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Proteomic Profiling Comparing the Effects of Different Heat Treatments on Camel (Camelus dromedarius) Milk Whey Proteins

Camel milk is consumed in the Middle East because of its high nutritional value. Traditional heating methods and the duration of heating affect the protein content and nutritional quality of the milk. We examined the denaturation of whey proteins in camel milk by assessing the effects of temperature on the whey protein profile at room temperature (RT), moderate heating at 63 °C, and at 98 °C, for 1 h. The qualitative and quantitative variations in the whey proteins before and after heat treatments were determined using quantitative 2D-difference in gel electrophoresis (DIGE)-mass spectrometry. Qualitative gel image analysis revealed a similar spot distribution between samples at RT and those heated at 63 °C, while the spot distribution between RT and samples heated at 98 °C differed. One hundred sixteen protein spots were determined to be significantly different (p < 0.05 and a fold change of ≥1.2) between the non-heated and heated milk samples. Eighty protein spots were decreased in common in both the heat-treated samples and an additional 25 spots were further decreased in the 98 °C sample. The proteins with decreased abundance included serum albumin, lactadherin, fibrinogen β and γ chain, lactotransferrin, active receptor type-2A, arginase-1, glutathione peroxidase-1 and, thiopurine S, etc. Eight protein spots were increased in common to both the samples when compared to RT and included α-lactalbumin, a glycosylation-dependent cell adhesion molecule. Whey proteins present in camel milk were less affected by heating at 63 °C than at 98 °C. This experimental study showed that denaturation increased significantly as the temperature increased from 63 to 98 °C.


Introduction
The one-humped camel (Camelus dromedarius) lives in the arid and semi-arid desert regions of the Middle East and produces a considerable amount of milk despite this hot and dry environment. It is thus a valuable source of nutrition, providing both milk and meat in these areas of the world [1,2].
The 2D-DIGE experiments were carried out in triplicate and yielded reproducible spot patterns for all the milk samples, i.e., those at-RT, 63 and 98 °C. Approximately 1300 spots were mapped to the gels. The protein spots for the samples at room temperature (red/green), those at 63 °C (red), and those at 98 °C (green) were compared ( Figure 1A,B,D,E). The gel images showed a high degree of protein similarity between the RT and those heated at 63 °C, as shown by the number of yellow spots ( Figure 1C); protein similarity was minimal between the samples maintained at room temperature and those heated at 98 °C ( Figure 1F); yellow spots represent proteins with the same isoelectric point, molecular weight, and nearly equal protein fluorescence intensity. Statistical analysis of the gels was conducted using the Progenesis statistical software v.3.3 (Nonlinear Dynamics, Newcastle, UK). One hundred and ninety protein spots, exhibiting a fold change of ≥1.2, and ANOVA p-value ≤ 0.05, were chosen for identification via mass spectrometry.

PCA Analysis
The unsupervised principal component analysis (PCA) bi-plot of gels and spots ( Figure 2B) shows distinct gel grouping that agrees with the experimental groups. PCA plot of the two first principal components explained 85.15% of the selected spot's variability within the three groups. The differentially abundant spots showed expression pattern clusters according to their abundant patterns based on a hierarchical clustering analysis (Figure 2A). The clustering pattern showed

PCA Analysis
The unsupervised principal component analysis (PCA) bi-plot of gels and spots ( Figure 2B) shows distinct gel grouping that agrees with the experimental groups. PCA plot of the two first principal components explained 85.15% of the selected spot's variability within the three groups. The differentially abundant spots showed expression pattern clusters according to their abundant patterns based on a hierarchical clustering analysis (Figure 2A). The clustering pattern showed minimum amount of change in the protein intensities for the selected spots between RT and 63 • C while the difference for the same spots between RT and 98 • C was higher and significantly different.  Colored dots and numbers represent gels and spots, respectively.

MALDI-TOF-MS
The 190 protein spots of interest were excised from the preparative 2-D gels. After trypsin digestion, the digested spots were analyzed by MALDI-TOF-MS; then, 116 protein spots, isolated using MALDI-TOF-MS analysis, were successfully identified using the MASCOT Swiss Prot database. The database search results, with the coverage and score, are listed in Table S1. Thirty-one proteins were successfully matched with high certainty with entries in the C. dromedarius database, and an additional 85 assignments were made by matching to known homologous peptides identified in other mammalian databases (Table 1). Not all spots of interest could be identified because some proteins were low in abundance and did not yield sufficiently intense mass of fingerprints; other spots were mixtures of multiple proteins.

