We also noticed that signals for Orc[1-11] were also reduced with inclusion of the inhibitor cocktail. Upon carrying out multiple trials making use of the inhibitor cocktail, we consistently found reduced levels of both Orc[1-11]-OMe and Orc[1-11] when the inhibitor was present; however, inhibition was never complete. Regardless, these results provide evidence to support the hypothesis that an enzyme

participates in production of the Orc[1-11]-OMe product. Heat has been used an effective means to reduce proteolytic degradation of proteins when processing vertebrate tissue samples [9], Sirolimus manufacturer [12], [13] and [44]. Working from the hypothesis that an enzyme plays a role in promoting the formation of Orc[1-11]-OMe during extraction of eyestalk tissues, we attempted to deactivate enzymatic components with heat. To test this approach we removed the paired eyestalk ganglia from one lobster. The ganglion from a single eyestalk was placed in a microcentrifuge tube with 50 μL of extraction solvent and the tightly capped tube was placed in a boiling Olaparib mw water bath for 5 min. Concurrently, the ganglia from the second

eyestalk of the same lobster were placed in extraction solvent and left at room temperature for 5 min. Both eyestalk tissue samples were then homogenized, sonicated, and centrifuged prior to MALDI-FTMS analysis. While the control eyestalk extract showed the Orc[1-11]-OMe-derived peaks at m/z 1270.57, 1253.54, 894.43, 876.42, and 537.28 (see Fig. 11A), no evidence for these peaks was found for the tissue/extraction solvent mixture that was placed in the boiling water bath for 5 min ( Fig. 11B and C). We also did not detect

IKBKE the truncated peptide, Orc[1-11]. When this approach was replicated (n > 6), the treatment consistently eliminated the production of Orc[1-11]-OMe and Orc[1-11]. We also tried freezing eyestalk ganglion tissues in liquid nitrogen before homogenizing and adding extraction solvent, but found that this treatment did not measurably reduce production of Orc[1-11]-OMe. Many enzymes are known to function in aqueous-methanolic solvent mixtures [2] and [38]; however, enzymatic activity is generally reduced or eliminated when the water content is reduced [22] and [25]. We hypothesized that, if an enzyme plays a role in the production of Orc[1-11]-OMe, production of the peptide would be reduced if the extraction solvent contained a lower percentage of water. To determine if the percentage of water in the extraction solvent influenced the extent of methylation, we extracted eyestalk ganglia in solvents containing 1–30% water.