Casocidin-1

Bovine mastitis is usually caused by either Gram positive or Gram negative bacteria, reducing the quantity and quality of milk produced. This investigation using capillary electrophoresis and mass spectroscopy, studied peptides in milk from cows with clinical mastitis in comparison to milk from healthy cows to identify biomarkers for mastitis. In addition, the milk peptidome from udders infected with Gram positive Staphylococcus aureus (S. aureus) or with Gram negative Escherichia coli (E. coli), was examined to assess differential diagnosis between the causative agent. Comparison of the peptidome between healthy (n=10) and mastitic milk (n=27) identified 154 peptides for a biomarker panel which in a model for diagnosis of mastitis showed 100% sensitivity and specificity. β-casein and α(s1) casein provided the majority of peptides identified in this model. The peptidome comparison of milk from mastitis cases caused by S. aureus (n=8) or E. coli (n=11) revealed a biomarker panel of 47 peptides which discriminated between cause of infection with a sensitivity of 75% and a specificity of 100%. β-casein fragments were the most common of the peptides in this model. Peptide biomarkers of milk could be used in the diagnosis of mastitis and can discriminate between these two bacterial causes. Biological significance: The paper describes an innovative approach to the use of gel free proteomics to identify the peptides that are present in milk during clinical mastitis, which is a major cause of loss of production to dairy farmers worldwide. The use of capillary electrophoresis, liquid chromatography and mass spectrometry has been able to identify panels of peptides which can be used for disease diagnosis and for differential diagnosis of the causative bacteria of the infections of the mammary gland. As well as contributing to our knowledge of the pathophysiology of bovine mastitis the results could be the basis of improved detection and differential diagnosis of the disease.

Under the traditional grazing system on the Qinghai-Tibetan Plateau, the amount of milk in domesticated yak (Bos grunniens) with clinical mastitis decreases and the milk composition is altered. To understand the mechanisms of mammary gland secreted milk and disease infection, changes in the protein composition of milk during clinical mastitis were investigated using a proteomic approach. Milk whey from yak with clinical mastitis was compared to whey from healthy animals with two-dimensional gel electrophoresis using a mass spectrometer. Thirteen protein spots were identified to be four differentially expressed proteins. Increases in the concentrations of proteins of blood serum origin, including lactoferrin, were identified in mastitic whey compared to normal whey, while concentrations of the major whey proteins, casocidin-I, a-lactalbumin, and b-lactoglobulin, were downregulated in mastitic whey. These results indicated significant differences in protein expression between healthy yaks and those with clinical mastitis, and they may provide valuable information for finding new regulation markers and potential protein targets for the treatment of mastitis.

Surface active maghemite nanoparticles (SAMNs) are able to recognize and bind selected proteins in complex biological systems, forming a hard protein corona. Upon a 5-min incubation in bovine whey from mastitis-affected cows, a significant enrichment of a single peptide characterized by a molecular weight at 4338 Da originated from the proteolysis of aS1-casein was observed. Notably, among the large number of macromolecules in bovine milk, the detection of this specific peptide can hardly be accomplished by conventional analytical techniques. The selective formation of a stable binding between the peptide and SAMNs is due to the stability gained by adsorption-induced surface restructuration of the nanomaterial. We attributed the surface recognition properties of SAMNs to the chelation of iron(III) sites on their surface by sterically compatible carboxylic groups of the peptide. The specific peptide recognition by SAMNs allows its easy determination by MALDI-TOF mass spectrometry, and a threshold value of its normalized peak intensity was identified by a logistic regression approach and suggested for the rapid diagnosis of the pathology. Thus, the present report proposes the analysis of hard protein corona on nanomaterials as a perspective for developing fast analytical procedures for the diagnosis of mastitis in cows. Moreover, the huge simplification of proteome complexity by exploiting the selectivity derived by the peculiar SAMN surface topography, due to the iron(III) distribution pattern, could be of general interest, leading to competitive applications in food science and in biomedicine, allowing the rapid determination of hidden biomarkers by a cutting edge diagnostic strategy. Graphical abstract The topography of iron(III) sites on surface active maghemite nanoparticles (SAMNs) allows the recognition of sterically compatible carboxylic groups on proteins and peptides in complex biological matrixes. The analysis of hard protein corona on SAMNs led to the determination of a biomarker for cow mastitis in milk by MALDI-TOF mass spectrometry.