Quantitation of cardioexcitatory Asn‐D‐Trp‐Phe‐NH₂ diastereomers in Aplysia's central nervous system by nanoscale liquid chromatography‐tandem mass spectrometry

Abstract

A tripeptide, Asn-D-Trp-Phe-NH(2) (NdWFa) that contains a D-amino acid residue (i.e. D-tryptophan) was previously identified in Aplysia's central nervous system (CNS) and found to be cardioexcitatory. However, the occurrence of its diastereomers including NWFa, theoretically the precursor of NdWFa, remains largely unknown. In this work, a nanoscale liquid chromatographic/tandem mass spectrometric (nano-LC-MS/MS) method was developed for a sensitive determination of the diastereomers of NWFa. Resolution of the diastereomers including NWFa, NdWFa, NWdFa, and dNWFa was achieved on capillary columns packed with C(18) silica particles with an MS detection-friendly mobile phase consisting of water, acetonitrile, and formic acid. Columns of different internal diameters (IDs) ranging from 75 to 250 microm were evaluated to achieve the best sensitivity. With the use of a 75 microm ID column integrated with a nanoelectrospray emitter, the method had limits of detection (LOD) of 0.21 nM (or 0.49 pg on column, 5 microl injected) NdWFa in tissue homogenate (S/N=3). The five major ganglia in Aplysia californica's CNS (i.e. buccal, cerebral, abdominal, plural, and pedal) were analyzed. NdWFa was detected only in abdominal ganglion at the ng/g tissue level. Further, its diastereomer, NWFa, was also detected for the first time and also only in abdominal ganglion at a significantly lower level. The levels of both NWFa and NdWFa varied from animal to animal in the range from 0 to 81 ng/g tissue.

Analysis of Peptide Stereochemistry in Single Cells by Capillary Electrophoresis–Trapped Ion Mobility Spectrometry Mass Spectrometry

“…For example, the relative abundance of another known DAACP in A. californica , NdWF-NH 2 , has been previously quantified in both single cells and bulk tissue extracts. It was found that the relative abundance of NdWF-NH 2 in whole ganglia homogenates was between 60 and 80%, whereas in single cells it was closer to 90%. , …”

Preparation and Evaluation of Long Chain Alkyl Methacrylate Monoliths for Capillary Chromatography

“…Methacrylates are now one of the most widely popular monoliths used as chromatographic separation media [21,22]. There are several advantages associated with using methacrylate-based polymers as monolithic stationary phases, including high stability in a wide range of mobile phase pH (2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12), fast and simple preparation and easy functionalization. The methacrylate monolithic columns have also various selectivities towards monomers with wide ranging polarities [23,24].…”

“…Capillary HPLC offer several advantages over conventional normal scale HPLC. The advantages include increased chromatographic resolution, higher efficiency, lower sample and solvent consumption, the ability to analyze and isolate rare compounds of interest, greater mass sensitivity and ease of on-line connection to mass spectrometer [1][2][3].…”

Surfactant-bound monolithic columns for separation of proteins in capillary high performance liquid chromatography

“…The advantages include increased chromatographic resolution, higher efficiency, lower sample consumption, the ability to analyze and isolate rare compounds of interest, reduced solvent consumption, greater mass sensitivity [1–4] and convenient online connection to mass spectrometer [5–7]. …”