Quantitative Proteomic Analysis Protocol

A total of 6 animal per group was used in these analyses. The methodology was carried out in biological triplicate, after pooling two animals from the same group into one single sample. After, the samples were submitted to a cryogenic mill, followed by protein extraction by buffer lysis under constant stirring and 4 °C. Then, a standardized protein concentration was determined by Bradford’s method (1 μg/μL) and a fixed protein amount (50 μg) was used for the following steps: alkylation, digestion, desalting and elution. The reading and identification of the peptides was performed by using the nanoAcquity UPLC-Xevo QTof MS system (Waters Co., UK). Detailed methodology is available elsewhere^{23 (link),26 (link)}.