047 Da) is accounted for by an alanine (A) residue (71.037 Da) and water (18.011 Da), consistent with the sequence for putative Orc[Ala11]; however, we were surprised that the mass spectrum did not show a [b4+H2O]+ product ion at m/z 466.24. The [bn+H2O]+ ion is a C-terminal fragment that we have detected at >30% abundance in the SORI-CID spectra of yn+1 ions derived from many orcokinin family peptides, including Orc[1-12] [43], [Val13] [43], and [Ala13] (data not shown). The absence of this characteristic peak led us to question the sequence assigned to putative Orc[Ala11]. To more conclusively establish if the m  /z   1270.57 peptide is, in fact, Orc[Ala11], we measured

SORI-CID mass spectra for a synthetic form of the peptide. As shown CYC202 cell line in Fig. 4B, SORI-CID of the m  /z   1270.57, [M+H]+, peak yields a spectrum that closely resembles that of the eyestalk extract-derived peptide; however, we note that the intensity Anti-infection Compound Library of the y8 peak (m  /z   894.43) for the standard ( Fig. 4B) is consistently lower than that observed for the putative Orc[Ala11] peptide ( Fig. 4A). While this mass spectral difference was reproducible, the fact that the mass spectrum is dominated by Asp-Xxx cleavage ions (y8, y8o, and y5) limited our ability to carry out a more detailed comparison

of structural features. In contrast, SORI-CID of the y5 peak at m/z 537.28 proved to be more revealing. While similar ions and ion intensities were detected in the lower m/z range of the spectrum, more significant differences in ion intensities and ion identity were observed at higher m/z values ( Fig. 5B). Most notably, the SORI-CID spectrum of the Orc[Ala11]-derived y5 peak ( Fig. 5B) shows an abundant [b4+H2O]+ product ion at m/z 466.24, which was not detected in the spectrum of the eyestalk extract-derived peptide ( Fig. 5A). Production of the [b4+H2O]+ ion from the Orc[Ala11]-derived Lck y5 ion is congruent with predicted fragmentation behavior, based upon studies of

other orcokinin peptides (described above). The fact that this peak is not detected in the spectrum of putative Orc[Ala11] provides defining evidence that our eyestalk extract-derived peptide is not Orc[Ala11]. Furthermore, when structural elements that would block formation of the [b4+H2O]+ ion are considered, we are able to propose a sequence for the eyestalk-derived m/z 1270.56 orcokinin peptide. Specifically, the mechanism responsible for the production of [bn−1+H2O]+ product ions has been investigated, and it is known that ion formation involves a rearrangement at the C-terminus that requires a free C-terminal carboxyl group [45]. This rearrangement is prevented when the C-terminus is blocked by amidation or esterification. Based upon this information, we hypothesized that the m/z 1270.57, eyestalk extract-derived peptide was not Orc[Ala11], but was, instead, NFDEIDRSGFG-OMe (also m/z 1270.