Proteolytic processing of the
            <i>Aplysia</i>
            egg-laying hormone prohormone

Garden, Rebecca W.; Shippy, Scott A.; Li, Lingjun; Moroz, Tatiana P.; Sweedler, Jonathan V.

doi:10.1073/pnas.95.7.3972

Cited by 89 publications

(118 citation statements)

References 37 publications

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“…Because some physiological saline is required to allow for adequate adhesion to the substrate, the MALDI spectra tend to contain a large number of salt adducts. As shown in Figure 6, a MALDI mass spectrum generated from a 15 m abdominal ganglion section with SA matrix micro-spotted in the region between R3-14 neurons and bag cell clusters reveal their combined peptide profiles [4,5] but with excessive cationation adducts (sodiated, potassiated, and combinations of both). Further work is necessary to investigate methods of reducing physiological saline prior to adhesion to the cellular substrate.…”

Section: Resultsmentioning

confidence: 99%

“…Even with the wealth of genomic information available, direct biochemical measures are often required to determine the presence and particular form of the neuropeptides present throughout the central nervous system (CNS). Direct MALDI MS analysis of molluscan and insect neurons [1][2][3][4][5][6][7][8][9][10][11][12][13], connective nerve tissues [14], and even single peptidergic vesicles [15] provides information about gene products and their processing to bioactive neuropeptides. Mass profiling individually isolated neurons is the most common method for obtaining spatial information such as the cell-specific distribution of peptides within brain regions known as ganglia.…”

mentioning

confidence: 99%

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Spatial profiling invertebrate ganglia using MALDI MS

Kruse

Sweedler

2003

J. Am. Soc. Mass Spectrom.

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108

100

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The ability of MALDI TOF MS to spatially map peptides and proteins directly from a tissue is an exciting advance to imaging mass spectrometry. Recent advances in instrumentation for MS have resulted in instruments capable of achieving several micron spatial resolution while acquiring high-resolution mass spectra. Currently, the ability to obtain high quality mass spectrometric images depends on sample preparation protocols that often result in limited spatial resolution. A number of sample preparation and matrix deposition protocols are evaluated for spatial profiling of Aplysia californica exocrine gland and neuronal tissues. Such samples are different from mammalian tissues, but make good targets for method optimization because of the wealth of biochemical information available on neuropeptide processing and distribution. Electrospray matrix deposition and a variety of freezing methods have been found to be optimum for these invertebrate tissues,

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Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

Spatial profiling invertebrate ganglia using MALDI MS

Kruse

Sweedler

2003

J. Am. Soc. Mass Spectrom.

Self Cite

108

100

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“…16 Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry has been used recently to investigate proteolytic processing of peptides in single neurons from Aplysia. 17 Interest in analyzing individual biological entities has extended from the realm of the single cell to that of subcellular components. Microelectrodes, when placed in proximity to the surface of a cell, have been used to detect released components, including those corresponding to single vesicles.…”

mentioning

confidence: 99%

Separation and Characterization of Amines from Individual Atrial Gland Vesicles ofAplysiacalifornica

Lillard¹,

Chiu²,

Scheller³

et al. 1998

Anal. Chem.

