Proteomics of human and the domestic bovine and caprine milk

Transcription

1 AsPac J. Mol. Biol. Biotechnol Vol. 19 (1), 2011 Vol. 19 (1) : 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 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 ( -casein, -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 consumption. 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, -casein) and whey proteins such as α-lactalbumin, lactoferrin, immunoglobulins (siga, IgM, IgG), serum albumin, and lysozyme (de Wit, 1998; Hamosh, 2002; Lonnerdal, 2003). Other minor forms of milk proteins, including bile salt-stimulated lipase, amylase, α 1 -antitrypsin, insulin-like growth factor-binding proteins, folate binding protein, haptocorrin, and lactoperoxidase have also been reported to be present in human milk (Lonnerdal, 2003). Approximately % of ordinary bovine milk is made up of protein (Phadungath, 2005). The caseins present in cow milk include all the four subtype, namely - casein, -casein, -casein, and K-casein (Swaisgood, 1992). -lactoglobulin and α-lactalbumin are the two main whey proteins present in cow milk, the rest being immunoglobulins, bovine serum albumin, and trace amount of enzymes and proteins with specific metabolic functions e.g. lactoferrin 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 ( -casein, -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 circumstances 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 protein 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 compare them with human milk; the data obtained will be useful in understanding the nutritional value of milk from domes- * Author for correspondence: Dr Lay-Harn Gam, School of Pharmaceutical Sciences, Universiti Sains Malaysia, USM, Penang, Malaysia. Tel: ext 2208, Fax no: , layharn@usm.my.

2 46 AsPac J. Mol. Biol. Biotechnol. Vol. 19 (1), 2011 tic ruminant species. MATERIALS AND METHODS Sample Preparation. Milk samples collected were of mature milk, not colostrums. Breast milk samples were collected 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 samples were pasteurised at 63 o C for 30 minutes. The pasteurised milk samples were then centrifuged at 3000 rpm (20 o C) for 15 minutes to remove milk fat which appeared at the upper phase as white cream. The skimmed milk was aliquoted and kept at 4 o C for immediate use, and then deposited at -70 o C for long term storage. Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis (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 dimension 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 Imaging 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 according 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 further analysis using a mass spectrometry method. High Performance Liquid Chromatography/Mass Spectrometry (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 supernatant 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 interfaced to a mass spectrometer (Agilent Technologies). A data dependent mass spectrometric analysis method was created. 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 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 software (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 using 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 statistical 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 similar 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. Protein band 1 for human milk is an example of unresolved proteins separation in SDS-PAGE, where two immunoglobulin subtypes, α 1 and α 2, a hypothetical protein and a secretary component were detected in this band. However, the limitation of gel resolution was compensated for by using tandem mass spectrometry (LC/MS/MS) analysis, which can identify 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. Figure 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 human 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-

3 AsPac J. Mol. Biol. Biotechnol. Vol. 19 (1), Figure 1. SDS-PAGE profiles of proteins from non-reduced human 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%), immunoglobulin (~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 approximation of the ratio during the normal course of lactation. Nevertheless, 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; Goldfarb, 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. -casein, -casein, -casein and K-casein, were detected with -casein and -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 contained all the four caseins subtypes, i.e. -casein, -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 relatively 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 reverse 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, and respectively, which give rise to a total protein 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 finding 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 experiment 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-

4 48 AsPac J. Mol. Biol. Biotechnol. Vol. 19 (1), 2011 (a) (b) (c) (d) Figure 3. Mass spectrometric analysis and protein identification 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 precursor 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 database 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 spectrometric analysis was used to determine the identity of proteins found in the milk. Human milk obviously contained more types 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 representation 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 human milk were carboxyl ester lipase, serum albumin, immunoglobulin, lysozyme, and lactoferrin and mannose receptor. 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 milkbased 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 between 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

5 AsPac J. Mol. Biol. Biotechnol. Vol. 19 (1), 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. R f Molecular Weight Trace Intensity Relative Quantity Proteins Ig α 1 Ig α 2 Hypothetical protein (Ig-like) Secretory component Ig α 1 Ig α 2 Ig γ Hypothetical protein (Ig-like) Hypothetical protein (similar to Ig, IgK) CDNA (highly similar to Human (hybridoma H210) anti-hepatitis A IgG) Ig α 2 Ig Y Ig Y 1 Ig Y 2 Ig Hypothetical protein (Ig-like) Hypothetical protein ( involved in spermatogenesis) Carboxyl ester lipase Factor VII active site mutant immunoconjugate Mannose receptor Carboxyl ester lipase Lactoferrin Lactoferrin Serum albumin Carboxyl ester lipase Lysozyme α-lactalbumin Total intensity

