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45AsPac J. Mol. Biol. Biotechnol. Vol. 19 (1), 2011 Proteomics of Human, Cow and Goat milkAsPac J. Mol. Biol. Biotechnol. 2011Vol. 19 (1) : 45 - 53

Proteomics of human and the domestic bovine and caprine milk

Eek-Poei Tay and Lay-Harn Gam*

School of Pharmaceutical Sciences, Universiti Sains Malaysia, Penang, Malaysia

Received 9th February 2011/ Accepted 17th March 2011

* Author for correspondence:Dr Lay-Harn Gam, School of Pharmaceutical Sciences, Universiti Sains Malaysia, 11800 USM, Penang, Malaysia. Tel: 604-6533888 ext 2208, Fax no: 604-6570017, E-mail: [email protected].

Abstract. The application of proteomics gives a unique opportunity for understanding of dairy protein science. In this study, we analysed the milk protein composition of domestic breeds of cows and goats and compared them with human milk. Although human milk is known to be superior over cow or goat milk for newborns, cow and goat milk are shown to be the most consumed types of milk. Upon analysis of protein from milk obtained from nursing mothers, cows and goats from local farms, we found that the ruminants’ milk has extensive similarity in protein content, consisting of casein proteins (αs1-casein, αs2-casein, -casein and K- casein), -lactoglobulin and α-lactalbumin. Nevertheless, the quantities of these proteins in the milk differed. Human milk, on the other hand, contained more types of proteins, mostly made up of whey proteins that were absent in the animals’ milk. The proteins identified in human milk included -casein and the whey proteins comprised of α-lactalbumin, carboxyl ester lipase, serum albumin, immunoglobulin, lysozyme, lactoferrin and mannose receptor. In term of total protein content, goat milk contained the highest amount of protein per volume of milk followed by cow milk and human milk. Human milk’s casein to whey ratio was 30:70 compared to 85:15 in cow milk and 80:20 in goat milk.

Keywords: Cow milk, Goat milk, Human milk, Proteomics.

INTRODUCTION

Milk is a nutritious product secreted by the mammary gland. It comprises a complex biological fluid containing a high amount of proteins, lipids and minerals. Milk serves an important role in the human daily diet, particularly in the provision of balanced nutrients to the young. Milk from all species is dominated by the presence of a few primary proteins (O’Donnell et al., 2004) that contribute to the nutritional value of milk-originated products for consump-tion. The nutritional value of milk varies and depends on the composition of milk, which in turn relies on several factors such as the stage of lactation and the nutritional status of the lactating mother (Lonnerdal, 2003).

Human milk contains a wide variety of proteins that contribute to its unique quality. These include the caseins ( -casein, K- casein, αs1-casein) and whey proteins such as α-lactalbumin, lactoferrin, immunoglobulins (sIgA, IgM, IgG), serum albumin, and lysozyme (de Wit, 1998; Ha-mosh, 2002; Lonnerdal, 2003). Other minor forms of milk proteins, including bile salt-stimulated lipase, amy-lase, α1-antitrypsin, insulin-like growth factor-binding pro-teins, folate binding protein, haptocorrin, and lactoperoxi-dase have also been reported to be present in human milk (Lonnerdal, 2003).

Approximately 3.0 - 3.5% of ordinary bovine milk is made up of protein (Phadungath, 2005). The caseins present in cow milk include all the four subtype, namely αs1-

casein, αs2-casein, -casein, and K-casein (Swaisgood, 1992). -lactoglobulin and α-lactalbumin are the two main whey

proteins present in cow milk, the rest being immunoglobu-lins, bovine serum albumin, and trace amount of enzymes and proteins with specific metabolic functions e.g. lactofer-rin and lysozyme (Brunner, 1977; Andrews, 1992; de Wit, 1998; Walstra, 1999; Lindmark-Mansson et al., 2005).