MALDI-TOF-MS
The 190 protein spots of interest were excised from the preparative 2-D gels. After trypsin digestion, the digested spots were analyzed by MALDI-TOF-MS; then, 116 protein spots, isolated using MALDI-TOF-MS analysis, were successfully identified using the MASCOT Swiss Prot database. The database search results, with the coverage and score, are listed in Table S1. Thirty-one proteins were successfully matched with high certainty with entries in the C. dromedarius database, and an additional 85 assignments were made by matching to known homologous peptides identified in other mammalian databases (Table 1). Not all spots of interest could be identified because some proteins were low in abundance and did not yield sufficiently intense mass of fingerprints; other spots were mixtures of multiple proteins.

Changes in Abundance of Proteins after Application of Different Heating Strategies
Heat treatment of milk subjects it to the highly complex Maillard reaction, which greatly affects the structure and properties of its constituents including the whey proteins [21]. The reaction commonly occurs between the milk sugar, lactose, and the lysine residues of the milk proteins, leading to formation of large high molecular weight aggregates. This reaction is mostly seen to take place between the different casein fractions and lactoglobulin. β-Lactoglobulin is present in the milk of other dairy animals, such as the cow and the buffalo, but is characteristically absent from camel milk; this renders the composition of camel milk similar to that of human milk and accounts for the decreased allergenic property of camel milk. Camel milk is also known to have a lower amount of lactose in comparison to bovine milk. The major whey proteins in camel milk are α-lactoglobulin, lactoferrin, lactoperoxidase, serum albumin, immunoglobulin G, and secretory immunoglobulin A. Thermal denaturing, and aggregation or gelation, in the bovine milk has been extensively studied, while only a few studies have examined it in camel milk [16,22]. We found that the major fraction of proteins affected by heat treatment included 61% enzymes, 20% binding proteins, 10% cell adhesion proteins, 5% proteins involved in the immune response, 2% transport proteins, and 2% others.

Proteins That Showed Decreased Abundance after Heating at 63 or 98 • C, Compared to Room Temperature
We found that a total of 80 protein spots, corresponding to samples heated at 63 and 98 • C, were decreased, with an appreciable difference in fold change. Statistical analysis revealed a significant decrease in the levels of these spots, which related to proteins including: lactotransferrin, spot #348  Table 1). Intensive heat treatment affected the functional properties [15] and solubility of milk serum proteins [23,24]. Caseins and whey proteins are engaged in protein aggregates, found in heat-treated milk; the formation of intermolecular disulfide bonds is mostly responsible for this heat-induced protein association. Proteins, especially enzymes, are heat-sensitive, and are denatured at higher temperatures. Therefore, moderate and high heating temperatures eradicated 68% of the protein spots in our dataset, in which enzymes accounted for 61% of the spots. Spots relating to mitochondrial enzymes, such as cytochrome P450 11B2 mitochondrial spot and succinate dehydrogenase cytochrome b560, were completely absent at 98 • C. Spots relating to lactoferrin, a protein involved in iron metabolism and having antioxidant activity, were found to have variable intensities, this was likely caused by the different temperatures, because some of the spots representing the isoforms of lactoferrin were absent at 63 • C, while others were present.

Proteins That Increased in Abundance after Heating at 63 or 98 • C, Compared to Room Temperature
We found that eight protein spots had increase in abundance after heating at 63 and 98 • C; the fold change was greater at the latter temperature. These spots related to: α-lactalbumin, spot #1069 (fold change of 1.4/11.9 at 63/98 • C, respectively); copper chaperone for superoxide dismutase, spot #1041 (fold change of 1.5/1.6 at 63/98 • C, respectively); ERI1 exoribonuclease 2, spot #1167 (fold change of 1.2/15.6 at 63/98 • C, respectively); and glycosylation-dependent cell adhesion molecule 1, spot #985 (fold change of 1.3/3.9 at 63/98 • C, respectively). Using SDS-PAGE, Farah showed that the heat denaturation of the individual camel whey proteins is not as pronounced as that in bovine whey proteins; more intensive heat treatment of camel milk is necessary to obtain the same degree of denaturation as that observed in bovine milk [22]. Heat treatment at more than 63 • C results in unfolding of the globular structure of whey proteins and they denature [30,31]. These non-native structures can then form aggregates with other unfolded monomers or aggregate with other types of protein molecules [32,33], forming co-aggregates [34], which is a characteristic feature of the Maillard reaction.

