Agilent 1100 chromatograph

The peptide profile of the WSEs was analyzed by liquid chromatography–tandem mass spectrometry (LC-MS/MS) in a system consisting of an Agilent 1100 chromatograph (Agilent Technologies, Santa Clara, CA, USA) and an LTQ-FT Ultra mass spectrometer (Thermo Scientific, Waltham, MA, USA) as described in Ref. [13 (link)]. The data are available in Supplementary Materials, Table S1.
The set analysis of peptidomics data was performed using “UpSetR” and “eulerr” R packages. The heat maps of peptidomics data were constructed using Peptigram—a web-based application for peptidomics data visualization [14 (link)]. The search for peptides with previously reported antioxidant and ACE-I activities was performed in the Milk Bioactive Peptide Database [15 (link)] and BIOPEP-UWM Database [16 (link)].

Acetonitrile (ACN) for HPLC (Sigma Aldrich, St. Louis, MO, USA) and trifluoroacetic acid (TFA) for mass spectrometry (Fisher Scientific International, Hampton, NH, USA) were used in the present studies. Standards of dairy proteins (bovine origin) were β-lg (variants A and B, 90% of protein), α-la (85% of protein), BSA (90% of protein), casein (88% of protein), and Ig G (90% of protein) from Sigma (St. Louis, MO, USA).
HPLC analysis was performed on Agilent 1100 chromatograph (Agilent, Santa Clara, CA, USA) using Zorbax–300SB C8 column (4.6 × 250 mm, 5 µm, Agilent, Santa Clara, CA, USA). The column was equilibrated with 0.1% aqueous TFA solution. The samples were eluted using acetonitrile gradient (ACN-water-TFA = 95:5:0.1 mL/100 mL): 0–5 min, 5%; 5–10 min, 5–10%; 10–30 min, 10–40%; 30–32 min, 40%; 32–40 min, 40–50%; 40–45 min, 50%; 45–50 min, 50–10%. Separation was performed at room temperature with a flow of 1.0 mL/min for 50 min, and detection was performed at 214 nm. HPLC profiles were analyzed using specialized software ChemStation for LC 3D systems Rev.B.04.01 (Agilent, Santa Clara, CA, USA).

Sugars and tartaric acid were quantified using an Agilent 1100 chromatograph (Santa Clara, CA, USA) equipped with an evaporative light scattering detector (ELSD) (Sedex, Sedere, Olivet, France). The LC separation was obtained on a Hi-plex H column (300 × 7.7 mm), as previously reported [34 (link)], as mobile phases of water 0.002% trichloride acetic acid were used. The flow rate was 0.6 mL min⁻¹ and the column temperature was set at 75 °C. The ELSD detector parameter was 70 °C, 2.5 bar, and gain 7. Glucose, fructose, and tartaric acid were used as the reference compounds and standard solutions were prepared by exactly weighing 30 mg of each compound in 10 mL of water, and then diluted to obtain a calibration curve.