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 (original) (raw)
Related papers
Expanding the Crustacean Neuropeptidome Using a Multifaceted Mass Spectrometric Approach
Journal of Proteome Research, 2009
Jonah crab Cancer borealis is an excellent model organism long served for many areas of physiology, including the study of endocrinology and neurobiology. Characterizing the neuropeptides present in its nervous system provides the first critical step toward understanding the physiological roles of these complex molecules. Multiple mass spectral techniques were used to comprehensively characterize the neuropeptidome in C. borealis, including matrix assisted laser desorption/ionization Fourier transform mass spectrometry (MALDI FTMS), MALDI time of flight (TOF)/TOF MS and nanoflow liquid chromatography coupled to electrospray ionization quadrupole time of flight tandem mass spectrometry (nanoLC ESI Q TOF MS/MS). In order to enhance the detection signals and expand the dynamic range, direct tissue analysis, tissue extraction, capillary electrophoresis (CE) and off-line HPLC separation have also been employed. In total, 142 peptides were identified, including 85 previously known C. borealis peptides, 22 peptides characterized previously from other decapods, but new to this species, and 35 new peptides de novo sequenced for the first time in this study. Seventeen neuropeptide families were revealed including RFamide, allatostatin (A and B type), RYamide, orcokinin, orcomyotropin, proctolin, crustacean cardioactive peptide (CCAP), crustacean hyperglycemic hormone precursor-related peptide (CPRP), crustacean hyperglycemic hormone (CHH), corazonin, pigment-dispersing hormone (PDH), tachykinin, pyrokinin, SIFamide, red pigment concentrating hormone (RPCH) and HISGLYRamide. Collectively, our results greatly increase the number and expand the coverage of known C. borealis neuropeptides, and thus provide a stronger framework for future studies on the physiological roles played by these molecules in this important model organism.
Analytical Chemistry, 1998
The combination of two sensitive and powerful analytical techniques on the same biological sample was examined: (i) matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), which gives informative peptide profiling on complex samples such as organs or cells; (ii) immunological tools such as enzyme-linked immunosorbent assay (ELISA) and immunocytochemistry to probe for specific peptides in biological extracts or cells. The cellular expression of the two precursors of the hyperglycemic hormone (cHH) was analyzed in neurosecretory cells (30-micron diameter) from the crayfish Orconectes limosus. Neurohemal organs were used to optimize the sample preparation and to demonstrate that, after peptide fingerprinting by MALDI-TOF MS, the sample can be recovered from the MALDI plate for further immunological analysis by ELISA. It was also established that, after immunocytochemistry following 4% paraformaldehyde fixation of the organ, the stained tissue could be recovered for further MALDI-TOF MS analysis. This dual characterization was successfully scaled down to the level of a single crayfish neurosecretory cell. Direct peptide profiling by MALDI-TOF MS on a single cHH-producing cell previously identified by immunocytochemistry demonstrated that both procHH isoforms were expressed in each cell analyzed.
Measurement of neuropeptides in crustacean hemolymph via MALDI mass spectrometry
Journal of the American Society for Mass Spectrometry, 2009
Neuropeptides are often released into circulatory fluid (hemolymph) to act as circulating hormones and regulate many physiological processes. However, the detection of these low-level peptide hormones in circulation is often complicated by high salt interference and rapid degradation of proteins and peptides in crude hemolymph extracts. In this study, we systematically evaluated three different neuropeptide extraction protocols and developed a simple and effective hemolymph preparation method suitable for MALDI MS profiling of neuropeptides by combining acid-induced abundant protein precipitation/depletion, ultrafiltration, and C 18 micro-column desalting. In hemolymph samples collected from the crab Cancer borealis, several secreted neuropeptides have been detected, including members from at least five neuropeptide families, such as RFamide, allatostatin, orcokinin, tachykinin-related peptide (TRP), and crustacean cardioactive peptide (CCAP). Furthermore, two TRPs were detected in the hemolymph collected from food-deprived animals, suggesting the potential role of these neuropeptides in feeding regulation. In addition, a novel peptide with a Lys-Phe-amide C-terminus was identified and de novo sequenced directly from the Cancer borealis hemolymph sample. To better characterize the hemolymph peptidome, we also identified several abundant peptide signals in C. borealis hemolymph that were assigned to protein degradation products. Collectively, our study describes a simple and effective sample preparation method for neuropeptide analysis directly from crude crustacean hemolymph. Numerous endogenous neuropeptides were detected, including both known ones and new peptides whose functions remain to be characterized.
Mass spectrometric elucidation of the neuropeptidome of a crustacean neuroendocrine organ
Peptides, 2012
The blue crab Callinectes sapidus has been used as an experimental model organism for the study of regulation of cardiac activity and other physiological processes. Moreover, it is an economically and ecologically important crustacean species. However, there was no previous report on the characterization of its neuropeptidome. To fill in this gap, we employed multiple sample preparation methods including direct tissue profiling, crude tissue extraction and tissue extract fractionation by HPLC to obtain a complete description of the neuropeptidome of C. sapidus. Matrix-assisted laser desorption/ionization (MALDI)-Fourier transform mass spectrometry (FTMS) and MALDI-time-of-flight (TOF)/TOF were utilized initially to obtain a quick snapshot of the neuropeptide profile, and subsequently nanoflow liquid chromatography (nanoLC) coupled with electrospray ionization quadrupole time-of-flight (ESI-Q-TOF) tandem MS analysis of neuropeptide extracts was conducted for de novo sequencing. Simultaneously, the pericardial organ (PO) tissue extract was labeled by a novel N, N-dimethylated leucine (DiLeu) reagent, offering enhanced fragmentation efficiency of peptides. In total, 130 peptide sequences belonging to 11 known neuropeptide families including orcomyotropin, pyrokinin, allatostatin A (AST-A), allatostatin B (AST-B), FMRFamide-like peptides (FLPs), and orcokinin were identified. Among these 130 sequences, 44 are novel peptides and 86 are previously identified. Overall, our results lay the groundwork for future physiological studies of neuropeptides in C. sapidus and other crustaceans.