Proteins with an Increase in Abundance at 63 • C but a Decrease at 98 • C and RT
We found that three protein spots, relating to hemopexin spot, #413 (fold change of 1.258/−16.162 at 63/98 • C, respectively); lactate dehydrogenase, spot #856 (fold change of 1.923/−1.445 at 63/98 • C, respectively); and ADP-ribosylation factor GTPase-activating, spot #796 (fold change of 1.280/−2.509 at 63/98 • C, respectively), were found to be differentially regulated between the two heating temperatures. These spots increased in abundance at 63 • C but decreased significantly, or were undetectable at, 98 • C. When subjected to different temperatures at low pH, milk forms different types of gels, causing the whey proteins to partially unfold and adopt an intermediate state, depending on the degree of heating [35]. Hattem et al. showed that the highest level of denaturation occurs after heating 90 • C for 30 min, and the lowest at 63 • C for 30 min; the rate of protein denaturation is proportional to increases in temperature [36]. Moderate thermal treatment (60-70 • C) induces structural unfolding in milk proteins, whereas at higher temperatures, protein aggregation occurs [37]. Lactate dehydrogenase is part of the glycolytic pathway mediating the oxidative/reductive connection between pyruvate and lactic acid. Avallone et al. found that lactate dehydrogenase is heat stable and retains most of its activity up to 70 • C; it, then, loses its activity upon further heating. This agrees with the presence of the spots indicating lactate dehydrogenase, where we observed a band at 63 • C, which was absent at 98 • C [38]. The enzyme levels in buffalo milk are used as markers for pasteurization because of their sensitivity to heat inactivation [39]. Hemopexin is a plasma glycoprotein present in mature camel milk; it acts as a binding protein for iron and possesses antioxidant activity. In their proteomic study, Le et al. used ion exchange fractionation to identify hemopexin in the colostrum and mature bovine milk, with a higher abundance in the colostrum. The presence of this protein in camel milk signifies the important properties of camel milk [40].

Animals and Sample Collection
Camel milk was obtained from three different healthy camels from Saudi Arabia (cream, tan, and black subtypes of Camelus dromedarius), fed annually by grazing, from a local farm in the city of Al Majmaah, Saudi Arabia. Approximately 600 mL of milk, from each subtype of camel, was collected at 5:00 p.m. by milking into sterile cans and transported to the ORC (Obesity Research Center), College of Medicine, King Saud University proteomic lab in an ice box at 4 • C within 4 h of collection. Upon reaching the laboratory, the pH of the milk samples was determined to be 6.6 with a pH meter (PB11, Sartorius, Gottingen, Germany).

Sample Preparation
The milk samples from the three different subtypes of the same breed of camel were pooled together to produce a homogenous sample. The pooled sample was then triplicated to represent three independent biological samples for the application of heat treatment. The initial step, conducted on the samples, involved the removal of fat by centrifugation at 400× g and 4 • C for 30 min to obtain skim milk (Figure 3). hemopexin in the colostrum and mature bovine milk, with a higher abundance in the colostrum. The presence of this protein in camel milk signifies the important properties of camel milk [40].

Animals and Sample Collection
Camel milk was obtained from three different healthy camels from Saudi Arabia (cream, tan, and black subtypes of Camelus dromedarius), fed annually by grazing, from a local farm in the city of Al Majmaah, Saudi Arabia. Approximately 600 mL of milk, from each subtype of camel, was collected at 5:00 p.m. by milking into sterile cans and transported to the ORC (Obesity Research Center), College of Medicine, King Saud University proteomic lab in an ice box at 4 °C within 4 h of collection. Upon reaching the laboratory, the pH of the milk samples was determined to be 6.6 with a pH meter (PB11, Sartorius, Gottingen, Germany).

Sample Preparation
The milk samples from the three different subtypes of the same breed of camel were pooled together to produce a homogenous sample. The pooled sample was then triplicated to represent three independent biological samples for the application of heat treatment. The initial step, conducted on the samples, involved the removal of fat by centrifugation at 400× g and 4 °C for 30 min to obtain skim milk (Figure 3).

