Land Snail Proteomics Analysis

Land snail samples were reduced with 5 Mm DTT for 20 min at RT and subsequently alkylated with iodoacetamide for 20 min at 37°. The samples (15 µL) were analyzed using an LTQ Orbitrap Velos Pro system (Thermo Fisher Scientific) online coupled to a U3000 RSLCnano (Thermo Fisher Scientific) Uplc as described previously [26 (link)].
De novo sequencing and database searches were performed with PEAKs X software (http://www.bioinfor.com/peaks-software/, accessed on 13 February 2022) using its current workflow comprising de novo sequencing as well as database searches with user-defined modifications (PEAKs DB), post-translational modifications (PEAKs PTM) from the Unimod database “http://www.unimod.org/modifications_list.php accesed on 13.02.2023” and mutations (Spider). For peptide identification, the MS/MS spectra were correlated with the UniProt reviewed Mollusca proteins (http://www.uniprot.org, accessed on 13 February 2022) and the APD3 antimicrobial peptide database (http://aps.unmc.edu/AP/, accessed on 13 February 2022) [6 (link)]. For the database search with PEAKs DB, carbamidomethylated cysteine was considered as a fixed modification along with oxidation (M) as a variable modification. False discovery rates were set on both peptide and protein levels to 1%. On the other hand, only those de novo peptides with an average local confidence higher (ALC) than 80% were selected.