Shotgun Proteomics Characterizes Coelomic Fluid Found in Purple Sea Urchin

The purple sea urchin, Strongylocentrotus purpuratus, has been studied previously as a model organism and in phylogenetic studies. As a part of this research, the S. purpuratus genome has been sequenced.^1,2 Sequencing of the S. purpuratus genome has revealed a highly complex immune system, including the presence of a complement system,³ antibacterial molecules,⁴ and scavenger receptor cysteine-rich proteins (SRCRs).⁵

To better understand the immunological functions of S. purpuratus, Dheilly et al. employed shotgun proteomics techniques to characterize proteins found within the coelomic fluid of S. purpuratus.⁶ Animals were obtained and kept in the laboratory for six months, and the coelomic fluid was tested to ensure the immune system of each animal remained active, according previous studies.⁷ For protein characterization, coelomic fluid was drawn from the peristomeum, the samples were freeze-dried, and the proteins were extracted.

Proteins were characterized using LC/MS/MS on a Thermo LCQ Deca ion trap mass spectrometer (Thermo Scientific). MS data was searched against the combined Strongylocentrotus database, which contained 44,037 downloaded protein sequences from NCBI. To be included in the final totals, results required tolerances of 2 Da or 0.2 Da, containing up to three missed tryptic cleavages and K/R-P cleavages, and the proteins needed to be present in at minimum two of the three animals analyzed. In addition, a protein required a log(e) value <-9 and a total of four spectral counts over the three samples. These criteria indicated a FDR of <1%.

Shotgun proteomics analysis was able to characterize 307 proteins, with 267 proteins predicted based on the genomic analysis of S. purpuratus. Proteins were grouped into 13 functional categories based on the NCBI database, blast searches, and gene ontology. These included proteins responsible for maintaining cell structure, shape, mobility, adhesion, proliferation, reproduction, and development. Some proteins also played a role in immune response, cell signaling, intracellular transport. In addition, there were also proteins functioning in stress response and detoxification, nucleic acid and protein metabolism, and processing detected.

The types of proteins included lysosomal proteins, proteases, and peptidases, as well as exchangers and ATPases. Three proteins did not fit into these categories and were labeled as “other,” and six proteins were previously undescribed with unknown function. The Dheilly group is now completing additional studies to further understand the interactions between coelomocytes (sea urchin blood cells), the specialized proteins found in this publication, and how the immune system of S. purpuratus might be affected.

References

1. Sodergren, E., et al. (2006) ‘The genome of the sea urchin Strongylocentrotus purpuratus‘, Science, 314 (5801), (pp. 941-952)

2. Tu, Q., et al. (2012) ‘Gene structure in the sea urchin Strongylocentrotus purpuratus based on transcriptome analysis‘, Genome Research, Oct 22 (10), (pp. 2079-2087)

3. Smith, L.C., et al. (2006) ‘The sea urchin immune system‘, Invertebrate Survival Journal, 3 (1) (pp. 25-39)

4. Li, C. (2009) ‘Two recombinant peptides, SpStrongylocins 1 and 2, from Strongylocentrotus purpuratus, show antimicrobial activity against Gram-positive and Gram-negative bacteria‘, Developmental and Comparative Immunology, 34 (3), (pp. 286-392)

5. Pancer, Z. (2000) ‘Dynamic expression of multiple scavenger receptor cysteine-rich genes in coelomocytes of the purple sea urchin‘, Proceedings of the National Academy of Sciences of the United States of America, 97 (24), (pp. 13156-13161)

6. Dheilly, N.M., et al. (2013) ‘Shotgun proteomics of coelomic fluid from the purple sea urchin, Strongylocentrotus purpuratus‘, Developmental and Comparative Immunology, published online January 23, 2013. doi: 10.1016/j.dci.2013.01.007

7. Clow, L.A., et al. (2000) ‘Expression of SpC3, the sea urchin complement component, in response to lipopolysaccharide‘, Immunogenetics, 51 (12), (pp. 1021-1033)