Probing Single Secretory Vesicles with Capillary Electrophoresis

Chiu, Daniel T.; Lillard, Sheri J.; Scheller, Richard H.; Zare, Richard N.; Rodriguez‐Cruz, Sandra E.; Williams, Evan R.; Orwar, Owe; Sandberg, Mats; Lundqvist, J A

doi:10.1126/science.279.5354.1190

Cited by 99 publications

(97 citation statements)

References 17 publications

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“…Low-Mass Amines.-Previously, we found by HPLC analysis of a lysed vesicle extract, compared to a mixture of amino acid standards, that taurine was the major low-mass component of an atrial gland sample of a population of vesicles. 24 The presence of taurine was confirmed by spiking the vesicle sample with a taurine standard, and recoveries of 104-105% were obtained. Further confirmation of taurine was achieved by recording 1 H NMR spectra of a vesicle lysate sample fractionated with size exclusion chromatography and comparing this to a taurine standard.…”

Section: Resultsmentioning

confidence: 97%

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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Section: Resultsmentioning

confidence: 97%

Separation and Characterization of Amines from Individual Atrial Gland Vesicles ofAplysiacalifornica

Lillard¹,

Chiu²,

Scheller³

et al. 1998

Anal. Chem.

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“…To fuse a cell or vesicle to another cell, individual manipulation of at least one of the fusion partners is required for alignment. It is well documented that individual cells, as well as other biological structures, including organelles of small dimension, can be manipulated and moved at will in solution by using optical trapping with highly focused laser beams (10)(11)(12)(13). Here, a cell in solution was trapped by an IR laser beam and brought into contact with an adherent cell, by using the setup schematically shown in Fig.…”

Section: Resultsmentioning

confidence: 99%

Manipulating the genetic identity and biochemical surface properties of individual cells with electric-field-induced fusion

Strömberg

Ryttsén

Chiu

et al. 2000

Proc. Natl. Acad. Sci. U.S.A.

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“…This volume compares favorably with the nanoliter volumes reported for derivatization reactions in capillary-based chemical cytometry methods (1)(2)(3)(4)(5), although smaller volumes have been reported (26). The capacity to confine a lysing, reacting plug of cell and reagents is an advantage that motivated prior work by us (8) and others (18) to use valves formed by multilayer soft lithography to confine approximately nanoliter volumes.…”

Section: Integrated Microfluidic Chip For Manipulation and Analysis Omentioning

confidence: 64%

Chemical cytometry on a picoliter-scale integrated microfluidic chip

Wheeler

Zare

2004

Proc. Natl. Acad. Sci. U.S.A.

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239

186

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Probing Single Secretory Vesicles with Capillary Electrophoresis

Cited by 99 publications

References 17 publications

Separation and Characterization of Amines from Individual Atrial Gland Vesicles ofAplysiacalifornica

Separation and Characterization of Amines from Individual Atrial Gland Vesicles ofAplysiacalifornica

Manipulating the genetic identity and biochemical surface properties of individual cells with electric-field-induced fusion

Chemical cytometry on a picoliter-scale integrated microfluidic chip

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