Aeromonas hydrophila (Ah) is an opportunistic Gram-negative bacterium and a serious global pathogen causing Motile Aeromonas Septicaemia (MAS) in fish and many other vertebrates. This study aimed at identifying the cellular proteins and processes altered by Ah infection in liver tissue of Labeo rohita, an important aquaculture species. Discovery based proteomics data could identify a panel of differentially expressed proteins (DEPs) between Control and AH condition (Ah infected group). Protein abundance changes in a few selected DEPs was then validated using Selected reaction monitoring (SRM) approach.

The experiment was carried out on a TSQ Altis Mass Spectrometer linked with an HPLC-Dionex Ultimate 3000 system (Thermo Fisher Scientific, USA) A Hypersil Gold C-18 column was used to separate the peptides for 10 minutes at a flow rate of 0.45 mL/min. In the binary buffer system, buffer A was 0.1 % Formic acid (FA), while buffer B was 80 % Acetonitrile in 0.1 % FA. A background proteome was employed, which consisted of the Uniprot protein database for Labeo rohita (ProteomeID- UP000290572, Taxonomy ID- 84645, downloaded on June 18, 2021). The collision energy values used in the experiment were determined by Skyline software (Ver 20.1.1.196). The first round of optimization was carried out with pooled peptide samples. Consequently, the list was refined based on consistency of the spectral data. For the final SRM run, the data was acquired for all samples using two transition lists (two SRM methods) consisting of 599 transitions and 90 peptides corresponding to 27 proteins. All the samples were spiked in with equal amount or heavy labelled synthetic peptide DIFTGLIGPMK (C-terminus lysine labelled) before injecting to mass spectrometer. Synthetic peptide was added to monitor the consistency of the mass spectrometric run, for which 18 transitions were added to the final transition list. After data acquisition, it was analyzed using Skyline software. [The skyline file shared here is the analyzed file consisting of 328 transitions, 45 peptides in which 18 transitions are for spiked in peptide].

The SRM data was acquired for a total of 11 samples belonging to two conditions, AH (N=5) and Control (N=6). The data was acquired for two transition lists for all the samples resulting in 22 raw files. One μg of peptides from each sample were injected and run against the list. After data acquisition, condition wise analysis was performed in Skyline.