2A); most peptides eluted within a narrow range

of retention times in reversed-phase chromatography, approximately 17–27 min (20–33% acetonitrile). A total of 113 peptide components were found ( Table 1, Fig. 3A), ranging from 1275.9 Da to 8615.5 Da, with the highest frequency between 1500 and 2000 Da ( Fig. 3D). On the other hand B. granulifera (Bg-3-4) yielded 53 fractions from a more complex reversed-phase profile ( Fig. 2B), exhibiting a richer elution pattern in relation to S. helianthus, in the range 10–35 min (12–42% acetonitrile). The B. granulifera neurotoxic fraction ( Table 2, Fig. 3E) also yielded a larger number of peptide components (156), with molecular masses from 1221.6 Da to 6983.1 Da, but more frequently within the range of 4500–5000 Da ( Fig. 3E). B. granulifera and B. cangicum [85], which belong to the same genus, share a similar complexity regarding their reversed-phase profiles ( Fig. 2B and C), being the INNO-406 in vivo compound screening assay group of highly abundant and hydrophobic 4–5 kDa peptides with tR > 25 min (>32% acetonitrile) their most distinguishable feature. However, only 81 different molecular masses were found in B. cangicum, 78 of them above 1000 Da; with the highest occurrence within the range of 4500–5000 Da ( Fig. 3F), similarly to B. granulifera, mainly due to the

last eluting intense peaks mentioned above. On the contrary, such cluster of abundant and hydrophobic 4–5 kDa peptides is absent in S. helianthus. A common feature of these sea anemone SSR128129E species is the presence of a notable peptide population in the range of 1.5–2 kDa (Fig. 3D–F). In both Bunodosoma species these peptides are present among the early eluting fractions ( Fig. 3B and C), whereas in S. helianthus they can be found scattered throughout the reversed-phase profile ( Fig. 3A). Known sea anemone peptides isolated from S. helianthus

and B. granulifera were identified by comparing their molecular masses with our experimental values. Thus ShI (5136.8 Da) [43] was located in fraction Sh 27.26 (5139.1 Da), ShPI-2 (6197.0 Da) [22] in fraction Sh 17.55 (6196.2 Da), BgII (5071.6 Da) and BgIII (5072.6 Da) [52] in fractions Bg 26.91a (5068.9 Da) and Bg 26.91b (5071.9 Da), respectively, and BgK (4275.9 Da) [2] and [18] in fraction Bg 16.07a (4275.8 Da). ShK (4054.8 Da) [14] and ShPI-1 (6109.9 Da) [22] could not be identified among the reversed-phase fractions. Unlike other venomous animals [19], [27] and [29], not a single sea anemone neurotoxin has been found in two or more species even belonging to the same genus. In the previous peptidomic study of a sea anemone, the peptides Bcg 25.96 (B. cangicum) and BcIII (Bunodosoma caissarum) exhibited identical reversed-phase chromatographic behavior and molecular masses, but it still remains to be confirmed whether these two peptides are the same toxin. In the present work we found a total of 269 peptides, most of them presumably new.