Figure 1.
Separation of protein lysates of tardigrades in three different states by one-dimensional polyacrylamide gel electrophoresis.
Lane 1: Rainbow molecular weight marker. Lane 2: Protein extract of adult tardigrades in active state (AS). Lane 3: Protein extract of adult tardigrades in tun state (TS). Lane 4: Protein extract of tardigrades in early embryonic state (EES). Bottom. SEM-images of M. tardigradum in the corresponding states.
Table 1.
Semi-quantitative analysis of selected proteins associated with diverse processes such as response to stimulus, protection and development.
Table 2.
Major protein components in early embryonic state (EES).
Table 3.
Major protein components in adult tardigrades in active and tun state. 53 proteins were found as major components in adult tardigrades in AS and 49 in TS.
Figure 2.
Comparative proteome analysis of proteins identified in different states.
The Venn diagram illustrates the number of protein identifications in EES, AS and TS. A total of 1301 proteins were found in all three states, 472 proteins are found only in EES (a) and 680 proteins are found only in adult tardigrades in TS and AS (f). Proteins which are non-overlapping (a, b, c) or partially overlapping (d, e, f) between the different states are analyzed using Blast2GO program to determine the involved biological processes. The ten major biological processes for non-overlapping proteins are listed in 2a-2c and for partially overlapping proteins in 2d–2f.
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.
Figure 4.
Comparative expression analysis of selected proteins.
Total emPAI is the sum of emPAI of each protein member. Large lipid transfer protein (LLTP) superfamily belong to major components in all three states. This protein family is highly expressed in EES compared to adults. Semi-quantitative analysis of proteins contributing to the structural integrity of ribosome indicates a significant up-regulation of ribosomal proteins in EES. In contrast proteins involved in cytoskeletal-, muscle- and vitellin membrane structure are not highly abundant at this state compared to adults in AS and TS. Proteins with antioxidant activity such as GSTs are approximately 3 fold higher in adults compared to EES. In contrast Cu-Zn SODs are upregulated in EES.