦C, in centrifugal filter units with nominal molecular weight limit (NMWL) of 30 kDa (MRCF0R030, Millipore, Billerica, MA, USA). Peptides were subsequently recovered by centrifugal filtration, acidified with formic acid (FA) (10%, *v*/*v*), desalted and concentrated by reversed-phase extraction (C18 Tips, 100 μL, Thermo Scientific, 87784) using acetonitrile (ACN) (70%, *v*/*v*) and TFA (0.1%, *v*/*v*) for peptide elution. Before LC–MS/MS, the peptides were recovered in 0.1% (*v*/*v*) Formic acid (FA) to the concentration of 0.04–0.06 μg/μL.

FASP protein digests (duplicate samples) were analyzed by nano-LC coupled to a hybrid Ion-trap mass spectrometer (LTQ Orbitrap Velos Pro—ETD, Thermo Scientific) as described previously [68]. Peptides were separated by reverse-phase chromatography (20 mm × 100 μm C18 precolumn followed by a 100 mm × 75 μm C18 column with particle size 5 μm, NanoSeparations, Nieuwkoop, The Netherlands) using a linear ascending gradient of buffer B (ACN + FA, 0.1%, *v*/*v*), being buffer A TFA, 0.1%, *v*/*v* in water. The gradient started from 2% B to 30% B in 40 min and to 95% B (*v*/*v*) in 30 min, at a flow rate of 0.3 μL/min (total elution time 70 min). Peptides were analyzed by on-line nano-electrospray ionization (easy nano-ESI) in positive mode, with Xcalibur software (version 2.6, Thermo Scientific). Full scans were performed at a resolution of 30,000 with scan ranges of 380–2000 *<sup>m</sup>*/*<sup>z</sup>*. The top 20 most intense ions were isolated and fragmented with CID by applying normalized collision energy of 30% value, isolation width of 2.0, activation time of 10 milliseconds and Q-value of 0.25. In total 4 nano-LC-MS/MS runs were performed.