Direct Sequencing of Neuropeptides in Biological Tissue by MALDIāPSD Mass Spectrometry
Analytical Chemistry, 1999
Dissected tissue pieces of the pituitary pars intermedia from the amphibian Xenopus laevis was directly subjected to matrix-assisted laser desorption/ionization (MALDI) mass analysis. The obtained MALDI peptide profile revealed both previously known and unexpected processing products of the proopiomelanocortin gene. Mass spectrometric peptide sequencing of a few of these neuropeptides was performed by employing MALDI combined with postsource decay (PSD) fragment ion mass analysis. The potential of MALDI-PSD for sequence analysis of peptides directly from unfractionated tissue samples was examined for the first time for the known desacetyl-r-MSH-NH 2 and the presumed vasotocin neuropeptide. In addition, the sequence of an unknown peptide which was present in the pars intermedia tissue sample at mass 1392.7 u was determined. The MALDI-PSD mass spectrum of precursor ion 1392.7 u contained sufficient structural information to uniquely identify the sequence by searching protein sequence databases. The determined amino acid sequence corresponds to the vasotocin peptide with a C-terminal extension of Gly-Lys-Arg ("vasotocinyl-GKR"), indicating incomplete processing of the vasotocin precursor protein in the pituitary pars intermediate of X. laevis. Both vasotocin and vasotocinyl-GKR are nonlinear peptides containing a disulfide (S-S) bridge between two cysteine residues. Interpretation of the spectra of these two peptides reveals three different forms of characteristic fragment ions of the cysteine side chain: peptide-CH 2 -SH (regular mass of Cys-containing fragment ions), peptide-CH 2 -S-SH (regular mass + 32 u) and peptidedCH 2 (regular mass -34 u) due to cleavage on either side of the sulfur atoms.
1991
Capillary zone electrophoresis is fast becoming one of the most sensitive separation schemes for sampling complex mnicroenvironmnents. A unique detection scheme is developed in which a chargecoupled device (CCD) detects laser induced fluorescence from an axially illuminated electrophoresis capillary. The fluorescence from an analyte band is measured over a several centimeter section of the capillary, greatly increasing the observation time of the fluorescently tagged band. The sensitivity of the system is in the 1-8 x 1O20 mnol range for derivatized amino acids and peptides. Subattomnole quantities of bag cell neuropeptides collected from the giant marine mollusk Aplysia californica can be measured.
Imaging Mass Spectrometry of Neuropeptides in Decapod Crustacean Neuronal Tissues
Journal of Proteome Research, 2007
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 postsource 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.
ELECTROPHORESIS, 2008
In this study, the suitability of SPE coupled on-line to CE-electrospray-MS (SPE-CE-ESI-MS) was evaluated for the analysis of neuropeptides in human plasma. First, CE-ESI-MS was investigated and a sample pretreatment based on precipitation with ACN was used for cleanup of plasma samples. The main quality parameters were determined and were consistent with those previously obtained for the analysis of standard mixtures, e.g., the LODs were of around 1 mg/mL. SPE-CE-ESI-MS employing microcartridges containing a C18 stationary phase was explored in order to decrease the LODs. A double-step sample cleanup pretreatment consisting of precipitation with ACN and ultrafiltration through 3000 Da MW cut-off membranes was needed to prevent the microcartridge saturation. Repeatability, reproducibility, linearity, and LODs of the SPE-CE-ESI-MS method and the durability of the microcartridges were studied. The LODs were markedly improved, ranging between 10 and 0.1 ng/mL depending on the peptide.
Enhanced neuropeptide profiling via capillary electrophoresis off-line coupled with MALDI FTMS
Analytical chemistry, 2008
An off-line interface incorporating sheathless flow and counter-flow balance is developed to couple capillary electrophoresis (CE) to matrix-assisted laser desorption ionization Fourier transform mass spectrometry (MALDI FTMS) for neuropeptide analysis of complex tissue samples. The new interface provides excellent performance due to the integration of three aspects: (1) A porous polymer joint constructed near the capillary outlet for the electrical circuit completion has simplified the CE interface by eliminating a coaxial sheath liquid and enables independent optimization of separation and deposition. (2) The electroosmotic flow at reversed polarity (negative) mode CE is balanced and reversed by a pressure-initiated capillary siphoning (PICS) phenomenon, which offers improved CE resolution and simultaneously generates a low flow (<100 nL/min) for fraction collection. (3) The predeposited nanoliter volume 2,5-dihydroxybenzoic acid (DHB) spots on a Parafilm-coated MALDI sample pl...