Ex vivo proteomics of Mycobacterium marinum

Proteins expressed and exported out of the mycobacterial biofilm cells into granulomatous environment during infection were identified by LC-MS/MS. To this end, adult 5- to 10-month-old female AB wild-type zebrafish (Danio rerio) were infected with 4-day old M. marinum ATCC927 (carrying the pTEC27 plasmid expressing the tdTomato fluorescent protein) cells. After 8 weeks post infection, ten zebrafish were euthanized, and red fluorescent mycobacterial granulomas were carefully dissected from the zebrafish ovaries. Ten granulomas were collected per tube, frozen on dry ice and stored at -80 °C until preparation for proteomics. Altogether, ten replicate samples, each with ten mycobacterial granulomas, were subjected to protein extraction and LC-MS/MS identification.