The major milk proteins detected in goat milk and cow milk are similar in term of general classifications of caseins (αs1-casein, αs2-casein, -casein, K-casein) and α-lactalbumin (Jenness, 1980; Martin, 1993; Kim and Jimenez-Flores, 1994; Grousclaude, 1995; Haenlein, 2004). Nevertheless, many unique differences between the two species have also been documented, where goat milk was reported to contain higher protein content than cow milk; besides, in many cir-cumstances goat milk is used as an alternative milk product for those who suffer from cow milk allergy (Jenness, 1980; Haenlein, 1996; 2001).

In Malaysia, there is little information on the milk pro-tein composition of domestic breeds of cows and goats. Therefore, the aim of this study was to analyse the protein profiles of domestic breeds of cow and goat milk and com-pare them with human milk; the data obtained will be useful in understanding the nutritional value of milk from domes-

46 AsPac J. Mol. Biol. Biotechnol. Vol. 19 (1), 2011 Proteomics of Human, Cow and Goat milk

tic ruminant species.

MATERIALS AND METHODS

Sample Preparation. Milk samples collected were of ma-ture milk, not colostrums. Breast milk samples were collect-ed from 3 Malay donors who had delivered their babies two months earlier. Three samplings each of cow (local hybird) and goat (local hybrid) milk were purchased fresh from the local farms on three different occasions. Obtained milk sam-ples were pasteurised at 63oC for 30 minutes. The pasteur-ised milk samples were then centrifuged at 3000 rpm (20oC) for 15 minutes to remove milk fat which appeared at the up-per phase as white cream. The skimmed milk was aliquoted and kept at 4oC for immediate use, and then deposited at -70oC for long term storage.

Sodium Dodecyl Sulphate-Polyacrylamide Gel Electro-phoresis (SDS-PAGE). The discontinuous gel system used was as described by Laemmli et al. (1970). The gel consisted of 12% resolving gel and 4% stacking gel. The gel dimen-sion was 16x20 cm. Ten microliters each of homogenous skimmed milk samples were mixed with 5 μL sample buffers (0.5M Tris-HCl pH6.7, 20% (v/v) glycerol, 10% (w/v) SDS and 0.5% (w/v) Bromophenol Blue) and gently vortexed prior to being loaded into wells on a polyacrylamide gel. Electrophoresis was carried out at a constant voltage of 200 V throughout the 3 hour separation. The gel was Coomassie blue stained and an image was captured using Versa doc Im-aging System Model 4000 (BioRad). Subsequently, analysis of gel images, including molecular weight determination and band intensity analysis, were performed using Quantity One Analysis Software (BioRad, version 4.5.1).

In-Gel Digestion. In-gel digestion was performed accord-ing to Gam and Aishah (2001). In summary, the proteins in the gel were reduced, alkylated and digested using trypsin. The peptides were eluted from the gel and subjected to fur-ther analysis using a mass spectrometry method.

High Performance Liquid Chromatography/Mass Spec-trometry (HPLC/MS). The peptides were suspended in 15 μL of distilled water, then vortexed briefly followed by centrifugation at 500xg (20°C) for 5 min. 5 μL of the super-natant was injected into a RP-column (5 μm particle, 300Å, 250 mm x 1 mm) running at 5% - 90% B gradient for 55 minutes with flow rate of 15 μL/min. Mobile phase A was 0.05% formic acid in distilled water and mobile phase B was 0.05% formic acid in acetonitrile. The HPLC was in-terfaced to a mass spectrometer (Agilent Technologies). A data dependent mass spectrometric analysis method was cre-ated. The parameters for mass spectrometer (MS) analysis using Electrospray Ionization Mode (ESI) were as follows: nebulizer at 25 psi; dry gas at 8 L/min; dry temperature at 300oC; scan range 200-1800 m/z with positive polarity.

The parameters for MS/MS analysis were: collision energy (voltage) 1.10 V; charge state 2; minimum threshold 2000 counts; and the isolation width 2 m/z. The spectrometry data were collected and analysed using LC/MSD Trap soft-ware (Agilent Technologies, version 4.2).