6 50 AsPac J. Mol. Biol. Biotechnol. Vol. 19 (1), 2011 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. Band no. R f Molecular Weight Trace Intensity Relative Quantity Proteins Lactoglobulin A K K K K Lactoglobulin A Lactoglobulin A Total intensity K α-lactalbumin

7 AsPac J. Mol. Biol. Biotechnol. Vol. 19 (1), 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. Band no. R f Molecular Weight Trace Intensity Relative Quantity Proteins K K K K K K K K K K K K K K αs1 K Lactophorin Lactoglobulin α-lactalbumin Total intensity

8 52 AsPac J. Mol. Biol. Biotechnol. Vol. 19 (1), 2011 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. Caseins: Protein type Human milk Cow milk Goat milk beta K 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, domestic 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 appropriate cow milk substitute for children with IgE-mediated cow milk allergenicity, where the allergen is -casein. As shown in this study, both cow milk and goat milk contained - casein. However, -casein, -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 domestic 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 allergy. 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.

9 AsPac J. Mol. Biol. Biotechnol. Vol. 19 (1), ACKNOWLEDGEMENTS We would like to thank Institute of Neutraceutical and Pharmaceutical Malaysia for providing mass spectrometer for LC/MS/MS analysis. REFERENCES Andrews, A.T Indigenous enzymes in milk: General introduction. In Advanced dairy chemistry 1: proteins, ed. P. F. Fox, pp Elsevier Science Publisher Ltd. Bellioni-Businco, B., Paganelli, R., Lucenti, P., Giampietro, P.G., Perborn, H. and Businco, L Allergenicity of goat s milk in children with cow s milk allergy. Journal of Allergy and Clinical Immunology 103: Brunner, J.R Milk proteins. In Food proteins, ed. J.R. Whitaker and S.R. Tannenbaum, pp Connecticut: AVI Publishing Company Inc. de Wit, J.N Nutritional and functional characteristics of whey proteins in food products. Journal of Dairy Science 81: Gam, L.H. and Aishah, L Identification and confirmation of urinary human chorionic gonadotrophin using in-gel digestion and HPLC/MS/MS analysis. Journal of Biosciences 13: Goldfarb, M Two-dimensional electrophoresis and computer imaging: quantitation of human milk casein. Electrophoresis 20: Grosclaude, F Genetic polymorphisms of milk proteins. In Proceedings of the IDF seminar on implications of genetic polymorphism of milk proteins on production and processing of milk, Zurich, Switzerland. pp Brussels: International Dairy Federation Publications. Haenlein, G.F.W Nutritional value of dairy products of ewe and goat milk. In Proceedings of the IDF/ CIRVAL seminar production and utilization of ewe and goat milk. pp Brussels: International Dairy Federation Publications. Haenlein, G.F.W Past, present, and future perspectives of small ruminant research. Journal of Dairy Science 84: Haenlein, G.F.W Goat milk in human nutrition. Small Ruminant Research 51: Hamosh, M Bioactive components in human milk more than just nutrition. Pediatric Basics the Journal of Pediatric Nutrition and Development 99: Høst, A Development of atopy in childhood. Allergy 52: Kim, H.H.Y. and Jimenez-Flores, R Comparison of milk proteins using preparative isoelectric focusing followed by polyacrylamide gel electrophoresis. Journal of Dairy Science 77: Izani, O., Majid, M.I.A., Manjit, S., Gooi, Subatra, S., Seng, L. and Gam, L.H Proteomics of grade three infiltrating ductal carcinoma in Malaysian chinese breast cancer patients. Biotechnology and Applied Biochemistry 52: Laemmli, U.K Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: Lindmark-Mansson, H., Timgren, A., Alden, G. and Paulsson, M Two-dimensional gel electrophoresis of proteins and peptides in bovine milk. International Dairy Journal 15: Lonnerdal, B Nutritional and physiologic significance of human milk proteins. American Journal of Clinical Nutrition 77: 1537s-1543s. Martin, P Polymorphisme genetique des lactoproteines caprines. Lait 73: Miranda, G., Mahé, M.F., Leroux, C. and Martin, P Proteomic tools to characterize the protein fraction of Equidae milk. Proteomics 4: O Donnell, R., Holland, J.W., Deeth, H.C. and Alewood, P Milk proteomics. International Dairy Journal 14: Park, Y.W Hypo-allergenic and therapeutic significance of goat milk. Small Ruminant Research 14: Phadungath, C Casein micelle structure: a concise review. Songklanakarin Journal of Science and Technology 27: Swaisgood, H. E Chemistry of the caseins. In Advanced dairy chemistry 1: proteins, ed. P. F. Fox, pp Elsevier Science Publisher Ltd. Walstra, P Casein sub-micelles: do they exist? International Dairy Journal 9: