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The Milky500: five hundred worthy proteins

    NASCAR with logos to represent the play on the Indy 500 with the Milk 500 for the 500 MFGM proteins discovered.

    Written by: Daniela Barile, Ph.D., Matthew Lange | Issue # 2 | 2012

    The Indy 500 is perhaps the most famous car race in the United States. Unlike every other sporting competition in the world, the legendary 500 mile car race, held annually in Indianapolis on the last weekend in May, is celebrated with the victor drinking not Champagne, but rather a bottle of fresh milk! Cutting edge research suggests that the term “500” may have more to do with the milk than with the miles.

    The quest to completely annotate the milk proteome began nearly a decade ago. The wide dynamic range of milk protein concentrations has so far hindered a comprehensive understanding of its biological role. Impressive analytical advances combined with new separation methods have recently been published in renowned scientific journals and represent a culmination of these efforts, leading to the most comprehensive inventory of milk proteins in history.

    It has been known for decades that milk proteins are excellent sources of essential amino-acids, but milk proteins can actually deliver more than just basic nutrition. Many people are aware of bovine and human milk proteins such as immunoglobulins, alpha-lactoalbumin, caseins, and lactoferrin. Recently, increasing numbers of less abundant—but biologically pertinent—proteins are being discovered in other milk “compartments,” within the milk fat globule membrane, for example.

    Two new studies published in the last few months deserve special attention considering that when we combined their findings, over 500 proteins were identified in human milk.

    First, the study by Gianluca Picariello and colleagues1 appeared in the “Journal of Chromatography A” in January 2012. This was rapidly followed by the article by Claire Molinari2 in the “Journal of Proteome Research” in February 2012.

    Piccariello et al. provided a yet unmatched method to profile low-abundance proteins in human milk. Instead of using the classical electrophoresis-based approach, they first isolated the milk fat globule membrane (MFGM) – a fraction of the milk containing lipids as well as proteins that is typically problematic to study – along with whey protein fractions. Piccariello et al. subsequently employed a shotgun strategy with targeted mass spectrometry. Because of their pre-fractionation strategy, they were able to unambiguously identify 296 proteins, including 165 exclusively present in the MFGM fraction. Because the MFGM proteins are the most genetically conserved milk proteins across species, they are of particular interest from a biological point of view. The identified proteins, which were derived from multiple metabolic pathways, are involved in different physiological functions, such as membrane trafficking, cell signaling, fat metabolism and transport, metabolite delivery, protein synthesis/proteolysis or folding, and immunity-related actions.

    The study by Molinari et al.2 investigated the low-abundant proteins of term and preterm human milk. Similarly to the Piccariello study, they also used various pre-fractionation methods, including a combination of casein depletion and a novel kit designed to enrich low abundance proteins called ProteoMiner, to ensure detection and characterization of the minor protein components. Their use of electrophoretic and separative techniques, combined with advanced analytics (two-dimensional liquid chromatography and MALDI ToF mass spectrometry), led to the identification of 415 unique proteins, 261 of which had not been previously found in human skim milk. The vast majority of proteins identified participate in either immune response or cellular metabolism/growth; some minor components are involved in lipid metabolism and/or nutrient delivery. Among the newly discovered proteins, several growth factors identified may induce nutritional and developmental advantages, but further studies are necessary to assess whether these proteins retain their activity after digestion.

    When we superimpose the results from these two studies, we find only 123 proteins in common, bringing a total of 588 different and unique protein entities in human milk.

    Recently, D’Alessandro and colleagues3 provided an updated synopsis of the known bovine milk proteins, merging data from independent studies. After manual removal of redundant duplicates and incomplete proteins, the authors obtained a final list containing 573 non-redundant annotated protein entries, resulting in the broadest bovine milk protein inventory reported to date.

    Moving forward, it is noteworthy that all current methods for descriptive analysis of complex proteomes lack protocols that provide a comprehensive snapshot of complex biological systems in a single run. For this reason, specifically designed strategies are required to target common post-translational modifications (e.g. glycosylation) in mammalian milk proteins. Indeed, all studies revealed the protein sequence of numerous previously undiscovered proteins, but to do so, used special enzymes to cleave the carbohydrates (N-glycans) that were coating the proteins. This process, known as N-deglycosylation, is unfortunately accompanied by a significant loss of information. That is, once this method is performed, it is not possible to know neither the extent nor the type of glycosylation. This is a significant drawback because an increasing number of reports demonstrate that protein glycosylation plays an important role in the function of cellular components and processes.

    A recent publication by Charles Nwosu and colleagues4 in the current issue of “Journal of Proteome Research” endorses the fact that it is indeed important to look into the details of glycans attached to the milk proteins. The researchers used nanoflow liquid chromatography coupled with time-of-flight high-resolution mass spectrometry to reveal the first comprehensive N-glycan repertoire of milk. This enables the direct comparison of bovine and human milk glycoproteins as never before. Immunoglobulins, lactoferrin, and casein are all glycosylated proteins, present both in human and bovine milk. They are all thought to have a protective function against pathogenic organisms, therefore playing important roles in preventing gastrointestinal disease. In addition to preventing the proteolytic digestion of proteins in the stomach, it is thought that the glycans participate in additional biological functions.

    Nwosu and colleagues were able to determine that human and bovine milk contain over 100 different structures of glycans, corresponding to 38 and 51 unique compositions of isomeric glycan structures, respectively. They observed a pronounced diversity in structures, with only 20 glycan compositions found to be exactly identical in the two milks.

    It is important to note that studying glycans is more difficult than studying proteins alone since glycans lack a genetic template, and their compositions are not predictable by bioinformatics methods alone. Studying glycans therefore requires several additional analytical techniques combined with more rigorous analyses.

    The combination of all the aforementioned analytical tools for protein and glycan determination may allow for the advancement of our current knowledge of milk bioactives and the characterization of new components, both of which are vital scientific endeavors for deepening our understanding of the biologically significant components in human and bovine milk. Unbeknownst to them, the Indy500 drivers get a nutritional boost for every mile driven.


    1. Picariello, G., et al., Gel-free shotgun proteomic analysis of human milk. Journal of Chromatography A, 2012. 1227: p. 219-233.

    2. Molinari, C.E., et al., Proteome mapping of human skim milk proteins in term and preterm milk. J Proteome Res, 2012. 11(3): p. 1696-714.

    3. D’Alessandro, A., L. Zolla, and A. Scaloni, The bovine milk proteome: cherishing, nourishing and fostering molecular complexity. An interactomics and functional overview. Mol. BioSyst., 2011. 7(3): p. 579-597.

    4. Nwosu, C.C., et al., Comparison of the Human and Bovine Milk N-Glycome via High-Performance Microfluidic Chip Liquid Chromatography and Tandem Mass Spectrometry. J Proteome Res, 2012.