For pathway A, selective detection of Orc[1-11]-OMe, but not Orc[1-12]-OMe, Erastin supplier would require a kinetic effect favoring water addition (hydrolysis) to form Orc[1-12], with methanol able to compete effectively following loss of the phenylalanine (F) residue. A second hypothesis, pathway B in Fig. 16, invokes an endopeptidase with specificity toward cleavage between the Gly-Phe peptide bond. Again, to rationalize production of Orc[1-11]-OMe, methylation would occur in conjunction with the enzymatic cleavage of the peptide bond. Finally, we note that an amidated orcokinin, NFDEIDRSGFamide (Orc[1-10]-NH2), has

been reported in the literature for H. americanus [30] and detected in our lab (data not shown). In this peptide, the C-terminal Gly11 residue (methylated in Orc[1-11]-OMe) is the residue targeted by the peptidylglycine enzymes responsible for converting Gly11 into an amide group. The specificity associated with methylation of the Gly11 residue may be related to formation

of an activated intermediate that is formed in the possible conversion of Gly11 to the amidated, Orc[1-10]-NH2 product. Further experimentation is clearly needed to determine if any of these speculations about the highly specific conversion observed in this study have merit. The truncated and C-terminally modified orcokinins, NFDEIDRSGFG-OMe (Orc[1-11]-OMe) and SSEDMDRLGFG-OMe

were identified in eyestalk tissue extracts and the conditions responsible for production were explored using mass spectrometry. We found that the truncation Osimertinib cell line not with C-terminal methyl esterification occurs as a result of the extraction procedure, but the reaction is not a simple chemical acid-catalyzed esterification. Experiments with enzyme inhibitors and the use of heat for enzyme deactivation supported an enzymatically mediated conversion of full-length orcokinins to the truncated, methylated NFDEIDRSGFG-OMe (Orc[1-11]-OMe) and SSEDMDRLGFG-OMe product. These products were not detected when tissues were analyzed directly. This study should heighten awareness regarding unexpected structural perturbations that may occur when neuropeptides are extracted from biological tissues. This project was supported by the National Science FoundationMRI-0116416 (E.A.S), CHE-1126657 (E.A.S.), and IBN-0111040 (P.S.D.); National Center for Research Resources (5P20RR016463-12) and the National Institute of General Medical Sciences (8 P20 GM103423-12) from the National Institutes of Health, institutional funds provided to A.E.C by MDIBL, the Surdna Foundation (fellowship to D.A.P.); the Merck Foundation and Henry L. and Grace Doherty Charitable Foundation Coastal Studies Research Fellowship (to E.B.). We thank Rachel Ackerman for her MALDI-FTMS analysis of H.