Protein Identification. Data from all the MS/MS scans was used for elucidation of protein identities via the Mascot protein database search engine. Searches was conducted us-ing an MS/MS ion search, whereby the trypsin enzyme and fixed modification site of carboxymethyl (C) were selected. A maximum of one missed cleavage was allowed. Protein identification was based on standard Mascot criteria for sta-tistical analysis of MS/MS data.

RESULTS AND DISCUSSION

A good separation of protein mixture using SDS-PAGE should produce bands where each containing a single type of protein. However, good protein separation by SDS-PAGE is rarely possible due to the limitation of gel resolution. This limitation often results in co-migration of proteins of simi-lar molecular weights (Izani et al., 2009). In this study, a number of gels were run to determine the profile of milk proteins; a consistent protein profile was obtained from each type of milk, as represented in Figure 1 for human, cow and goat milk, respectively. All the protein bands were subjected to in-gel digestion and the protein identities listed in Table 1, 2 and 3 for human, cow and goat milk, respectively. Pro-tein band 1 for human milk is an example of unresolved pro-teins separation in SDS-PAGE, where two immunoglobulin subtypes, α1 and α2, a hypothetical protein and a secretary component were detected in this band. However, the limita-tion of gel resolution was compensated for by using tandem mass spectrometry (LC/MS/MS) analysis, which can iden-tify all the proteins in a single band. The relative intensity of each protein in a single band was further quantified using peptide extracted-ion chromatograms for the proteins. Fig-ure 2 shows the peak intensity of each type of milk.

Tables 1, 2 and 3 also show information on the trace intensity and relative quantity of each protein bands in hu-man milk, cow milk and goat milk, respectively. The trace intensity is defined as the intensity of a band as measured by the area under its intensity profile curve. On the other hand, the relative quantity represents the intensity of a protein band as measured by its intensity, expressed as a percentage to the total intensity of all protein bands in the same lane i.e. for each milk type. Trace intensity and relative quantity can be used to estimate the amount of each protein type in the milk. Since identical volumes of homogenous skimmed milk samples were loaded on the gel, the protein content of different types of milk could be compared and analyzed simultaneously.

In this study, we have found that human milk contained approximately 30% casein ( -casein) and 70% whey pro-

47AsPac J. Mol. Biol. Biotechnol. Vol. 19 (1), 2011 Proteomics of Human, Cow and Goat milk

Figure 1. SDS-PAGE profiles of proteins from non-reduced hu-man milk, cow milk and goat milk. Protein marker with known molecular weights was used. The gel was stained using Coomassie blue following separation on 12% SDS-PAGE gel.

teins, which comprised of α-lactalbumin (~24%), carboxyl ester lipase (~15%), serum albumin (~13%), immunoglob-ulin (~8%), lysozyme (~8%), and lactoferrin (~1%) and mannose receptor (~1%) (Table 4). de Wit (1998) reported a ratio of 40:60 for caseins to whey proteins in human milk. This frequently cited ratio of 40:60 serves as an approxima-tion of the ratio during the normal course of lactation. Nev-ertheless, the ratio of whey to casein in human milk varies throughout lactation (Lonnerdal, 2003). Although human milk has been revealed to be completely different from those of ruminant species (Kim and Jimenez-Flores, 1994; Gold-farb, 1999), ruminant milk has been widely consumed as infant formula. Interest has grown in the past few decades to improve the nutritional and biological performance of animal milk to increase similarity to human milk in edible products such as infant formula.

The ratio of caseins to whey proteins in domestic breed cow milk was seen to be approximately 85:15 compared to the 80:20 found by de Wit (1998). The four major casein components, i.e. αs1-casein, αs2-casein, -casein and K-ca-sein, were detected with αs1-casein and αs2-casein being the dominant proteins. Other proteins detected were two major whey proteins, -lactoglobulin (~11%) and α-lactalbumin (< 4%).

