In this study, we examined the kidney proteome of Labeo rohita infected intraperitoneally with Edwardsiella tarda (Et). A comparative proteomic analysis of Control and Et infected kidney fwas performed. High-resolution mass spectrometry was used for gut data using label-free quantification. Discovery based proteomics data could identify a panel of differentially expressed proteins (DEPs) between Control and ET condition (Et infected group). Protein abundance changes in a few selected DEPs was then validated using selected/multiple reaction monitoring (S/MRM) 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 100 % 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). The collision energy values used in the experiment were determined by Skyline software. 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 MRM run, the data was acquired for all samples with final method consisting of 471 transitions corresponding to 14 proteins. The samples (6 healthy and 6 Et infected kidney samples) with a pool sample injection after every 4 samples (to monitor consistency of runs) were run against this list in a scheduled run. The raw files were further analysed in Skyline and MSstats (integrated in Skyline) along with the use of Prosit based spectral library. The skyline file shared here is the analyzed file consisting of 394 transitions, 50 peptides.

The MRM data was acquired for a total of 12 samples belonging to two conditions, ET (n=6) and Control (n=6) and a pool peptide sample. 1.4 μg of peptides from each sample were injected and run against the final transition list. A total of 12 result files were obtained after the experiment.