2. Materials and methods2.1. Milk s

2. Materials and methods
2.1. Milk samples

Camel milk was collected from reared camels by been directly milked into a sterile milking can. The camel milk used was pooled milk samples assembled from 20 different healthy camels (Camelus dromedarius) that ranged between 2 and 12 months in lactation. Cow milk was collected from 20 different Holstein cows. Both milk samples were derived from a local breeding located in the south of Tunisia. The milk samples were immediately transported to our laboratory using an ice box within 4 h. Once reaching the laboratory at 4 °C, pH (744-pH meter, Metrohm SA CH-9101, Herisau, Switzerland) was determined. Subsequently, both milks were skimmed by centrifugation at 3000g during 20 min at 4 °C (Gyrozen 1580MGR, Multi-purpose Centrifuge, Daejeon, Korea).2.2. Heat treatment experiments

Heat treatment experiments of camel and bovine milks were conducted in a stainless steel recipient (316L; Total volume = 500 mL) at 80 °C for 60 min. Heat treatment consisted in heating over a hot plate without agitation (Felfoul, Lopez, Gaucheron, Attia, & Ayadi, 2015). The experiments were reproduced at least 3 times.

2.3. LC/MS analysis

100 μL of fresh and heat-treated skimmed milk samples were diluted in 400 μL buffer Urea 4 M/Tris 25 mM pH8, and then placed at 37 °C for 1 h 15 min in a water bath with regular manual agitation. All solutions were diluted 2 × in 0.5% TFA solution, afterwards, filtered through Millipore® Millex®filters HVPVDF membrane, 0.45 μm Pore Size before injection.

Mass spectrometry (MS) experiments were performed using an on-line liquid chromatography mass spectrometry setup via an Agilent 1100 HPLC system coupled to an AB Sciex QSTAR XL Quadrupole-Time Of Flight (QTOF) mass spectrometer. The separation of proteins was performed with a separation column C4 VYDAC Grace, reference 214TP5215, 150 mm length × 2.1 mm inner diameter (i.d.) using solvent A (0.106% TFA in deionized water) and solvent B (80% (v/v) acetonitrile and 0.1% (v/v) TFA in deionized water). A linear gradient from 37 to 60% of solvent B in 40 min was applied for the elution at a flow rate of 0.25 mL/min. Protein signal was recorded by both UV detection at 214 nm wavelength and electrospray mass spectrometry. Eluted proteins were directly electrosprayed into the mass spectrometer operated in positive mode. Mass spectra were collected in the selected mass range from 800 to 3000 Thomson. The instrument was calibrated by multipoint calibration using fragment ions that resulted from the collision-induced decomposition of a peptide from β-casein, β-CN (193–209). The quantification data of the studied camel and cow milk protein fractions (expressed in μg/μL) was estimated basing on the peak areas of the chromatographic profiles as well as both milks compositional data. Measurements were performed in duplicate with independent samples.

2.4. SDS-PAGE

Skimmed camel and cow milks, before and after heat treatment at 80 °C for 60 min (fresh camel, heat-treated camel, fresh cow and heat-treated cow milks), were diluted with deionized water. Protein concentrations of skimmed milks were determined by dint of the Bradford method (1976) and adjusted to be the same among samples equal to 5 g/L. The proteins were then separated by polyacrylamide gel electrophoresis containing 0.1% sodium dodecyl sulphate (SDS-PAGE) in non-reducing conditions. Electrophoresis experiments were carried out using a Bio-Rad apparatus (Mini-Protean Tetra Cell, Bio-Rad Laboratories, Hercules, USA) of gels in vertical slabs. 50 μg of protein was loaded for each sample onto 12% acrylamide resolving gel. As described by Laemmli (1970), electrophoresis was run at 120 V/20 mA until the marker color (bromophenol blue, Sigma-Aldrich Chemie S.a.r.l., Saint-Quentin Fallavier, France) was 0.5 cm from the anode end of the block (approximately 3 h). The molecular weight of the different protein fractions were estimated by comparing their electrophoretic mobilities with those of marker proteins having known molecular weights. The electrophoresis experiment was repeated 3 times. All gels were photographed and the most significant one was presented.

2.5. Protein bands identification by tandem mass spectrometry

After separation by SDS-PAGE, the proteins were identified by mass spectrometry (MS) after an in-gel trypsin digestion according to Shevchenko, Wilm, Vorm, and Mann (1996). Gel pieces were excised from the gel, placed in eppendorf tubes of 0.5 mL, washed with 100 μL acetonitrile and NH4HCO3 solution (50%/50%) 1–3 times based on their coloration intensity. Subsequently, the supernatants were removed using a small benchtop centrifuge. Finally, the gel pieces were dried under vacuum in a SpeedVac concentrator (SVC100H-200; Savant, Thermo Fisher Scientific, Waltham, MA) for 15 min. In-gel trypsin digestion was performed overnight at 37 °C and stopped with spectrophotometric-grade trifluoroacetic acid (TFA) (Sigma-Aldrich Chemie S.a.r.