Imaging mass spectrometry (IMS) of neuropeptides in crustacean neuronal tissues was performed on a MALDI-TOF/TOF instrument. Sample preparation protocols were developed for the sensitive detection of these highly complex endogenous signaling molecules. The neuromodulatory complements of the pericardial organ (PO) and brain of the Jonah crab, Cancer borealis, were mapped. Distributions of peptide isoforms belonging to 10 neuropeptide families were investigated using the IMS technique. Often, neuropeptides of high sequence homology were similarly located. However, two RFamide-family peptides and a truncated orcokinin peptide were mapped to locations distinct from other members of their respective families. Over 30 previously sequenced neuropeptides were identified based on mass measurement. For increased confidence of identification, select peptides were fragmented by post-source decay (PSD) and collisional-induced dissociation (CID). Collectively, this organ-level IMS study elucidates the spatial relationships between multiple neuropeptide isoforms of the same family as well as the relative distributions of neuropeptide families.

Herein we describe a novel method for quantitation using a Fourier transform mass spectrometer (FTMS) equipped with a MALDI ion source. The unique instrumental configuration of FTMS and its ion trapping and storing capabilities enable ion packets originating from two physically distinct samples to be combined in the ion cyclotron resonance (ICR) cell prior to detection. These features are exploited to combine analyte ions from two differentially labeled samples spotted separately and then combined in the ICR cell to generate a single mass spectrum containing isotopically paired peaks for quantitative comparison of relative ion abundances. The utility of this new quantitation via in cell combination (QUICC) approach is explored using peptide standards, a bovine serum albumin tryptic digest, and a crude neuronal tissue extract. We show that spectra acquired using the QUICC scheme are comparable to those obtained from premixing the isotopically labeled samples in solution. In addition, we show direct tissue in situ isotopic formaldehyde labeling of a crustacean neuroendocrine organ, thus demonstrating the potential application of the QUICC methodology for direct tissue quantitative analysis.

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In the extraction of oils from seeds of the genus Coriandrum, GC separations of petroselinate from oleate often gave poor resolution of these two isomers. Oleic and petroselinic acids were esterified with a series of alcohols (methanol, ethanol, 1-propanol, 2-propanol, 2-methyl-1-propanol, 1-butanol, 3-methyl-1-butanol, and 2-ethyl-1-hexanol). GC resolution of the ∆6 from the ∆9 and ∆11 octadecenoates was examined for all ester derivatives on a polar phase column. The ∆6 and ∆9 isomers were unresolved as methyl esters; however, the 2-ethyl-1-hexyl esters gave baseline separation of all three isomers under temperature programming conditions. When isothermal conditions were optimized for each ester, separation of these isomers was possible with good resolution values (>89%) for all the alcohols except methanol, which had a partial resolution of 51%. The rates of esterification of all the alcohols were determined for reactions with both oleic acid and triolein using potassium hydroxide as the esterification catalyst. Methanol gave the largest rate constant in both acid and oil esterification reactions with a rate constant 10-fold better than all of the other alcohols. Based on rates of reaction, resolution of petroselinate from oleate, and removal of residual alcohol, the ethyl ester derivative appears to be the best choice for seed oils containing petroselinic acid.Petroselinic acid is present in a number of seed oils but is most commonly found in the Umbelliferae family. One interest in this acid is that it could serve as a useful raw material for producing adipic and lauric acids via oxidative cleavage of the ∆6 double bond in petroselinic acid. Adipic acid is commonly used as a monomer for nylon synthesis, and lauric acid is used in detergent applications. Analysis of petroselinic acid in natural oils by GC is typically confounded by the presence of oleic acid. When methyl esters of petroselinic and oleic acids are synthesized, the GC separation of these two isomers is incomplete (1,2). Separation of petroselinate from oleate was reported to have only a 0.04 equivalent chain length (ECL) unit resolution, which made quantification of the individual isomers difficult.In an effort to overcome these quantification problems, several tedious chromatographic separation or derivatization techniques have been devised. Kleiman (3) used a combination of silver ion chromatography to isolate the monoene fraction of Umbelliflorae seed oils followed by ozonolysis and GC quantification of the cleavage products. Santinelli (4) epoxidized the olefins and then converted the epoxides to chlorohydrins via a hydrochloric acid-mediated ring opening. The resulting chlorohydrins were converted to their trimethylsilyl ethers, which were partially resolved by GC. Thies (5) treated the FA with dimethyldisulfide to make the bis-methylthio derivatives, which gave only partial separation by GC.Good separation of the ∆6 and ∆9 isomers has been reported using phenylethyl (6), isopropyl (7), and butyl (8-10) esters. The phenylethyl ester gives n...

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