*2.5. Purification of Peptide Fractions*

In order to increase the resolution in peptide separation, the peptide enriched fractions obtained with solid phase extraction were further purified. 5–30% ACN fraction (35 mg) was dissolved in 2 mL of 0.1% TFA and further purified using preparative Reversed Phase-High Performance Liquid Chromatography (RP-HPLC) with a PrepNova-Pak ® HR C18 column (6 μm 60 Å 25 × 10 mm; Waters, Milford, MA, UK) with a linear gradient from 5% to 40% ACN with a flow rate of 12 mL/min. Briefly, after an isocratic step at 5% ACN for 5 min, ACN was linearly increased from 5% to 40% for 24 min and then to 100% within the following 5 min. The column e ffluent was monitored by UV detection (λ = 220 nm). Fractions were collected every 2 min and then lyophilized.

#### *2.6. Liquid Chromatography-Tandem Mass Spectrometry (LC-MS*/*MS) Analysis*

The fraction of interest was dissolved in H2O/0.1% formic acid (FA) and an amount of sample corresponding to 1 μg (2.5 μL) was subjected to LC-MS/MS analysis. For the MS analyses a LTQ-Orbitrap XL mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA) coupled online with a nano-HPLC Ultimate 3000 (Dionex-ThermoFisher Scientific, Waltham, MA, USA) was employed. A homemade 10 cm chromatographic column packed into a pico-frit (75 μm internal diameter (I.D.), 15 μm tip, New Objective, Woburn, MA, USA) with C18 material (Aeris Peptide 3.6 μm XB-C18, Phenomenex Torrance, CA, USA) was utilized for sample loading. A linear gradient from 3% to 40% of ACN, 0.1% FA in 20 min at a flow rate of 250 nL/min was chosen for peptide elution from the original sample. Data of the LC-MS/MS analysis were obtained performing on the Orbitrap a full scan at high resolution (60,000). The MS-MS fragmentation scans were conducted on the ten most intense ions acquired with collision-induced dissociation (CID) fragmentation in the linear trap (data-dependent acquisition—DDA). Furthermore, the following parameters were set for this analysis: capillary voltage 1.2 kV; source temperature 200 ◦C.