The ratio of caseins to whey proteins in domestic breed goat milk was approximately 80:20 compared to the 80:15

Figure 2. Representation of protein bands intensity profiles derived from SDS-PAGE profile for (a) human milk; (b) cow milk; (c) goat milk as in Figure 1. Each peak refers to a protein band. The y-axis represents the intensity of protein bands; the x-axis represents the Rf of protein bands. The number for each peaks correspond to the number for each protein band as in Figure 1.

of Miranda et al. (2004). Domestic breed goat milk con-tained all the four caseins subtypes, i.e. αs1-casein, αs2-casein,

-casein, and K-casein. Besides, an approximation of 16% -lactoglobulin and less than 7% α-lactalbumin were also

found. When comparing the three types of milk, the ratio of

caseins to whey proteins of cow milk and goat milk was rela-tively similar (85:15 for domestic breed cow milk and 80:20 for domestic breed goat milk) despite the differences in their protein profiles. On the other hand, human milk showed a distinctly different protein profiles, where a ratio of 30:70 caseins to whey proteins was identified, a ratio that was re-verse to those of the animals’ milk. This ratio was relatively similar to that of de Wit (1998), who noted a ratio of 40:60.

The total intensity of protein bands per volume of milk in human, domestic breeds cow and goat milk were 25953, 40664 and 59702 respectively, which give rise to a total pro-tein ratio of 1:1.6:2.3 for human to cow to goat milk. This ratio indicated that domestic breed goat milk contained the highest protein content per volume of milk compared to domestic breed cow milk and human breast milk. Our find-ing is similar to that of Miranda et al. (2004), where goat milk was found to contain the highest amount of protein followed by cow and human milk.

The fundamental data from the mass spectrometry ex-periment is shown in Figure 3. Figure 3a shows the base peak chromatogram (BPC) that was extracted from the total ion chromatogram (TIC). The peaks represent the signal in-


Figure 3. Mass spectrometric analysis and protein identifica-tion of protein band no. 5 from cow milk (C5). (a) Base peak chromatogram of tryptic peptides from C5; (b) Full scan MS view of peptide at retention time 37.6; (c) MS/MS mass spectra of pre-cursor ion m/z 685.0; (d) peptide sequence derived from data in (c). The protein identified is alpha-s2 casein precursor (bovine).

tensity of the most abundant ions eluted form HPLC at each retention time. The most intense peptide ion in a MS scan (Figure 3b) was isolated and excited to MS/MS scan (Figure 3c). The collision of the peptide ion with the argon gas in the ion trap resulted in its fragmentation to produce a series of fragment ions as shown in the MS/MS scan. The MS/MS scan contained information about the peptide sequence, which was used as the basis for protein identification. The MS/MS data was subjected to the Mascot protein data-base search engine where matching of the experimental and theoretical fragment ion masses was carried out for protein identification. A match was presented in the form of score number, a score greater than 41 shows extensive homology or significance at p<0.05 confident level. In this study, all the proteins had scores greater than the significance value. In addition, manual inspection of MS/MS data was carried out to ensure the reliability of the data. Mass spectromet-ric analysis was used to determine the identity of proteins

found in the milk. Human milk obviously contained moretypes of protein than the animals’ milk. Table 4 summarises the types of protein found in human milk, domestic breed cow and goat milk. Figure 4 shows a diagrammatic repre-sentation of the proteins found in each protein band. In this study, apart from the common milk proteins i.e. caseins and α-lactalbumin, other proteins found exclusively in hu-man milk were carboxyl ester lipase, serum albumin, immu-noglobulin, lysozyme, and lactoferrin and mannose recep-tor. Although bovine milk was reported to contain bovine serum albumin, immunoglobulin, lactoferrin and lysozyme (de Wit 1998), we did not detect any of these proteins in the domestic breed cow milk (Table 2).

The major difference between human milk and ruminant milk was the absence of -lactoglobulin in human milk. -lactoglobulin was suspected to be the major whey protein in cow milk, the protein group which has been said to be responsible for cow milk allergies (Park, 1994). Cow milk-based formula, when used as a substitute for breast-feeding, induces allergy in 2 to 3% of infants (Host, 1997).

