Detection of casein phosphopeptides in goat milk.

The aims of this study were to profile casein phosphopeptides in goat milk, to accurately determine the site of phosphorylation, and to evaluate whether or not any of the casein phosphorylation patterns were specific to a given physiological condition. Goat milk, collected before and after experimental induction of endotoxin mastitis, was separated by SDS-PAGE. Casein bands were digested with trypsin and the resulting peptides were analyzed by nLC-MS/MS. Eight out of nine predicted tryptic phosphopeptides corresponding to 18 different phosphorylation sites were detected in αS1-, αS2-, and ß-casein. Characterization of the phosphorylation sites illustrated the capability of tandem MS to accurately localize phosphorylated residues among a number of other putative sites. Despite an apparent lower abundance, almost all of the phosphopeptides were also detected in milk samples obtained from the goats following experimental induction of endotoxin mastitis. However, a tetra-phosphopeptide in αS2-casein was only observed in the milk samples obtained from healthy animals. The absence of this multiphosphopeptide in the mastitic goat milk samples could indicate changes in phosphorylation as a result of disease and potentially be used as a marker for milk quality. This study represents the first comprehensive analysis of casein phosphoproteome and reveals a much higher level of phosphorylation than previously demonstrated in goat milk.

Proteomic analyses of host and pathogen responses during bovine mastitis.

The pursuit of biomarkers for use as clinical screening tools, measures for early detection, disease monitoring, and as a means for assessing therapeutic responses has steadily evolved in human and veterinary medicine over the past two decades. Concurrently, advances in mass spectrometry have markedly expanded proteomic capabilities for biomarker discovery. While initial mass spectrometric biomarker discovery endeavors focused primarily on the detection of modulated proteins in human tissues and fluids, recent efforts have shifted to include proteomic analyses of biological samples from food animal species. Mastitis continues to garner attention in veterinary research due mainly to affiliated financial losses and food safety concerns over antimicrobial use, but also because there are only a limited number of efficacious mastitis treatment options. Accordingly, comparative proteomic analyses of bovine milk have emerged in recent years. Efforts to prevent agricultural-related food-borne illness have likewise fueled an interest in the proteomic evaluation of several prominent strains of bacteria, including common mastitis pathogens. The interest in establishing biomarkers of the host and pathogen responses during bovine mastitis stems largely from the need to better characterize mechanisms of the disease, to identify reliable biomarkers for use as measures of early detection and drug efficacy, and to uncover potentially novel targets for the development of alternative therapeutics. The following review focuses primarily on comparative proteomic analyses conducted on healthy versus mastitic bovine milk. However, a comparison of the host defense proteome of human and bovine milk and the proteomic analysis of common veterinary pathogens are likewise introduced.

Evaluation of protein expression in bovine bronchoalveolar fluid following challenge with Mannheimia haemolytica.

Proteomics analysis of bovine bronchoalveolar fluid (BAF) following induction of pneumonia with Mannheimia haemolytica using nanoflow liquid chromatography coupled with tandem mass spectrometry (nanoLC-MS/MS) resulted in the identification of 88 unique proteins. Proteins detected in BAF included antimicrobial peptides (AMPs), complement factors, acute-phase proteins, protease inhibitors, and proteins involved in oxidation-reduction. Notwithstanding biological variation, differences in relative protein abundance, determined using normalized peptide counts, were detected for select proteins in BAF from genuinely infected versus sham-infected animals. To demonstrate the applicability of using normalized peptide counts to assess protein expression trends, LC-MS/MS data for the acute-phase protein haptoglobin (HPT) were compared with ELISA data, and statistical evaluation of the relationship between the data revealed a strong measure of association. Differences were detected between sham- and genuinely infected animals for haptoglobin, as well as the AMPs cathelicidin-1 and cathelicidin-4, and inter-α-trypsin inhibitor heavy chain-4, a fairly novel protein involved in the acute phase response. Though the small sample size limited the scope of the inferences, the results indicate the likely importance of AMPs and acute-phase proteins during respiratory infection, and provide additional information regarding potential mechanisms involved in the bovine mucosal barrier defense.

A No Observable Adverse Effects Level (NOAEL) for pigs fed melamine and cyanuric acid.

