Combining tissue extraction and off-line capillary electrophoresis matrix-assisted laser desorption/ionization Fourier transform mass spectrometry for neuropeptide analysis in individual neuronal organs using 2,5-dihydroxybenzoic acid as a multi-functional agent

Abstract

In this study we report an improved protocol that combines simplified sample preparation and microscale separation for mass spectrometric analysis of neuropeptides from individual neuroendocrine organs of crab Cancer borealis. A simple, one-step extraction method with commonly used matrixassisted laser desorption/ionization (MALDI) matrix, 2,5-dihydroxybenzoic acid (DHB), in saturated aqueous solution, is employed for improved extraction of neuropeptides. Furthermore, a novel use of DHB as background electrolyte for capillary electrophoresis (CE) separation in the off-line coupling of CE to MALDI-Fourier transform mass spectrometric (FT-MS) detection is also explored. The new CE electrolyte exhibits full compatibility with MALDI-MS analysis of neuropeptides in that both the peptide extraction process and MALDI detection utilize DHB. In addition, enhanced resolving power and improved sensitivity are also observed for CE-MALDI-MS of peptide mixture analysis. Collectively, the use of DHB has simplified the extraction and reduced the sample loss by elimination of homogenizing, drying, and desalting processes. In the mean time, the concurrent use of DHB as CE separation buffer and subsequent MALDI-MS detection offers improved spectral quality by eliminating the interferences from typical CE electrolyte in MALDI detection.

Crustacean neuropeptides

“…Capillary electrophoresis techniques coupled with MALDI-FTMS analysis have been applied to crustacean tissue extracts [70,71]. Li and co-workers have developed immunoaffinity-based enrichment techniques (immunoprecipitation and immunodot blot screening), coupled with MALDI-FTMS and nanoLC-ESI-Q-TOF MS/MS, for the targeted analysis of FMRFamide-related peptides in the PO of Cancer borealis [72].…”

Advancing Matrix-Assisted Laser Desorption/Ionization-Mass Spectrometric Imaging for Capillary Electrophoresis Analysis of Peptides

“…The same sample mixture was analyzed previously by discrete spotting and detection with MALDI-FTMS. 34 In that report, multiple peptides in one fraction or the splitting of single peptide into multiple fractions were common by using 1-min interval collection (data not shown here). It was noted that visible peak splitting was also observed for two peptides in the current study, as reflected in the electropherogram (peaks 3 and 5 in Figure 3A) or the imaging traces (Figure 2D and 2F).…”

Analysis of low molecular weight compounds by MALDI-FTICR-MS

“…When the ball has rotated one-half turn, it is in position for laser desorption and following analysis [57][58][59]. The combination of separation methods with MALDI-FTICR-MS has been wildly applied in various fields including comparative neuropeptidomic analysis in crustacean model organisms in conjunction with stable isotopic labeling [60], analysis of neuropeptides from individual neuroendocrine organs of crab cancer borealis [61,62], structural characterization of toxin-binding gangliosides [63,64] and the studies of isotope beating effects in the analysis of polymer distributions [65].…”