Goat milk has been recommended as a substitute for patients allergic to cow milk. Park (1994) reported that be-tween 40 and 100% of patients allergic to cow milk proteins could tolerate goat milk. Although some goat milk proteins have immunological cross-reactivity with cow milk proteins, infants suffering from gastrointestinal allergy and chronic

(a)

(b)

(c)

(d)


Table 1. Protein information, trace intensity and relative quantity of protein bands in human milk. The band numbering corresponds to band numbering in Figure 1.

Band no. Rf Molecular Weight Trace Intensity Relative Quantity Proteins

1 0.008 472.705 2002.986 7.718

Ig α1

Ig α2Hypothetical protein (Ig-like)

Secretory component

2 0.038 316.327 146.605 0.565

Ig α1

Ig α2

Ig γHypothetical protein (Ig-like)

Hypothetical protein (similar to Ig, IgK)

CDNA (highly similar to Human (hybridoma H210) anti-hepatitis A

IgG)

3 0.06 232.926 103.715 0.4

Ig α2Ig YIg Y1Ig Y2Ig

Hypothetical protein (Ig-like)Hypothetical protein ( involved in

spermatogenesis)Carboxyl ester lipase

Factor VII active site mutant im-munoconjugate

4 0.084 166.663 267.071 1.029 Mannose receptor

5 0.113 124.589 682.828 2.631 Carboxyl ester lipase

6 0.188 79.713 71.044 0.274 Lactoferrin

7 0.198 76.251 224.033 0.863 Lactoferrin

8 0.221 69.566 3333.56 12.845 Serum albumin

9 0.293 53.153 3294.521 12.694 -CaseinCarboxyl ester lipase

10 0.367 42.679 31.281 0.121 -Casein

11 0.439 35.431 14.801 0.057 -Casein

12 0.492 31.84 117.48 0.453 -Casein

13 0.514 30.421 2738.05 10.55 -Casein

14 0.531 29.356 61.393 0.237 -Casein

15 0.545 28.572 44.659 0.172 -Casein

16 0.58 26.569 3110.35 11.985 -Casein

17 0.621 24.305 372.045 1.434 -Casein

18 0.669 21.399 213.165 0.821 -Casein

19 0.756 18.229 57.444 0.221 -Casein

20 0.789 17.351 130.808 0.504 -Casein

21 0.802 17.027 73.331 0.283 -Casein

22 0.816 16.673 496.764 1.914 -Casein

23 0.916 14.349 2277.231 8.774 -CaseinLysozyme

24 0.981 13.001 6087.876 23.457 α-Lactalbumin

Total intensity 25953.041


Table 2. Protein information, trace intensity and relative quantity of protein bands in cow milk. The band numbering corresponds to band numbering in Figure 1.


1 0.004 505.3 138.204 0.34 -Lactoglobulin AK-Casein

2 0.025 378.651 971.57 2.389K-Caseinαs2-Caseinαs1-Casein

3 0.038 315.41 1239.225 3.047K-Caseinαs2-Casein


5 0.198 76.109 3876.201 9.532 αs2-Casein

6 0.228 67.803 2148.48 5.283 αs2-Casein

7 0.282 55.53 825.796 2.031 αs1-Casein

8 0.497 31.48 9373.996 23.052αs2-Caseinαs1-Casein


-Casein

10 0.614 24.761 1438.436 3.537 αs1-Casein-Casein

11 0.677 20.953 604.988 1.488 αs1-Casein-Casein

12 0.688 20.34 666.052 1.638 αs1-Casein-Casein

13 0.734 18.854 369.14 0.908 -Casein

14 0.749 18.441 128.71 0.317 -Casein

15 0.805 16.93 424.865 1.045 -Lactoglobulin A

16 0.853 15.774 4355.029 10.71 -Lactoglobulin A

17 0.945 13.722 674.74 1.659 -CaseinK-Casein

18 0.963 13.365 1509.537 3.712 α-Lactalbumin-Casein

19 0.975 13.114 1212.124 2.981 -Caseinαs2-Casein



Table 3. Protein information, trace intensity and relative quantity of protein bands in goat milk. The band numbering corresponds to band numbering in Figure 1.



