Hostname: page-component-78c5997874-94fs2 Total loading time: 0 Render date: 2024-11-19T13:37:19.223Z Has data issue: false hasContentIssue false
  • English
  • Français

Comparative proteomic analysis of casein and whey as prepared by chymosin-induced separation, isoelectric precipitation or ultracentrifugation

Published online by Cambridge University Press:  24 September 2012

Hanne B. Jensen ,
Nina A. Poulsen ,
Hanne S. Møller ,
Allan Stensballe  and
Lotte B. Larsen
Hanne B. Jensen
Affiliation:
Department of Food Science, Aarhus University, DK-8830 Tjele, Denmark
Nina A. Poulsen
Affiliation:
Department of Food Science, Aarhus University, DK-8830 Tjele, Denmark
Hanne S. Møller
Affiliation:
Department of Food Science, Aarhus University, DK-8830 Tjele, Denmark
Allan Stensballe
Affiliation:
Section of Biotechnology, University of Aalborg, DK-9000 Aalborg, Denmark
Lotte B. Larsen*
Affiliation:
Department of Food Science, Aarhus University, DK-8830 Tjele, Denmark
*
*For correspondence; e-mail: [email protected]
Get access Rights & Permissions [Opens in a new window]

Abstract

Fractionation of bovine milk was performed using chymosin-induced separation, isoelectric precipitation or ultracentrifugation as separation techniques prior to gel-based proteomic analysis. This approach allowed for comparative display and identification of proteins partitioned into casein and whey, respectively. Initially, three different staining methods (silver staining, colloidal Coomassie Blue G-250 or fluorescent Flamingo Pink staining) for two-dimensional gel electrophoresis (2-DGE) analysis were compared for their suitability as staining agent, especially in relation to their suitability to reveal differences in the casein fractions. Fluorescent staining proved to be the most appropriate for this purpose, giving a high sensitivity, and using this staining method, characteristic 2-DGE fingerprints were obtained for each casein and whey fraction from each separation method. A number of protein spots in both casein and whey fractions varied with separation method and these spots were subsequently identified using tandem mass spectrometry (MS). In rennet casein, proteolytic fragmentation of caseins (αs1-, αs2,-, β- and κ-) was identified as a result of chymosin hydrolysis, whereas the 2-DGE profile of acid and ultracentrifuged casein was dominated by the presence of multiple isoforms of κ-caseins. Furthermore, casein remnants were identified in milk serum after ultracentrifugation. This study shows that gel-based proteomic analysis is suitable for characterisation of subtle variations in protein composition of milk fractions that occur as a consequence of different milk fractionation strategies.

Keywords

Milk separation techniquegel electrophoresismass spectrometry
Type
Research Article
Information
Journal of Dairy Research , Volume 79 , Issue 4 , November 2012 , pp. 451 - 458
Copyright
Copyright © Proprietors of Journal of Dairy Research 2012

Access options

Get access to the full version of this content by using one of the access options below. (Log in options will check for institutional or personal access. Content may require purchase if you do not have access.)

