Qualitative and quantitative profiling of the bovine milk fat globule membrane proteome Michael Affolter a, ⁎ , Laetitia Grass a , Frank Vanrobaeys b , Begoña Casado c , Martin Kussmann a a Functional Genomics Group, Department of BioAnalytical Sciences, Nestlé Research Center, Lausanne, Switzerland b Nestlé Research Center, Beijing, China c Analytical Profiling Group, Department of Quality and Safety, Nestlé Research Center, Lausanne, Switzerland ARTICLE INFO ABSTRACT Milk is a biological fluid of unique quality and complexity. It has co-evolved with mammals and mankind to nourish offspring and contains macro- and micronutrients for growth and development of the newborn. The milk fat globule membrane (MFGM) represents an important milk fraction, which is rich in bioactive proteins. In order to better understand functionality of milk fractions and, thereby, enhance the benefits of milk products, detailed qualitative and quantitative protein knowledge of fractions such as MFGM is required. We report the qualitative and quantitative profiling of two MFGM-enriched milk fractions, a whey protein concentrate (WPC) and a buttermilk protein concentrate (BMP), as derived from three different analytical workflows. First, an LC-MS/MS-based shotgun approach revealed 244 protein identities in WPC and 133 in BMP, respectively, and provided an extensive characterisation of the protein content in those two fractions. Second, label-free profiling resulted in rapid and efficient semi-quantitative comparison and yielded valuable protein fingerprints. Third, absolute quantification of selected MFGM proteins was achieved by stable isotope dilution (SID)-MS, in combination with multiple reaction monitoring (MRM) detection. In summary, we provide new information on composition, quantity and possible health benefits of two MFGM-enriched milk fractions highly valuable for future nutritional applications. © 2009 Elsevier B.V. All rights reserved. Keywords: Proteomics Nutrigenomics Nutrition Milk Milk fat globule membrane 1. Introduction Milk is the ideal nutrition for newborns and contains macro- and micronutrients to nourish, protect and help develop infants. Milk has co-evolved with mammals and mankind to feed offspring and, not surprisingly, its composition is unique- ly adapted to the needs of each mammalian species. Exten- sive characterisation of milk from different species has been reported [1–3] and the composition in terms of major proteins, lipids, and carbohydrates has been established. Macro- and micronutrients in human milk provide bioactive factors with beneficial effects for the immune system, cognitive develop- ment, and prevention of pathogen colonisation and modula- tion of the intestinal microflora [4–6]. Most of the properties regarding preventing and protecting against infections have been attributed to the high levels of immunoglobulins (antibodies) present in milk. However, more recently, specific proteins such as lactoferrin and lactoperoxidase as well as complex carbohydrates have been recognised as bioactive elements, too [7]. JOURNAL OF PROTEOMICS 73 (2010) 1079 – 1088 ⁎ Corresponding author. Functional Genomics Group, Department of Bioanalytical Sciences, Nestlé Research Center, CH-1000 Lausanne, Switzerland. Tel.: +41 21 785 8966; fax: +41 21 785 9486. E-mail address: michael.affolter@rdls.nestle.com (M. Affolter). 1874-3919/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.jprot.2009.11.008 available at www.sciencedirect.com www.elsevier.com/locate/jprot