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

Administration of colostrum to the newborn calf before gut closure is pivotal to its health, because of the transfer of passive immunity. Traditionally, passive immunity has been attributed to the transfer of immunoglobulins although it is increasingly clear that multiple other factors contribute, including innate immune proteins, developmental factors, immunomodulatory factors, and the presence of cellular immunity. The objective of this study was to produce a comprehensive comparison of the bovine colostrum proteome and the milk proteome by applying 2-dimensional liquid chromatography-tandem mass spectrometry. Further, the objectives were to rank proteins mutually and generate protein ratios from the spectral counts of the 2 proteomes and ELISA to gain insight into which proteins could be of most relevance to neonatal calf health. To obtain an in-depth picture of the bovine colostrum and milk proteome, we compared the contents of different fractions from bovine colostrum and milk from our 2 previous studies. A total of 140 colostrum fluid-phase proteins and 103 milk fluid-phase proteins were detected. In the cellular fraction, 324 and 310 proteins were detected in colostrum and milk, respectively. In total, 514 proteins were detected, of which 162 were in the fluid phase. Of these, 50 proteins were exclusively seen in colostrum, 13 were exclusively seen in milk, and 99 were common to colostrum and milk. Ranking of proteins mutually and calculating protein ratios based on spectral counts and ELISA resulted in new information on how proteins were associated with the fluid or cellular fraction of the samples. Interestingly, despite lower counts/concentrations than the classical proteins such as immunoglobulins, ß-lactoglobulin, and lactotransferrin, several proteins appeared in higher or similar colostrum:milk spectral count ratios as these. Using this approach indicated, for example, that osteopontin, haptoglobin, milk amyloid A, and gelsolin may be interesting molecules to study in detail in their relation to calf health. Although the sensitivity, identification, and ranking of proteins varied between the 2 methods, and proteome analysis clearly suffers from low sensitivity, we believe that this idea and approach of generating ratios and ranking proteins can contribute new information and perspectives on how to prioritize the importance of multiple proteins, beyond immunoglobulins, in relation to neonatal calf health.

Expanding the bovine milk proteome through extensive fractionation.

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.

In-depth analysis of low abundant proteins in bovine colostrum using different fractionation techniques.

Bovine colostrum is well known for its large content of bioactive components and its importance for neonatal survival. Unfortunately, the colostrum proteome is complicated by a wide dynamic range, because of a few dominating proteins that hamper sensitivity and proteome coverage achieved on low abundant proteins. Moreover, the composition of colostrum is complex and the proteins are located within different physical fractions that make up the colostrum. To gain a more exhaustive picture of the bovine colostrum proteome and gather information on protein location, we performed an extensive pre-analysis fractionation of colostrum prior to 2D-LC-MS/MS analysis. Physical and chemical properties of the proteins and colostrum were used alone or in combination for the separation of proteins. ELISA was used to quantify and verify the presence of proteins in colostrum. In total, 403 proteins were identified in the nonfractionated colostrum (NF) and seven fractions (F1-F7) using six different fractionation techniques. Fractionation contributed with 69 additional proteins in the fluid phase compared with NF. Different fractionation techniques each resulted in detection of unique subsets of proteins. Whey production by high-speed centrifugation contributed most to detection of low abundant proteins. Hence, prefractionation of colostrum prior to 2D-LC-MS/MS analysis expanded our knowledge on the presence and location of low abundant proteins in bovine colostrum.

A simple two-step purification procedure for the iC3b binding collectin conglutinin.

Bovine conglutinin is a serum protein involved in innate immunity. It binds calcium dependently to iC3b, a product of the complement component C3 deposited on cell surfaces, immune complexes or artificial surfaces after complement activation. We here present a simple and efficient two-step procedure for the purification of conglutinin. In the first step, bovine serum is incubated with non-coupled chromatographic TSK beads at 37°C to allow complement activation and iC3b deposition on the beads and subsequent binding of conglutinin to iC3b. Conglutinin is then eluted from the beads by EDTA. In the second step, conglutinin is separated from iC3b and IgM by ion-exchange chromatography. This purification procedure yielded 81 µg of conglutinin per ml of serum with a recovery of 61.2%. Surface plasmon resonance analysis showed that the purified conglutinin had a high affinity for mannan (K(d)=2.3-3.2 nM). SDS-PAGE and time-resolved immunofluorometric assays showed that the conglutinin was not contaminated with other serum collectins such as collectin-43 or mannan-binding lectin.

To what extent do variabilities in hormones, metabolites and energy intake explain variability in milk yield?

The objective of the present paper is to describe the extent to which variability in milk yield can be explained by variability in plasma hormones, metabolites and DE intake. Results from a study including 317 cows and 634 lactations were used. Detailed registration of performance was carried out on these cows and 10,809 plasma samples analyzed for selected hormones and metabolites. Univariate analysis was carried out on energy-corrected milk yield and concentrations of selected plasma hormones (insulin, growth hormone (GH) and triiodothyronine (T3)), metabolites (non-esterified fatty acids (NEFA)), glucose, beta-hydroxybutyrate (BOHB) and urea nitrogen (BUN), and digestible energy intake (DE intake) to estimate between-cow variation through lactations. Partial least square (PLS) models were subsequently run to estimate the extent to which between-cow differences in energy-corrected milk yield could be explained by between-cow differences in hormone concentrations, metabolite concentration or DE intake. The between-cow variability in energy-corrected milk yield and the hormones and metabolites were generally found to be considerable and total variance changed through lactation, particularly for GH, T3, NEFA and BOHB. In this study, the total variance was highest in third lactation cows. When analyzed separately using partial least square models, hormones, metabolites and DE intake accounted for 24, 25 and 26% of the variability in ECM, respectively. Insulin and glucose were the single most important predictors among the selected hormones and metabolites. When including both the hormones and metabolites, the model explained 36% of the between-cow variability in ECM and this figure was increased to 53% if DE intake was also included. The lack of additivity in the variability explained shows that hormones, metabolites and DE intake were correlated illustrating the integration and orchestration of metabolism and intake. Perspectives of the analysis for use in prevention of diseases and reproduction are briefly discussed.