References

Chevalier, F, Hirtz, C, Sommerer, N & Kelley, AL 2009 Use of reducing/nonreducing two-dimensional electrophoresis for the study of disulfide-mediated interactions between proteins in raw and heated bovine milk. Journal of Agricultural and Food Chemistry 57 5948–5955CrossRefGoogle Scholar
Davies, DT & Law, AJR 1983 Variation in the protein-composition of bovine casein micelles and serum casein in relation to micellar size and milk temperature. Journal of Dairy Research 50 67–75Google Scholar
Fong, BY, Norris, CS & Palmano, KP 2008 Fractionation of bovine whey proteins and characterisation by proteomic techniques. International Dairy Journal 18 23–46Google Scholar
Fox, PF 2003 Milk proteins: general and historical aspects. In Advanced Dairy Chemistry, Vol. 1, part A, 3rd edition, pp. 1–41 (Eds Fox, PF & McSweeney, PLH). New York: Kluwer Academic/Plenum Publishers.Google Scholar
Griffin, CA 1988 The disaggregation of calcium-depleted casein micelles. European Journal of Biochemistry 174 339–343Google Scholar
Grove, H, Hollung, K, Uhlen, A, Martens, H & Færgestad, EM 2006 Challenges related to analysis of protein spot volume from two-dimensional gel electrophoresis as revealed by replicate gels. Journal of Proteome Research 5 3399–3410Google Scholar
Guillou, H, Miranda, G & Pelissier, JP 1991 Hydrolysis of beta-casein by gastric proteases. I. Comparison of proteolytic action of bovine chymosin and pepsin A. International Journal of Peptide and Protein Research 37 494–501CrossRefGoogle ScholarPubMed
Harris, LR, Churchward, MA, Butt, RH & Coorssen, JR 2007 Assessing detection methods for gel based proteomic analyses. Journal of Proteome Research 6 1418–1425Google Scholar
Holland, JW, Deeth, HC & Alewood, PF 2004 Proteomic analysis of κ-casein micro-heterogeneity. Proteomics 4 743–752Google Scholar
Holland, JW, Deeth, HC & Alewood, PF 2006 Resolution and characterisation of multiple isoforms of bovine κ-casein by 2-DE following a reversible cysteine-tagging enrichment strategy. Proteomics 6 3087–3095Google Scholar
Holt, C, Davies, DT & Law, AJR 1986 Effects of colloidal calcium phosphate content and free calcium ion concentration in the milk serum on the dissociation of bovine casein micelles. Journal of Dairy Research 53 557–572Google Scholar
Hyslop, DB 2003 Enzymatic coagulation of milk. In Advanced Dairy Chemistry Vol. 1, part B, 3rd edition, pp. 839–869 (Eds Fox, PF & McSweeney, PLH). New York: Kluwer Academic/Plenum PublishersGoogle Scholar
Jensen, ON, Larsen, MR & Roepstorff, P 1998 Mass spectrometric identification and microcharacterization of proteins from electrophoretic gels: strategies and applications. Proteins 2 74–89Google Scholar
Kang, D, Gho, YS, Suh, M & Kang, C 2002 Highly sensitive and fast protein detection with Coomassie brilliant blue in sodium dodecyl sulfate-polyacrylamide gel electrophoresis bull. Korean Chemical Society 23 1511–1512Google Scholar
Lametsch, R, Roepstorff, P & Bendixen, E 2002 Identification of protein degradation during post-mortem storage of pig meat. Journal of Agricultural and Food Chemistry 50 5508–5512Google Scholar
Larsen, LB, Wedholm-Pallas, A, Lindmark-Mansson, H & Andrén, A 2010 Different proteomic profiles of sweet whey and rennet casein obtained after preparation from raw versus heat-treated skimmed milk. Dairy Science and Technology 90 641–656Google Scholar
Lomholt, SB & Qvist, KB 1997 Relationship between rheological properties and degree of κ-casein proteolysis during renneting of milk. Journal of Dairy Research 64 541–549Google Scholar
Manso, M, Léonil, J, Jan, G, Gagnaire, VG & Jan, V 2005 Application of proteomics to the characterisation of milk and dairy products. International Dairy Journal 15 845–855CrossRefGoogle Scholar
McGann, TCA & Fox, PF 1974 Physico-chemical properties of casein micelles reformed form urea-treaded milk. Journal of Dairy Research 41 45–53Google Scholar
McSweeney, PLH, Olson, NF, Fox, PF, Healy, A & Højrup, P 1993 Proteolytic specificity of chymosin on bovine αs1,-casein. Journal of Dairy Research 60 401–412Google Scholar
McSweeney, PLH, Olson, NF, Fox, PF & Healy, A 1994 Proteolysis of bovine αs2-casein by chymosin. Zeitschrift für Lebensmittel-Untersuchung und -Forschung 199 429–432Google Scholar
Miller, I, Crawford, J & Gianazza, E 2006 Protein stains for proteomic applications: which, when, why? Proteomics 6 5385–5408Google Scholar
Murakami, K, Lagarde, M & Yuki, Y 1998 Identification of minor proteins of human colostrum and mature milk by two-dimensional electrophoresis. Electrophoresis 19 2521–2527Google Scholar
O'Connell, JE, Kelly, AL, Fox, PF & De Kruif, CG 2001 Mechanism for the ethanol-dependent, heat-induced dissociation of casein micelles. Journal of Agricultural and Food Chemistry 49 4424–4428Google Scholar
O'Donnell, R, Holland, JW, Deeth, HC & Alewood, P 2004 Milk proteomics. International Dairy Journal 14 1013–1023Google Scholar
Palmer, DJ, Kelly, VC, Smit, AM, Kuy, S, Knight, CG & Cooper, GJ 2006 Human colostrum: identification of minor proteins in the aqueous phase by proteomics. Proteomics 6 2208–2216Google Scholar
Shevchenko, A, Wilm, M, Vorm, O & Mann, M 1996 Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels. Analytical Chemistry 68 850–858Google Scholar
Wedholm, A, Larsen, LB, Lindmark-Månsson, H, Karlsson, AH & Andrén, A 2006 Effect of protein composition on the cheese-making properties of milk from individual dairy cows. Journal of Dairy Science 89 3296–3305CrossRefGoogle ScholarPubMed
Wedholm, A, Møller, SH, Steensballe, A, Lindmark-Månsson, H, Karlsson, AH, Andersson, R, Andrén, A & Larsen, LB 2008 Effect of minor milk proteins in chymosin separated whey and casein fractions on cheese yield as determined by proteomics and multivariate data analysis. Journal of Dairy Science 91 3787–3797Google Scholar
Yamada, M, Murakami, K, Wallingford, JC & Yuki, Y 2002 Identification of low-abundance proteins of bovine colostral and mature milk using two-dimensional electrophoresis followed by microsequencing and mass spectrometry. Electrophoresis 23 1153–1160Google Scholar
Zuo, X & Speicher, DW 2002 Comprehensive analysis of complex proteomes using microscale solution isoelectrofocusing prior to narrow pH range two-dimensional electrophoresis. Proteomics 2 58–68Google Scholar