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Mass spectrometry (MS)-based proteomic approaches have largely facilitated our systemic understanding of cellular processes and biological functions. Cutoffs in protein expression fold changes (FCs) are often arbitrarily determined in MS-based quantification with no demonstrable determination of small magnitude changes in protein expression. Therefore, many biological insights may remain veiled due to high FC cutoffs. Herein, we employ the intestinal epithelial cell (IEC) line Caco-2 as a model system to demonstrate the dynamicity of tandem-mass-tag (TMT) labeling over a range of 5-40% changes in protein abundance, with the variance controls of ± 5% FC for around 95% of TMT ratios when sampling 9-12 biological replicates. We further applied this procedure to examine the temporal proteome of Caco-2 cells upon exposure to human whey proteins (WP). Pathway assessments predict subtle effects due to WP in moderating xenobiotic metabolism, promoting proliferation and various other cellular functions in differentiating enterocyte-like Caco-2 cells. This demonstration of a sensitive MS approach may open up new perspectives in the system-wide exploration of elusive or transient biological effects by facilitating scrutiny of narrow windows of proteome abundance changes. Furthermore, we anticipate this study will encourage more investigations of WP on infant gastrointestinal tract development.

A key element in understanding how human milk proteins support the health and development of the neonate is to understand how individual proteins are affected during digestion. In the present study, a dynamic gastric model was used to simulate infant gastric digestion of human milk, and a subsequent proteomic approach was applied to study the behavior of individual proteins. A total of 413 human milk proteins were quantified in this study. This approach demonstrated a high degree of variability in the susceptibility of human milk proteins to gastric digestion. Specifically this study reports that lipoproteins are among the class of slowly digested proteins during gastric processes. The levels of integral lysozyme C and partial lactadherin in milk whey increase over digestion. Mucins, ribonuclease 4, and macrophage mannose receptor 1 are also resistant to gastric digestion. The retention or enhancement in whey protein abundance can be ascribed to the digestive release of milk-fat-globule-membrane or immune-cell enclosed proteins that are not initially accessible in milk. Immunoglobulins are more resistant to digestion compared to total milk proteins, and within the immunoglobulin class IgA and IgM are more resistant to digestion compared to IgG. The gastric digestion of milk proteins becomes more apparent from this study.

Human milk proteins provide essential nutrition for growth and development, and support a number of vital developmental processes in the neonate. A complete understanding of the possible functions of human milk proteins has been limited by incomplete knowledge of the human milk proteome. In this report, we have analyzed the proteomes of whey from human transitional and mature milk using ion-exchange and SDS-PAGE based protein fractionation methods. With a larger-than-normal sample loading approach, we are able to largely extend human milk proteome to 976 proteins. Among them, 152 proteins are found to render significant regulatory changes between transitional milk and mature milk. We further found that immunoglobulins sIgA and IgM are more abundant in transitional milk, whereas IgG is more abundant in mature milk, suggesting a transformation in defense mechanism from newborns to young infants. Additionally, we report a more comprehensive view of a complement system and associated regulatory apparatus in human milk, demonstrating the presence and function of a system similar to that found in the circulation but prevailed by alternative pathway in complement activation. Proteins involved in various aspects of carbohydrate metabolism are also described, revealing either a transition in milk functionality to accommodate carbohydrate-rich secretions as lactation progresses, or a potentially novel way of looking at the metabolic state of the mammary tissue. Lately, a number of extracellular matrix (ECM) proteins are found to be in higher abundance in transitional milk and may be relevant to the development of infants' gastrointestinal tract in early life. In contrast, the ECM protein fibronectin and several of the actin cytoskeleton proteins that it regulates are more abundant in mature milk, which may indicate the important functional role for milk in regulating reactive oxygen species.

In addition to milk's nutritional role, it contains immunoglobulins (antibodies) and immunoregulatory proteins that are active in the digestive tract of newborns. However, knowledge of the repertoire of milk proteins remains meager. In this work, we report an ion-exchange-based protein fractionation method that allows in-depth exploration of the whey proteome in bovine milk; 293 unique gene products were identified, of which 176 were newly identified in whey. This work also demonstrated qualitatively for the first time the consistency, albeit differing in protein levels, in milk proteome between colostrum and mature milk (3 mo. post calving). Semiquantitative analysis showed a number of up-regulated proteins in colostrum that may provide extra natural defenses for the neonate. Increased understanding of the composition and functions of bovine milk proteins and their potential health benefits may, in the future, play an important role in nutritional and biomedical applications as properly processed cow's milk proteins could potentially confer the same bioactivity as their human counterparts.