Expanding the bovine milk proteome through extensive fractionation

Abstract

Bovine milk is an agricultural product of tremendous value worldwide. It contains proteins, fat, lactose, vitamins, and minerals. It provides nutrition and immunological protection (e.g., in the gastrointestinal tract) to the newborn and young calf. It also forms an important part of human nutrition. The repertoire of proteins in milk (i.e., its proteome) is vast and complex. The milk proteome can be described in detail by mass spectrometry-based proteomics. However, the high concentration of dominating proteins in milk reduces mass spectrometry detection sensitivity and limits detection of low abundant proteins. Further, the general health and udder health of the dairy cows delivering the milk may influence the composition of the milk proteome. To gain a more exhaustive and true picture of the milk proteome, we performed an extensive preanalysis fractionation of raw composite milk collected from documented healthy cows in early lactation. Four simple and industrially applicable techniques exploring the physical and chemical properties of milk, including acidification, filtration, and centrifugation, were used for separation of the proteins. This resulted in 5 different fractions, whose content of proteins were compared with the proteins of nonfractionated milk using 2-dimensional liquid chromatography tandem mass spectrometry analysis. To validate the proteome analysis, spectral counts and ELISA were performed on 7 proteins using the ELISA for estimation of the detection sensitivity limit of the 2-dimensional liquid chromatography tandem mass spectrometry analysis. Each fractionation technique resulted in identification of a unique subset of proteins. However, high-speed centrifugation of milk to whey was by far the best method to achieve high and repeatable proteome coverage. The total number of milk proteins initially detected in nonfractionated milk and the fractions were 635 in 2 replicates. Removal of dominant proteins and filtering for redundancy across the different fractions reduced the number to 376 unique proteins in 2 replicates. In addition, 366 proteins were detected by this process in 1 replicate. Hence, by applying different fractionation techniques to milk, we expanded the milk proteome. The milk proteome map may serve as a reference for scientists working in the dairy sector.

Colostrum and milk protein rankings and ratios of importance to neonatal calf health using a proteomics approach

“…This investigation was based on the results previously reported for proteins identified in colostrum (Nissen et al, 2012) and milk (Nissen et al, 2013). Both publications contain a thorough description of the procedures of fractionation and analysis; therefore, only a brief summary and an illustration of the fractionation and sample processing is presented here (Figure 1).…”

“…The protein concentration was determined before pooling the samples (on a volume or protein weight basis) and in NF and fractions F1 to F5 before proteome analysis (Nissen et al, 2012(Nissen et al, , 2013. Further, colostrum and milk samples were collected and analyzed in a diagnostic dairy laboratory (Eurofins Steins Laboratory) for total protein content.…”

“…Indeed, cow milk also contains immunologically important proteins, although these are in relatively low amounts and are thus believed to have little biological significance compared with that of colostrum (Stelwagen et al, 2009;Nissen et al, 2013). The proposed primary immunological function of these proteins is to protect the dam's udder from developing mastitis caused by infection with extra-mammary pathogenic bacteria (Rainard and Riollet, 2006).…”

“…Previously, we published 2 comprehensive proteomebased studies of the of the proteins present in bovine colostrum sampled from the first milking after calving and in milk sampled on d 10 (Nissen et al, 2012(Nissen et al, , 2013. These results allow us to investigate whether biologically active proteins other than immunoglobulins were exclusive to colostrum or present in higher concentrations in colostrum than in milk.…”

Composition and properties of bovine colostrum: a review

“…Nissen et al (2012) identified 403 minor proteins in colostrum using 2D-LC-MS/MS, which, to the authors' knowledge, is the most comprehensive characterisation to date of whey proteins in colostrum. In a follow-on study, Nissen et al (2013), also using 2D-LC-MS/MS, identified a total of 742 minor proteins in milk; however, 366 of these proteins were identified only in one replicate and are therefore regarded as tentative results.…”