2 0.048 275.675 1155.083 1.935 K-Casein

3 0.06 231.853 1150.171 1.927 K-Casein




7 0.184 81.123 213.609 0.358 αs2-Casein

8 0.209 72.979 1578.179 2.643 K-Casein

9 0.275 56.859 2617.049 4.383 K-Casein

10 0.291 53.525 1374.426 2.302 K-Casein

11 0.307 50.519 210.367 0.352 K-Casein

12 0.351 44.557 56.317 0.094 K-Casein

13 0.381 41.051 131.911 0.221 K-Casein

14 0.406 38.267 38.2 0.064 K-Casein

15 0.478 32.722 4473.142 7.492K-Caseinαs2-Caseinαs1-Casein


17 0.614 24.761 1046.365 1.753αs2-CaseinK-Casein

Lactophorin

18 0.622 24.216 960.216 1.608 -Caseinαs2-Casein

19 0.666 21.544 955.419 1.6 -Caseinαs2-Casein

20 0.679 20.837 1482.666 2.483 -Caseinαs2-Casein

21 0.709 19.563 207.101 0.347 -Caseinαs2-Casein

22 0.747 18.48 265.294 0.444 -Caseinαs2-Casein

23 0.764 18.017 642.489 1.076 -Casein

24 0.813 16.752 88.733 0.149 αs1-Casein

25 0.888 14.963 9958.219 16.68 -Lactoglobulin

26 0.958 13.464 4566.787 7.649-Casein

αs2-Caseinα-Lactalbumin

27 0.976 13.1 604.618 1.013 -Casein



Figure 4. Diagrammatic SDS-PAGE profiles of proteins from (left panel) human milk; (centre panel) cow milk; (right panel) goat milk. Each horizontal line represents a protein band. The types of protein identified in each band are labeled.

Table 4. Types of milk protein identified in human milk, cow milk and goat milk.

Protein type Human milk

Cow milk

Goat milk

Caseins:

αs2-Casein - + +αs1-Casein - + +

beta-Casein + + +K-Casein - + +

beta-Lactoglobulin - + +α-Lactalbumin + + +

Immunoglobulin(including hypothetical protein) + - -

Lactoferrin + - +Serum albumin + - -

Lysozyme + - -Carboxyl ester lipase + - -Mannose receptor + - -

+: protein present; -: protein absent.

enteropathy as a result of cow milk were reportedly cured by goat milk therapy (Park, 1994). In this study, domes-tic breed goat milk was found to contain a relatively high amount of -lactoglobulin, which indicates that domestic breed goat milk may possibly induce cow milk allergy.

In addition, a study conducted by Bellioni-Businco et al. (1999) on the allergenicity of goat milk in children with a cow milk allergy indicated that goat milk is not an appropri-ate cow milk substitute for children with IgE-mediated cow milk allergenicity, where the allergen is αs1-casein. As shown in this study, both cow milk and goat milk contained αs1-casein. However, αs1-casein, αs2-casein and K- casein were not detected in human milk.

CONCLUSION

Human milk was shown to be unique, with the presence of a number of bioactive factors. On the other hand, there was extensive similarity between the protein content of domes-tic breed cow and goat milk. In addition, -lactoglobulin, which has been said to cause cow milk allergies, was detected in large quantity in the domestic breed goat milk, suggesting that domestic breed goat milk may induce cow milk-like al-lergy. Future work to validate the amount of protein present in the different milk via methods such as Western blotting or ELISA will be carried out.


ACKNOWLEDGEMENTS

We would like to thank Institute of Neutraceutical and Pharmaceutical Malaysia for providing mass spectrometer for LC/MS/MS analysis.

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