Figure 3. The experimental workflow to analyze the proteome of Milnesium tardigradum.

Tardigrades in different states were homogenized directly in lysis buffer. Total protein extracts of tardigrades in early embryonic state and adult tardigrades in active and tun state were separated by 1D gel electrophoresis. After staining gel lanes were sliced and proteins in-gel digested with trypsin. MS/MS data obtained by nanoLC-ESI-MS/MS analysis were searched against the tardigrade specific database. The database was developed by translating EST sequences of M. tardigradum, which were obtained by 454 sequencing. Identified proteins with annotation were classified in different functional groups using the Blast2GO program. Identified proteins without annotation were analyzed with the DomainSweep program to search for specific protein domains.