Figure 1.
SEM images of M. tardigradum in the active and tun state.
Tardigrades are in the active form when they are surrounded by at least a film of water. By loosing most of their free and bound water (>95%) anhydrobiosis occurs. Tardigrades begin to contract their bodies and change their body structure into a so-called tun.
Figure 2.
Comparison of different workup protocols for M. tardigradum.
Total protein extract of tardigrades in the active state was separated on a one-dimensional polyacrylamide gel. Lane 1: Rainbow molecular weight marker. Lane 2: Protein extract of whole tardigrades without any precipitation step. Lane 3: Protein extract after TCA precipitation. Lane 4: Protein extract after chloroform/methanol precipitation. Lane 5: Protein extract using clean-up kit.
Figure 3.
Analysis of protein degradation in total protein extracts of tardigrades by Western blot analysis.
Actin (A) and alpha tubulin (B) were used as marker proteins for the detection of proteolysis. Lane 1A and 1B: DualVue Western blotting marker. Lane 2A and 2B: Total protein extract of HeLa cells as control. Lane 3A and 3B: Total protein extract of M. tardigradum. Notably, no protein degradation was observed during the workup procedure.
Figure 4.
The experimental workflow to developing the proteome map.
Tardigrades were sonicated directly in lysis buffer. Total protein extracts were separated by two-dimensional gel electrophoresis. After silver staining protein spots were picked and in-gel digested with trypsin. MS/MS data obtained by LC-ESI-MS/MS analysis were searched against the NCBInr database, the clustered tardigrade EST database and the tardigrade protein database. Identified proteins with annotation were classified in different functional groups using the Blast2GO program. Identified proteins without annotation were analysed with the DomainSweep program to annotate protein domains.
Figure 5.
Image of a preparative 2D-gel with selected analysed protein spots.
Total protein extract of 400 tardigrades in the active state corresponding to 330 µg was separated by high resolution two-dimensional gel electrophoresis. Proteins were visualised by silver staining. Three different categories are shown: Identified proteins with functional annotation are indicated in green, identified proteins without annotation are indicated in blue and not yet identified proteins are indicated in red.
Figure 6.
Comparison of database performance for protein identification.
Protein spots were analysed by nanoLC-ESI-MS/MS and searched against the NCBInr database and the tardigrade protein database. The diagram illustrates the number of positive identifications in the respective database and the overlap between the two databases.
Table 1.
Overview of identified proteins classified in different functional groups.
Table 2.
Identified proteins with annotation.
Table 3.
Identified proteins without annotation.
Figure 7.
GO analysis of proteins identified in M. tardigradum.
A total of 271 spots representing 144 unique proteins was analysed with the Blast2GO program. The GO categories “molecular function” and “biological process” are shown as pie charts. A total of 9 different molecular function groups and 16 groups for biological processes are present in our result. The major parts of these categories (level 2) are shown in more detail (level 3) on the left and right side.
Figure 8.
Detection of hsp60 and hsp70 by Western blotting.
Total protein extract of M. tardigradum in the active state was separated on a one-dimensional polyacrylamide gel. Hsp60 (A) and hsp70 (B) could be immunodetected with high sensitivity. Lane 1A and 1B: DualVue Western blotting marker. Lane 2A and 2B: Total protein extract of HeLa cells. Lane 3A and 3B: Total protein extract of tardigrades. Notably, the protein bands in the HeLa control lysate show molecular weights of 60 and 70 kDa as expected. In contrast the detected protein band for hsp60 in M. tardigradum is considerably smaller. For hsp70 multiple bands are observed in M. tardigradum at higher as well as at lower molecular weights.
Figure 9.
Detection of hsp60 in six different tardigrade species by Western blotting.
Total protein extracts of tardigrades in the active state were separated on a one-dimensional polyacrylamide gel. Hsp60 (A) and actin (B) as loading control were immunodetected with high sensitivity. Lane 1: DualVue Western blotting marker. Lane 2: Total protein extract of HeLa cells. Lane 3: Total protein extract of M. tardigradum. Lane 4: Total protein extract of Paramacrobiotus richtersi. Lane 5: Total protein extract of Paramacrobiotus “richtersi group” 3. Lane 6: Total protein extract of Macrobiotus tonollii. Lane 7: Total protein extract of Paramacrobiotus “richtersi group” 2. Lane 8: Total protein extract of Paramacrobiotus “richtersi group” 1. Interestingly, the detected protein bands were ranging from 100 kDa to less than 24 kDa. Only hsp60 in the HeLa control lysate was detected at its expected position at 60 kDa.
Figure 10.
Statistical analysis of significant peptides found in the three different databases which were used to search the MS/MS data.
The number of significant peptide hits is compared between the different databases. When searching against the NCBInr database most proteins were identified with only one significant peptide hit. In contrast when using the tardigrade protein database most proteins were represented by two or more significant peptides.