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 gut 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). A panel of proteins from important pathways were selected for validation of differential abundances 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 23.1.1.268). 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 one transition lists (one SRM methods) consisting of 402 transitions and 62 peptides corresponding to 10 proteins. All the samples were spiked in with equal amount or heavy labelled synthetic peptide ENQTCDIYNGEGR (C-terminus Arginine labelled) before injecting to mass spectrometer. Synthetic peptide was added to monitor the consistency of the mass spectrometric run, for which 10 transitions were added to the final transition list. After data acquisition, it was analyzed using Skyline software. [Two Skyline files are uploaded here: 1. “20240101_SRM_Rohu_GutAH_vs_Control_Refined_ALL”- Consisting of 371 transitions, of all 59 peptides (after the analysis) including 10 transitions for spiked in peptide], 2. “20240101_SRM_Rohu_GutAH_vs_Control_Refined_Significant”- File contains 25 pepties which showed significant differential intensity/peak areas between Control and AH condition. A total of 161 transitions including 10 of the spiked-in peptide.

The SRM data was acquired for a total of 12 samples belonging to two conditions, six each for AH and Control. The data was acquired for one transition list for all the samples resulting in 12 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.