Ingesting melamine adulterated milk products led to kidney stones in many infants in 2008. This differs from the renal failure caused by intratubular crystal formation after co-ingestion of melamine (MEL) and cyanuric acid (CYA) in adulterated pet foods in 2007. To better understand the potential risk of developing crystal nephropathy following co-ingestion of MEL and CYA, we fed 16 weanling pigs 0, 1, 3.3, 10, 33, or 100 mg/kg bw/day of each MEL and CYA, or 200 mg/kg bw/day of either compound individually for 7 days. Crystals were found in the renal medulla and cortex and urine sediments of all pigs fed both MEL and CYA each at 10 mg/kg bw/day (or greater). Crystals were also found in one of the two pigs fed 200 mg/kg bw/day MEL-only. In a 28 day study, 36 weanling pigs were fed 0, 1, or 3.3 mg/kg bw/day of MEL and CYA or 200 mg/kg bw/day MEL-only. Only one of the 3.3 mg/kg MEL and CYA pig kidneys contained crystals. The no-observed-adverse-effect level (NOAEL) for pigs fed MEL and CYA for 28 days was concluded to be 1.0 mg/kg bw/day corresponding to 25 mg/kg (ppm) MEL and 25 mg/kg (ppm) CYA in dry feed.

The proteomic advantage: label-free quantification of proteins expressed in bovine milk during experimentally induced coliform mastitis.

Coliform mastitis remains a primary focus of dairy cattle disease research due in part to the lack of efficacious treatment options for the deleterious side effects of exposure to LPS, including profound intra-mammary inflammation. To facilitate new veterinary drug approvals, reliable biomarkers are needed to evaluate the efficacy of adjunctive therapies for the treatment of inflammation associated with coliform mastitis. Most attempts to characterize the host response to LPS, however, have been accomplished using ELISAs. Because a relatively limited number of bovine-specific antibodies are commercially available, reliance on antibodies can be very limiting for biomarker discovery. Conversely, proteomic approaches boast the capability to analyze an unlimited number of protein targets in a single experiment, independent of antibody availability. Liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS), a widely used proteomic strategy for the identification of proteins in complex mixtures, has gained popularity as a means to characterize proteins in various bovine milk fractions, both under normal physiological conditions as well as during clinical mastitis. The biological complexity of bovine milk has, however, precluded the complete annotation of the bovine milk proteome. Conventional approaches to reducing sample complexity, including fractionation and the removal of high abundance proteins, has improved proteome coverage, but the dynamic range of proteins present, and abundance of a relatively small number of proteins, continues to hinder comparative proteomic analyses of bovine milk. Nonetheless, advances in both liquid chromatography and mass spectrometry instrumentation, including nano-flow liquid chromatography (nano-LC), nano-spray ionization, and faster scanning speeds and ionization efficiency of mass spectrometers, have improved analyses of complex samples. In the current paper, we review the proteomic approaches used to conduct comparative analyses of milk from healthy cows and cows with clinical mastitis, as well as proteins related to the host response that have been identified in mastitic milk. Additionally, we present data that suggests the potential utility of LC-MS/MS label-free quantification as an alternative to costly labeling strategies for the relative quantification of individual proteins in complex mixtures. Temporal expression patterns generated using spectral counts, an LC-MS/MS label-free quantification strategy, corresponded well with ELISA data for acute phase proteins with commercially available antibodies. Combined, the capability to identify low abundance proteins, and the potential to generate temporal expression profiles, indicate the advantages of using proteomics as a screening tool in biomarker discovery analyses to assess biologically relevant proteins modulated during disease, including previously uncharacterized targets.

Evaluation of the renal effects of experimental feeding of melamine and cyanuric acid to fish and pigs.

OBJECTIVE: To determine whether renal crystals can be experimentally induced in animals fed melamine or the related triazine compound cyanuric acid, separately or in combination, and to compare experimentally induced crystals with those from a cat with triazine-related renal failure. ANIMALS: 75 fish (21 tilapia, 24 rainbow trout, 15 channel catfish, and 15 Atlantic salmon), 4 pigs, and 1 cat that was euthanatized because of renal failure. PROCEDURES: Fish and pigs were fed a target dosage of melamine (400 mg/kg), cyanuric acid (400 mg/kg), or melamine and cyanuric acid (400 mg of each compound/kg) daily for 3 days and were euthanatized 1, 3, 6, 10, or 14 days after administration ceased. Fresh, frozen, and formalin-fixed kidneys were examined for crystals. Edible tissues were collected for residue analysis. Crystals were examined for composition via Raman spectroscopy and hydrophilic-interaction liquid chromatography-tandem mass spectrometry. RESULTS: All animals fed the combination of melamine and cyanuric acid developed goldbrown renal crystals arranged in radial spheres (spherulites), similar to those detected in the cat. Spectral analyses of crystals from the cat, pigs, and fish were consistent with melamine-cyanurate complex crystals. Melamine and cyanuric acid residues were identified in edible tissues of fish. CONCLUSIONS AND CLINICAL RELEVANCE: Although melamine and cyanuric acid appeared to have low toxicity when administered separately, they induced extensive renal crystal formation when administered together. The subsequent renal failure may be similar to acute uric acid nephropathy in humans, in which crystal spherulites obstruct renal tubules.