4.5.2. LC-MS/MS

Samples were analyzed by liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) using an Orbitrap Fusion Lumos mass spectrometer coupled to an Easy nLC/Ultimate 3000 nano-HPLC chromatography system (Thermo Fisher Scientific, Waltham, MA, USA). In the process of separation, the column was equilibrated with 95% buffer A (0.1% formic acid). The digested proteins were desalted with a C18 pre-column (3 mm, 100 μm × 20 mm, Thermo Scientific, USA). After loading and washing the digested proteins, the separation was performed with an analytical C18 column (1.9 mm, 150 μm × 120 mm, Thermo Scientific, USA) at a flow rate of 600 nL/min for 75 min. The mobile phase was the mixture of the buffer A (0.1% FA, H2O) and the buffer B (0.1% formic acid (FA), 100% acetonitrile (ACN)). Elution gradient parameters were setup as follow: 0–14 min, 7–13% buffer B; 14–51 min, 13–23% buffer B; 51–68 min, 23–36% buffer B; 68–69 min, 36–100% buffer B; and 69–75 min, 100% buffer B. Mass spectrometric analyses were carried out by an automated data-dependent Tandem Mass Spectrometry (MS/MS) analysis with full scans (300–1400 *m*/*z*) that was acquired from proteins in the Orbitrap at a mass resolution of 120,000. The positive ion mode and the negative ion mode were separately employed with the spray voltage of the mass spectrometer at 2000 V and 600 V, and the spray temperature of 320 ◦C for peptides. Normalized collision energy was set to 35% and the stepped collision energy was 5%. Automatic gain control settings for Fourier Transform Mass Spectrometry (FTMS) survey scans were 500,000 and for FT MS/MS scans 5000. Maximum injection time was 50 ms for survey scans and 35 ms for MS/MS scans.

### 4.5.3. Database Search and Bioinformatics Analysis

All raw data were aligned from the *A. coerulea* transcriptomic database, which was previously built by us. Raw MS files were processed for the peptide analysis using the software Proteome Discoverer 1.4 (ver. 1.4.0.2888; Thermo Fisher Scientific, Waltham, MA, USA). The parameters used for data analysis were: enzyme = trypsin, max missed cleavages = 2, fix modifications = cysteine carbamido methylation, variable modifications = methionine oxidation, *N*-terminal acetylation, peptide mass tolerance = ±15 ppm, and fragment mass tolerance = 20 mmu. The false discovery rate (FDR) < 0.01 was selected for peptide and protein identification. Differential protein screening was performed at a threshold of 2 fold change (FC). FC ≥ 2, FC ≤ 0.5 and 0.5 < FC < 2 representing, up, down and no significant change in the data of protein expression, respectively.

The bioinformatics of Gene Ontology (http://www.geneontology.org, GO) was analyzed on the differentially expressed proteins with a 2 FC according to biological processes, cellular components and molecular functions. GO enrichment was analyzed on the differentially expressed proteins with *p* ≤ 0.05 that was calculated based on a hypergeometric distribution with the default database as the background. The signal pathways were analyzed by the primary public database Kyoto Encyclopedia of Genes and Genomes (KEGG) (http://www.kegg.jp/kegg/pathway.html), and enriched by the tool Pathway Maps. *p*-values were calculated based on a hypergeometric distribution with the default KEGG database as the background. Multiple sequence alignment analysis was performed with BioEdit software (http://www.mbio.ncsu.edu/bioedit/page2.html) under default parameters. RNA sequences of *A. coerulea* were translated into protein through ORFfinder online service (https: //www.ncbi.nlm.nih.gov/orffinder). Sequences used for multiple sequence alignment were collected from SwissProt (http://www.uniprot.org/uniprot) or NCBI (https://blast.ncbi.nlm.nih.gov/Blast.cgi) databases, and performed with CLUSTALW program using Bioedit software (version: 7.0.5.3). Finally, phylogenetic trees were constructed using MEGA7 using the Neighbor-Joining method, whereas the 3D modeling was carried out by the combination of the online service SWISS-MODEL (https://swissmodel.expasy.org/) and software Discovery studio 4.5.