Heat Treatment
From the previously skimmed milk samples, one group of the samples, kept at room temperature, served as the control. The other two groups served as thermally treated samples that were heated at 63 and 98 • C for 1 h each, using a thermostatically controlled water bath (Shaking Water Bath SHWB10, Cole Parmer, San Diego, CA, USA) with rapidly circulated water. The treated and untreated samples were again triplicated for assessing reproducibility.

Whey Protein Extraction
Following the individual heat treatments, casein was precipitated out, as described previously, by adding 10% acetic acid (10% v/v), followed by 10% 1 M NaOAc, with gentle shaking; the samples were allowed to rest for 30 min at 35 • C between each addition. After acidification, the samples were centrifuged at 20,000× g for 30 min at 5 • C and the supernatants were collected [41]. The acidified whey proteins were then precipitated out using the methanol/chloroform (4:4:1; v:v:v) precipitation method at room temperature, and centrifuged at 5000× g for 5 min to remove the upper phase. Another three volumes of methanol were added to the supernatant, and the samples were again mixed and centrifuged at 16,000× g for 5 min to obtain the protein pellets, which were dried at room temperature in a vacuum concentrator (Concentrator Plus, Eppendorf, Hamburg, Germany) [42].

Protein Labeling with Cyanine Dyes
The protein pellets were solubilized in a labeling buffer (7 M urea, 2 M thiourea, 30 mM Tris-HCl, 4% CHAPS, pH 8.5). The insoluble material was pelleted by centrifugation at 12,000× g at room temperature for 5 min. Protein concentrations were determined in triplicate using the 2D-Quantkit (GE Healthcare, Piscataway, NJ, USA), and the pH of the samples was adjusted to 8.5 using NaOH (100 mM). The proteins were labeled using 400 pmol CyDye™ DIGE Fluor dyes (GE Healthcare, Buckinghamshire, UK) in 1 µL of dimethylformamide (DMF) and then mixed with a sample containing 50 µg of protein. Samples were incubated on ice for 30 min in the dark. The labeling reaction was terminated by adding 1 µL of 10 mM lysine. Each sample was covalently labeled with a Cy3 or Cy5 fluorophore. A mixture of equal amounts of protein, isolated from each sample, was labeled with Cy2 and used as an internal standard (see Table S2).

Two-Dimensional Electrophoresis, Image Scanning, and Preparative Gel
First dimension analytical gel electrophoresis was performed as follows. Five Immobiline Dry Strips (24 cm, pH 3-11; GE Healthcare, Uppsala, Sweden) were passively re-hydrated at 30 V for 12 h. This was followed by isoelectric focusing using an Ettan IPGphor IEF unit (GE Healthcare, Sweden). Focusing was performed at 20 • C, at 50 µA per strip, according to the following hold sequence: (1) 500 V for 3 h, (2) 1000 V for 3 h, (3) 8000 V for 4 h, and (4) 8000 V for 6.25 h totalling up to 50,000 Vhrs. IPG (immobilized pH gradient) strips were then stored at −80 • C until second-dimension separation. Before the second-dimension separation, the IPG strips were first equilibrated with dithiothreitol for 15 min at room temperature with gentle stirring, then with 5 mM Tris-HCl (pH 8.8), 6 M urea, 30% glycerol, 2% SDS, and 65 mM DTT. The strips were then equilibrated for an additional 15 min in the same solution containing 250 mM iodoacetamide. Polyacrylamide fixed gels (12.5%) were prepared on low fluorescence glass using a 2-D Optimizer (NextGen Sciences, London, UK). Next, we performed second-dimension separation using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE; Ettan DALT six vertical units (GE Healthcare, Uppsala, Sweden), at 15 • C, 1 W per gel, for 1 h, and then at 2 W per gel until the bromophenol blue dye front reached the bottom of the gel. Then, the gels were scanned at the appropriate individual excitation and emission wavelengths using the Typhoon Trio Imager fluorescence gel scanner (GE Healthcare) with the values of filters and photomultiplier optimized for Cy3, Cy5, and Cy2.