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Several amine-containing components of individual vesicles from the atrial gland of Aplysia californica were identified with capillary electrophoresis (CE). On-line derivatization with naphthalene-2,3-dicarboxaldehyde was performed, and the derivatized amine-containing components were detected with laser-induced fluorescence (LIF). Amino acids, including taurine, that had not been determined previously in atrial gland vesicles were observed by using CE-LIF, and their identities were confirmed with CE, HPLC, NMR, and electrospray ionization mass spectrometry. The finding that taurine is packaged and stored into secretory vesicles supports the hypothesis that taurine may exhibit neuromodulatory activity. The bioactive peptides, well-known to be in atrial gland vesicles, were detected in lysed vesicle samples fractionated with HPLC and analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. These peptides were also observed in single-vesicle runs with CE-LIF. The atrial gland vesicles (ranging from 0.5 to 2 μm diameter and 65 aL to 4 fL volume, respectively) studied in this work represent the smallest biological entities to be analyzed chemically on an individual basis.The goal of analyzing individual biological cells has been realized with various microanalytical techniques. The motivation behind studying this specialized sample type is the heterogeneity that is present in the cell populations that comprise tissues. The measurement of an average value for a specific parameter of a population does not necessarily reflect the range of values resulting from individual members of the population. Although ensemble measurements can reveal abnormalities in a cell population, these measurements do not indicate intercellular differences that may provide more important clues about the history or future of a cell.Reports of single-cell analyses date back to the 1950s, in which single neurons were studied. 1-4 Since that time, several methods, including separations, have been used to analyze single cells. 5-23 Owing to its high sensitivity, laser-induced fluorescence (LIF) has played a significant role in the chemical analysis of individual cells with capillary electrophoresis (CE), 5,6 including wavelength-resolved LIF of neurotransmitters in single neurons. 7 Novel analyses of single cells with CE include cytoplasmic sampling from 8 and transporting solutes into 9 † Department of Chemistry, Stanford University. EXPERIMENTAL SECTIONReagents.All chemicals were from Mallinckrodt (Paris, KY) unless otherwise specified. KCN and KCl were from J. T. Baker, Inc. (Phillipsburg, NJ), and acetonitrile was obtained from Fisher (Fairlawn, NJ). Amino acids were from Sigma Chemical (St. Louis, MO). All solutions for CE-LIF, including buffers, naphthalene-2,3-dicarboxaldehyde (NDA) dilutions, vesicle extracts, and off-column derivatization reactions, were prepared and stored in glass vials. These glass vials were also used to contain the running buffer for CE-LIF. Sodium tetraborate solutions were...

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“…1 Numerous analytical techniques, including electrochemical detection, 2-6 separation-based methods, 7-9 fluorescence microscopy, 10-13 and mass spectrometry [14][15][16][17][18][19][20][21][22][23][24] have been used to surmount the inherent difficulties of measurements down to the μm-, pL-, and zmole-scale to study the intricate chemistry that exists within single cells.…”

Section: Introductionmentioning

confidence: 99%

Secondary Ion MS Imaging To Relatively Quantify Cholesterol in the Membranes of Individual Cells from Differentially Treated Populations

et al. 2007

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Time of flight secondary ion mass spectrometry (ToF-SIMS) is a well-established bioanalytical method for directly imaging the chemical distribution across single-cells. Here we report a protocol for the use of SIMS imaging to comparatively quantify the relative difference in lipid composition between the plasma membranes of two cells. This development enables direct comparison of the chemical effects of different drug treatments and incubation conditions in the plasma membrane at the single-cell level. Relative, quantitative ToF-SIMS imaging has been used here to compare macrophage cells treated to contain elevated levels of cholesterol with respect to control cells. Insitu fluorescence microscopy with two different membrane dyes was used to discriminate morphologically similar but differentially treated cells prior to SIMS analysis. SIMS images of fluorescently identified cells reveal that the two populations of cells have distinct outer leaflet membrane compositions with the membranes of the cholesterol-treated macrophages containing more than twice the amount of cholesterol of control macrophages. Relative quantification with SIMS to compare the chemical composition of single-cells can provide valuable information about normal biological functions, causative agents of diseases, and possible therapies for diseases.

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Proteolytic processing of the Aplysia egg-laying hormone prohormone

Cited by 89 publications

References 37 publications

Spatial profiling invertebrate ganglia using MALDI MS

Spatial profiling invertebrate ganglia using MALDI MS

Separation and Characterization of Amines from Individual Atrial Gland Vesicles ofAplysiacalifornica

Secondary Ion MS Imaging To Relatively Quantify Cholesterol in the Membranes of Individual Cells from Differentially Treated Populations

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