To determine if this was the case, we carried out tissue extractions using CD3OD, instead of CH3OH, in the extraction solvent. To eliminate variations that may result when comparing tissues extracted from different individuals, the paired eyestalk tissues were removed from one lobster. One eyestalk ganglion was placed in extraction solvent containing CH3OH and the other in extraction solvent containing CD3OD. The samples were homogenized, sonicated, and centrifuged, and the supernatant was separated from the tissue pellet. The samples were dried and reconstituted for analysis by MALDI-FTMS. The MALDI-FT mass spectra for the

two eyestalk tissue extracts show that the Orc[1-11]-OMe-derived peaks at m/z 1270.57, 1253.54, 894.43, 876.42, and 537.28 for the eyestalk extracted with CH3OH ( Fig. 8A and B) have all shifted by 3 Da to m/z 1273.59, 1256.56, 897.45, 879.44, and 540.30, for the eyestalk extracted with CD3OD ( Fig. 8C and D). MALDI-FT mass p38 MAPK apoptosis spectra and CID experiments carried out

on the Q-TOF also support our localization of the added methyl group at the C-terminus of the peptide. This localization is supported by the 3 Da mass shifts for the y8, y80, and y5 ion in the MALDI-FT mass spectra of eyestalks extracted with CD3OD (Fig. 8), which localizes the added methyl group to the C-terminal sequence, and by the 3 Da mass shift for y-type (but not b-type) ions produced in the selleck inhibitor Q-TOF MS/MS analysis (Fig. 7C and D). The most diagnostic fragment is the y1 peak, which undergoes a 3 Da mass shift (from m/z = 90.06 to 93.07) for the CD3-labeled peptide ( Fig. 7D). This Edoxaban fragment ion definitively localized the methyl addition to the C-terminus. These results document the incorporation of one CD3 group for Orc[1-11]-OMe

at the C-terminus and demonstrate that the methanolic extraction solvent is the source of the added methyl group. Acid-catalyzed esterifications have been recognized as the source of exogenous protein methylations that occur when methanol and acids are used, for example, in destaining SDS-PAGE gels [5], [17], [24] and [48]. In an early study by Haebel et al. [17], five test peptides were incubated in methanolic trichloroacetic acid (TCA) solutions (12.5:50:37.5, methanol:TCA:water) for 1–24 h to determine the propensity for methylation at different amino acid residues. The authors concluded that glutamate (E) undergoes the most rapid acid-catalyzed esterification, the C-terminus reacts with a rate that is lower by a factor of 2–6, and other groups (D, Q) are less reactive by at least a factor of 10 [17]. When acetic acid replaced TCA, methylation was not observed [17]. A direct chemical modification appeared to be unlikely as an explanation for the production of Orc[1-11]-OMe under our conditions, based upon the following observations and considering the work by Haebel et al. [17]. First, our data clearly show that methylation occurs, with 100% specificity, at the C-terminus.