Differential proteome analysis of bovine serum and associated changes related to facial eczema in dairy cattle using shotgun bottom-up methodology and high-resolution mass spectrometry

Abstract

Facial eczema (FE) is a secondary-photosensitisation ruminant disease caused by a fungal species Pithomyces chartarum. The fungus is known to release a mycotoxin called sporidesmin and tends to proliferate at the base of the pasture. Sporidesmin causes damage to the liver and the bile duct in the cattle. Chlorophyll a breakdown product, phylloerythrin is usually excreted from the liver to the bile. However, in FE affected cows the ability to excrete phylloerythrin from the liver to the bile is lost and phylloerythrin therefore starts to accumulate and spill over into the blood stream. The retention of phylloerythrin in the blood causes the cows to become photosensitive to sunlight and the condition gradually worsens to give rise to FE. In this project, we aimed to look for protein biomarker/s for early stage detection of the disease using mass spectrometry (MS). To examine serum samples from two distinct trials; an artificially-induced FE trial (2011) and a naturally-occurring FE trial (2015). 30 serum samples from 2011 artificially-induced FE trial were analysed on a TripleTOF 6600 to determine the serum proteome expression changes. We identified 221 proteins by performing LCMS on this instrument. Label free quantification of peptides were compared using MaxQuant and Perseus to statistically analyse the changes in expression of serum proteins before and after the exposure to sporidesmin. We found three proteins of interest; transthyretin, Ret-4 and conglutinin that seemed to be down regulated, possibly due to the action of sporidesmin. LC-MS on TripleTOF 6600 on samples from the naturally-occurring FE trial (2015), was able to detect 247 proteins. Samples were also analysed by direct infusion into a FTICR to generate MS and MS/MS spectra for the targeted peptides that showed differential expression in the TripleTOF 6600 analysis. The resolution offered by FTICR was around 100,000 at m/z 400, which was relatively higher than 35,000 seen in both MS and MS/MS modes on TripleTOF 6600.