Orcokinin family GW-572016 solubility dmso peptides have been characterized in many crustacean species. Of the many characterized,

full-length orcokinins [7], glycine, not alanine, is found exclusively at the 11th position in the sequence. Although genomic data for crustaceans is sparse, the available information documents no genes encoding full-length orcokinins with Ala11; furthermore, no genes have been found for any truncated orcokinin variants. This sequence analysis, coupled with our demonstration that isobaric Orc[1-11]-OMe is an extraction artifact, has led us to question the identity of previously published truncated orcokinin family peptides with an alanine, not glycine, at the 11th position. This concern has been supported by our analysis of H. americanus tissues using approaches that either exclude methanol or permit differentiation of Orc[1-11]-OMe/Orc[Ala11], where

we failed to find any evidence of putative Orc[Ala11]. The truncated orcokinin, Orc[Ala11], was first reported by Huybrechts et al. as a novel peptide de novo sequenced from the Jonah crab, C. borealis [21]. For that study, brain and thoracic ganglion BTK inhibitor tissues from 50 animals were extracted in methanol:water:acetic acid (90:9:1) and peptides were sequenced using ESI-Q-TOF MS/MS. As was the case for H. americanus, this peptide sequence, with an alanine appearing as the 11th residue, is at odds with the sequences of full-length C. borealis orcokinin peptides, which have been established by many MS studies [21] and [32]. We suggest that the peptide reported by Huybrechts et al. is, in fact, Orc[1-11]-OMe; this assertion is supported by work carried out in our laboratory, where we have evidence for the detection of Orc[1-11]-OMe in C. borealis brain tissue extracts

(data not shown), but not for any directly analyzed C. borealis tissues (CoG, SG, PO, brain) (data not shown). Because alanine (A) is isobaric with methylation 3-oxoacyl-(acyl-carrier-protein) reductase at a C-terminal glycine residue (G-OMe), this distinction would not have been revealed from mass measurements. Furthermore, the MS/MS technique used for de novo peptide sequencing in the Huybrechts et al. study would not have provided any obvious flags to distinguish the C-terminal alanine from G-OMe. The orcokinins NFDEIDRSGFA, SSEDMDRLGFA, and NFDEIDRSSFA, all with an alanine as the 11th residue and all detected in tissues that had been analyzed following extraction with acidified methanol, have been reported in other publications [15], [16], [21], [30], [31] and [32]. In summary, our work calls into question the identification of these truncated peptides, which may have Gly-OMe, not Ala, at the C-terminus. A unique issue for the in vitro modification detected in this study is the localized methylation at the C-terminus. Because the Gly-OMe modification is isobaric with Ala-OH, this structural change is not detectable via mass measurements.