Colloidal Coomassie Blue Staining of the Preparative Gel
Total protein (1 mg) was obtained from a pool of equal protein amounts of the nine camel milk samples. This was denatured in a lysis buffer, then mixed in a rehydration buffer. Gels were fixed in 40% (v/v) ethanol with 10% (v/v) acetic acid (overnight) and then washed (3×, 30 min each, ddH 2 O). The gels were incubated (1 h, 34% (v/v) methanol containing 17% (w/v) ammonium sulphate and 3% (v/v) phosphoric acid) prior to the addition of 0.5 g/L Coomassie G-250. After five days the stained gels were briefly rinsed with Milli-Q water and stored until the spots could be picked and identified by MS.

Protein Identification by MALDI-TOF MS
The spots from Coomassie-stained gels were excised manually, washed, and digested according to a previously published protocol [43]. The mixture of tryptic peptides (1 µL), derived from each protein, was spotted onto a MALDI (Matrix-assisted laser desorption/ionization) target (384 anchorchip MTP 800 µm Anchorchip; Bruker Daltonics, Bremen, Germany) together with 0.8 µL of matrix (10 mg α-cyano-4-hydroxycinnamic acid (CHCA) in 1 µL of 30% CH 3 CN and 0.1% aqueous CF 3 COOH) and then left to dry (RT) before MS analysis. Spectra were acquired using a MALDI-TOF MS (UltraFlexTrem, Bruker Daltonics, Bremen, Germany) in the positive mode with target voltage of 25 kV and pulsed ion extraction voltage of 20 kV. The reflector voltage was set to 21 kV and detector voltage to 17 kV. Peptide mass fingerprints (PMF) were calibrated against a standard (Peptide Calibration Standard II, Bruker Daltonics). The PMF were processed using the Flex AnalysisTM software (version 2.4, Bruker Daltonics). The MS data were interpreted using BioTools v3.2 (Bruker Daltonics), together with the MASCOT search algorithm (version 2.0.04 updated 09/05/2015; Matrix Science Ltd., London, UK). MASCOT search parameters were set as follows: fixed propionamide modification of cysteine, oxidation of methionine as variable modification, one missed cleavage site (such as in the case of incomplete trypsin hydrolysis), and a mass tolerance of 100 ppm. Identified proteins were accepted as correct if they showed a MASCOT score greater than 56 and p < 0.05. Not all spots of interest could be identified because some proteins were low in abundance and did not yield a sufficiently intense mass of fingerprints; other spots were mixtures of multiple proteins.

Image Acquisition
DIGE images were analyzed using the Progenesis Same Spots v3.3 software (Nonlinear Dynamics Ltd., Newcastle, UK). First, images were aligned. Then an automatic vector tool, using prominent spots, was employed to detect 400 total vectors for warping and aligning the gel images with a reference image of one internal standard across and within each gel. Gel groups were designated according to the experimental design; normalized spot volume was used to select statistically significant spots. The Progenesis Same Spots v3.3 software was used to calculate the normalized volume (NV) of each spot, on each gel, for Cy3 and Cy5, to Cy2 spot volume ratio. The software performs log transformations of the spot volumes to generate normally distributed data. Log normalized volume (LNV) was used to quantify differential expression. Independent direct comparisons were made between differently heat-treated milk samples. Fold differences and p-values were calculated using one-way ANOVA. All spots were pre-filtered and manually examined before applying the statistical criteria (ANOVA test, p ≤ 0.05 and fold ≥1.2). Instead of spot intensities, normalized spot volumes were used for statistical analysis. Only those spots that fulfilled the abovementioned statistical criteria were submitted for MS analysis.

Conclusions
This study aimed to identify the total protein composition during the heat treatment of camel milk whey proteins at a medium temperature of 63 • C and a high temperature of 98 • C for 60 min. A total of 116 protein were detected as significantly changing using 2-DIGE and identified by MALDI-MS.
The obtained results showed that camel whey proteins were significantly affected by heat treatment at 98 • C and several proteins disappeared completely from the gel patterns; however, the whey proteins remain slightly stable under heat treatment at 63 • C for 60 min. This experimental study showed that denaturation increased significantly as the temperature increased from 63 to 98 • C. The fold change in the abundance of proteins identified between RT and 63 • C ranged from 15%-61% and for RT and 98 • C from 79%-98%. Further studies are needed to elucidate the mechanism involved in the heat denaturation of camel milk whey proteins and study the mechanisms in further detail.