*3.5. Peptide Identification*

The resulting ion-trap raw data (LTQ Orbitrap) were searched against custom cnidarians protein databases using MaxQuant freeware software (version 1.5.5.1) with the Andromeda search engine. MS and MS/MS tolerances were set to 10 ppm and 0.6 Da, respectively. Trypsin was selected for protein cleavage allowing for one missed cleavage. Carbamidomethylation and oxidation were selected as static and dynamic modifications, respectively. Identifications were validated by performing a decoy database search for the estimation of False Discovery Rate (FDR) and peptide identifications were accepted if they could be established at a probability greater than 95.0%. Protein identifications were accepted if they could be established at a probability greater than 99.9% and contained at least two identified peptides (Razor + unique peptides) [116,117], based on Occam's razor principle). The protein database utilized was the locally stored NCBI copy of protein sequences of the genomes of the sea anemones *E. pallida* (26,042 protein count, GenBank accession: GCA\_001417965.1), *N. vectensis* (24,780 protein count, GenBank accession: GCA\_000209225.1), *Hydra vulgaris* (21,993 protein count, GenBank accession: GCF\_000004095.1) and *Acropora digitifera* (33,878 protein count, GenBank accession: GCF\_000222465.1). The identification of potential toxins was done against the manually reviewed venom proteins and toxins database, from the animal toxin annotation project of the UniProtKB/Swiss-Prot protein knowledgebase [118–120] (database size 1.20 MB, downloaded on 16 June 2016).

### *3.6. Protein Homology Search and GO Analysis*

Protein sequences with unknown function were annotated with a blast search in the National Centre for Biotechnology Information database (NCBI, http://www.ncbi.nlm.nih.gov/) using blastp algorithm employing a threshold e-value of 1 × 10−10. Total of proteins identified with Maxquant software, were also blasted and mapped using the Blast2Go software (version 2.4.4) [73]. Gene ontology (GO) terms were used to group proteins within the domains of BP, CC, andMF.
