Proteomic profiling of plasma-derived small extracellular vesicles: a novel tool for understanding the systemic...

Cattle ticks pose a significant threat to the health and profitability of cattle herds globally. The investigation of factors leading to natural tick resistance in cattle is directed toward targeted breeding strategies that may combat cattle tick infestation on the genetic level. Exosomes (EXs), small extracellular vesicles (EVs) of 50 to 150 nm diameter, are released from all cell types into biofluids such as blood plasma and milk, have been successfully used in diagnostic and prognostic studies in humans, and can provide essential information regarding the overall health state of animals. Mass spectrometry (MS) is a highly sensitive proteomics application that can be used to identify proteins in a complex mixture and is particularly useful for biomarker development. In this proof of principle study, EXs were isolated from the blood plasma of cattle (Bos taurus) with high (HTR) and low tick resistance (LTR) (n = 3/group). Cattle were classified as HTR or LTR using a tick scoring system, and EXs isolated from the cattle blood plasma using an established protocol. EXs were subjected to MS analysis in data-dependent acquisition mode and protein search performed using Protein Pilot against the B. taurus proteome. A total of 490 unique proteins were identified across all samples. Of these, proteins present in all replicates from each group were selected for further analysis (HTR = 121; LTR = 130). Gene ontology analysis was performed using PANTHER GO online software tool. Proteins unique to HTR and LTR cattle were divided by protein class, of which 50% were associated with immunity/defense in the HTR group, whereas this protein class was not detected in EXs from LTR cattle. Similarly, unique proteins in HTR cattle were associated with B-cell activation, immunoglobins, immune response, and cellular iron ion homeostasis. In LTR cattle, unique exosomal proteins were associated with actin filament binding, purine nucleotide binding, plasma membrane protein complex, and carbohydrate derivative binding. This is the first study to demonstrate that MS analysis of EXs derived from the blood plasma of HTR and LTR cattle can be successfully applied to profile the systemic effects of tick burden.