*2.7. Analysis of the Ser-Lacticaseicin 30 Amino Acid Sequence by Peptide Fingerprinting*

In total, 10 μL of native Ser-lacticaseicin 30, redissolved in 25 mM ammonium bicarbonate buffer, were hydrolyzed by 1 μL of trypsin/Lys-C mixture at 0.1 μg/μL for 3 h at 37 ◦C and again by adding another 1 μL of the enzyme mixture for 16 h at 37 ◦C and centrifuged for 10 min at 8000× *g*. The pellet was removed and the peptides in the supernatant were subjected to fingerprinting using the Q-TOF Synapt G2-Si™ spectrometer and the same HPLC and MS acquisition conditions as described above. A maximum of 10 precursor ions was chosen for MS/MS analysis with an intensity threshold of 10,000. MS/MS data were collected using the collision-induced dissociation (CID) mode and a scan time of 0.1 s at an energy collision of 8 V to 9 V for low *m*/*z* and at a range of 40 V to 90 V for high *m*/*z*.

For peptide identification, database searches were performed via PEAKS Studio Xpro software (Bioinformatics Solutions Inc., Waterloo, Canada) using the UniProtKB/Swiss-Prot-TrEMBL databases restricted to *Lactobacillus* (https://www.uniprot.org/, accessed on April 2022). Mass tolerance of 35 ppm and MS/MS tolerance of 0.2 Da were allowed. Data searches were performed, assigning trypsin as protease and three missed cleavage sites were allowed. Variable methionine oxidations were also considered. The relevance of peptide identities was judged according to their identification score returned by PEAKS Studio Xpro using a *p*-value of 0.05 (*p* < 0.05) and a false discovery rate (FDR) < 0.1%.
