americanus eyestalk tissues. The isobaric peptide Orc[Ala11] has been localized to H. americanus eyestalk ganglion and sinus glands by Li and co-workers [30]. The fact that Orc[1-11]-OMe is isobaric with

previously reported Orc[Ala11] lead us to wonder whether Orc[Ala11] was misidentified in previous studies or if Orc[Ala11] is a neuropeptide endogenous to the lobster eyestalk ganglia. Misidentification is a possibility, especially when considering the fact that most MS/MS measurements would not reveal a structural difference between the two peptides (described above). To address these concerns, we attempted to detect Orc[Ala11] using eyestalk extracts prepared using two non-methanolic extraction techniques, namely, extraction using HCl-acidified acetone [49] and extraction

with aqueous, saturated DHB [37]. These solvent systems should preclude the formation of Orc[1-11]-OMe and reveal any R428 Orc[Ala11] that may have been overshadowed by Orc[1-11]-OMe, particularly if that peptide was present at higher abundance. These approaches should then provide two additional measures, complementing our data on heat-deactivated methanolic extractions BIRB 796 where no evidence for Orc[Ala11]/Orc[1-11]-OMe was found (see Fig. 11B). In measurements with acidified acetone (see Fig. 14) and saturated DHB (data not shown), where we extracted single eyestalk ganglia from a minimum of three individuals, no peaks characteristic of Orc[Ala11] were detected by MALDI-FTMS. Because Orc[Ala11] was previously detected in the SG and stomatogastric nervous system of H. americanus [30], we carried out a detailed reexamination of MALDI-FTMS spectra generated using extracts from single sinus glands and paired commissural ganglia (CoGs), which were reported in a previous study from our laboratory [10]. In this study, sinus glands and CoGs were analyzed by MALDI-FTMS using direct tissue analysis, analysis of methanolic tissue extracts, and analysis of methyl-esterified tissue extracts. The last method of sample preparation,

see more in particular, allows the differentiation of Orc[1-11]-OMe and Orc[Ala11]. Specifically, while Orc[1-11]-OMe undergoes a mass shift to m/z 1312.62 following acid-catalyzed methyl esterification of the two aspartate and single glutamate residues, any Orc[Ala11], with a free C-terminal carboxylic acid, would undergo a mass shift to m/z 1326.63 resulting from the esterification of four, not three, acidic residues. A careful reexamination of these previously acquired data [10] showed no peaks characteristic of Orc[1-11]-OMe/Orc[Ala11] for direct tissue analysis, low abundance peaks in some, but not all, sinus gland and CoG extracts, low abundance peaks m/z = 1312.62 in some, but not all, sinus gland and CoG methyl esterified, and no peaks characteristic of methyl esterified Orc[Ala11] at m/z = 1326.63 in the methyl esterified extracts.