When a peptide sequence containing this amino acid combination is coupled with the energy imparted by the vacuum UV-MALDI ionization and the long trapping times required for FTMS analysis (>10 s), singly protonated orcokinin family peptides undergo so-called “Asp-Xxx cleavages” [47], which result in the production of characteristic C-terminal (y-type)

fragments (see Fig. 2B). Our identification of orcokinin family peptides by MALDI-FTMS relies on the detection of both the [M+H]+ ion and the observation of characteristic y-type ions resulting from Asp-Xxx cleavages. When we analyzed small pieces of eyestalk ganglion tissues directly by MALDI-FTMS, we detected neuropeptide peak profiles 17-AAG cost that reflected differential Gamma-secretase inhibitor distributions of neuropeptides in localized regions of the eyestalk ganglia. For example, the peptides CabTRP I (APSGFLGMRamide at m/z 934.49) and Val1-SIF (VYRKPPFNGSIFamide at m/z 1423.78) were detected in tissues from the LG, XO/MT, MI, and ME but not in the SG. Orcokinin family peptides were detected in many tissues, including the XO/MT, MI, ME, and SG. A representative spectrum from a small piece of XO/MT tissue is shown in Fig. 3A. In contrast with previous studies [10], where we found good agreement between single tissues analyzed directly and by single tissue extraction, our analysis

of 3-mercaptopyruvate sulfurtransferase extracts of tissues from the aforementioned regions of the eyestalk ganglion revealed the presence of a new peptide that had not been detected by direct tissue MALDI-FTMS. For example, Fig. 3C shows the spectrum observed when a small piece of XO/MT tissue was removed by microdissection techniques, placed in extraction solvent, homogenized, sonicated, and centrifuged. While we continue to detect peaks for CabTRP

I and Val1-SIF, an abundant signal at m  /z   1270.57 was observed, which was detected in combination with additional peaks showing the characteristic orcokinin family pattern. The full collection of peaks appeared at m  /z   1270.57, 1253.54, 894.43, 876.42, and 537.28; these peaks were assigned to [M+H]+, [MH−NH3]+, yn+4, yn+4o, and yn+1. Unexpectedly, these masses did not correspond to any orcokinin family members predicted from genomic information for H. americanus [10], nor did they correspond to masses expected from conventional post-translational modifications, including the truncation of full-length orcokinin family peptides. Instead, exact mass measurements (m/z 1270.5692, measured) were consistent with the orcokinin sequence, NFDEIDRSGFA (Orc[Ala11]; m/z 1270.5699, predicted). This peptide has been detected in other studies [4], [16], [19], [30] and [31] with the first characterization, from the crab, C. borealis, reported in a study by Huybrechts et al.