*2.6. Reverse Phase-High Performance Liquid Chromatography (RP-HPLC) Coupled to Electrospray Ionization-Mass Spectrometry (ESI-MS)*

Chloroform/methanol precipitation was performed, according to [34], from 200 μL of desalted Ser-lacticaseicin 30 suspension before RP-HPLC-ESI-MS analysis. Precipitated Serlacticaseicin 30 was redissolved in 40 μL of 25 mM ammonium bicarbonate buffer and 10 μL was chromatographically separated on an ACQUITY UPLC system (Waters Corporation) using a C18 column (150 × 3.0 mm, 2.6 μm, Uptisphere CS Evolution, Interchim, Montluçon, France). Eluent A was milliQ H2O containing formic acid (0.1%, *v*/*v*) and eluent B was acetonitrile (ACN) containing formic acid (0.1%, *v*/*v*). The ACN gradient (flow rate 0.5 mL/min) was as follows: from 5% to 40% eluent B over 45 min, from 40% to 95% eluent B over 5 min, followed by washing and equilibrating procedures with 95% and 5% solvent B for 5 min each, respectively. The eluate was directed into the electrospray ionization source of the Q-TOF Synapt G2-Si™ (Waters Corporation, Manchester, UK). MS analysis was performed in sensitivity, positive ion and data-dependent analysis (DDA) modes. The source temperature was set at 150 ◦C and the capillary and cone voltages were set to 3000 and 60 V, respectively. MS data were collected for *m*/*z* values in the range of 50 to 2000 Da with a scan time of 0.2 s and a lock mass correction with 556.632 *m*/*z*, corresponding to simply charged leucine encephalin. The mass spectrum corresponding to the sum of 400 ms acquisition was deconvoluted using UniDec software [35]. The molecular mass range was fixed to an upper limit of 20 kDa while the charge range was fixed between 1 and 50.
