Milk Fat Globule Membrane Proteins Detected Using Mass Spectrometry-Based Proteomics

The milk fat globule membrane (MFGM) are thin films that surrounds milk fat globules. These globules contain a triglyceride core that is protected by the MFGM. The MFGM contains 3 lipid layers consisting of polar lipids and embedded with trans-membrane proteins. While MFGM proteins only make up roughly 1-2% of milk content¹ they are of interest to researchers because their presence includes documented health benefits, such as antibacterial effects, preventing pathogens from adhering to the gastrointestinal tract.² In their recent publication, Le, et al used mass spectrometry to identify new MFGM-associated proteins present in raw bulk cow’s milk and milk fractions which included both buttermilk and butter serum fractions.³ Previous methods of characterizing the protein composition of MFGM have used n sodium dodecyl sulphate polyacrylamide gel electrophoresis, however low abundance proteins are much more detectable using higher resolution proteomics methods, such as mass spectrometry. Researchers used a previously described method to isolate MFGM proteins.⁴ Once the proteins were isolated, the proteins were reduced, alkylated and an in-gel trypsin digestion was performed prior to liquid chromatography and tandem mass spectrometry on an LTQ-FT Ultra mass spectrometer (Thermo Scientiﬁc). As a result of the analysis using mass spectrometry, the researchers were able to identify the largest amount of proteins identified to date. 265 proteins were identiﬁed in all samples, which included distinct proteins, and 260 distinct proteins identified in raw milk. To ensure the largest coverage of proteins, researchers used microﬁltration on samples of industrial buttermilk. A total of 225 distinct proteins (out 771 proteins detected) were identiﬁed from MFGM-enriched buttermilk samples. This resulted in a total of just 40 proteins that did not represent MFGM proteins, and still detected more MFGM proteins than the highest number (138 proteins) that had been reported previously.⁵ The most plentiful proteins detected in MFGM from raw milk included were butyrophilin, lactadherin, perilipin-2, dehydrogenase/oxidase, and aS1-casein. The top proteins present in buttermilk included were butyrophilin, lactadherin, b-casein, dehydrogenase/oxidase, and b-lactoglobulin. Using gene ontology, the researchers were able to classify MFGM proteins according to functional significance. The researchers found the highest percentage of proteins were involved in biological regulation, localization and transport. Interestingly, they also found that many of these proteins are also produced in the ileum of the intestinal tract. The researchers attribute their success in identifying and characterizing MFGM to their methods of isolating, fractionation and analyzing proteins. A key aspect to this investigation was the process of isolating the MFGM fraction. Now that protein composition can be determined, potential pharmaceutical and food processing applications can be carried out. References 1. Riccio, P. (2004). “The proteins of the milk fat globule membrane in the balance.”,Trends in Food Science & Technology, 15, (pp. 458-461) 2. Inagaki, M, et al. (2010) “The bovine lactophorin C-terminal fragment and PAS6/7 were both potent in the inhibition of human rotavirus replication in cultured epithelial cells and the prevention of experimental gastroenteritis.”, Bioscience, Biotechnology, and Biochemistry, 74, (pp. 1386-1390) 3. Le, T. T., et al (2013) “Distribution and isolation of milk fat globule membrane proteins during dairy processing as revealed by proteomic analysis.”, International Dairy Journal , 32 (pp. 110-120) 4. Le, T. T., et al (2009) “Effect of washing conditions on the recovery of milk fat globule membrane proteins during the isolation of milk fat globule membrane from milk.”, Journal of Dairy Science, 92, (pp. 3592-3603) 5. Reinhardt, T. A., & Lippolis, J. D. (2008) “Developmental changes in the milk fat globule membrane proteome during the transition from colostrum to milk.”, Journal of Dairy Science, 91, (pp. 2307-2318.)