FR AB - Science Reference

ABRF 2001 ABSTRACTS
S8
Assaying single cells to single organelles using mass spectrometry.
J.V. Sweedler; Univ. of Illinois, Urbana, 600 S. Mathews Ave. 63-5,
Urbana, IL 61801
Understanding the interactions of relatively simple networks of neurons is
hampered by a lack of knowledge of the full complement of neuropeptides
involved in most neuronal systems. Using matrix-assisted laser desorption/ionization
(MALDI) time-of-flight mass spectrometry, neuropeptides can
be identified in single cells and even in individual neuronal processes. Mass
spectrometric imaging methods are described that can provide spatial “maps”
of the neuropeptides found in simple invertebrate networks, as well as identify
new neuropeptides. Using these techniques, multiple novel neuropeptides
have been discovered in the common neuronal model Aplysia californica.
Approaches for direct de novo sequencing of peptides in single cells
are demonstrated. A unique sampling protocol has been developed that
allows the peptides in single attoliter to femtoliter volume vesicles to be measured
using mass spectrometry. Using the atrial gland of Aplysia as a model,
more than ten bioactive peptides are found in individual vesicles indicating
the complexity of such hormonal signaling. Methods which combine capillary
electrophoresis, fluorescence detection and mass spectrometry on the
same sample are described.
S10
Chemical sensors for monitoring secretory and metabolic dynamics
at single cells: application to pancreatic beta cells.
R.T. Kennedy, W-j. Qian, M.G. Roper, L.S. Kauri, G.D. Dalgren;
Univ. of Florida, PO Box 117200, Gainesville, FL 32611-7200
Microscale electrochemical sensors that allow detection of insulin, glucose,
and oxygen at single cells or small groups of cells have been developed. The
insulin electrodes have been used to detect insulin secretion at the level of
single exocytosis events while the glucose and oxygen electrodes have been
used to monitor the dynamics of glucose and oxygen consumption. Such
measurements, in combination with pharmacological probes and gene
knock-outs, have been used to characterize secretory pathways and the
interaction of metabolism with secretion. In one study, it was demonstrated
that glyoclytic and respiratory oscillations occur in single islets of Langerhans
and these oscillations require Ca 2� entry into the cell for proper feedback.
Such oscillations provide a mechanism for oscillatory insulin secretion seen
in vivo. In another study, using the single cell approach, we have shown that
activation of insulin receptors leads to insulin secretion suggesting that positive
feedback contributes to the mechanism for the first phase of insulin
secretion. The signaling pathway by which insulin stimulates insulin secretion
has been further studied revealing critical roles for IRS-1 and PI3-K in
mediating insulin-stimulated insulin secretion. These studies have demonstrated
that temporally resolved measurements at single cells or cell clusters
are useful in evaluating mechanisms of signal transduction. The temporal
measurements, especially of multiple key analytes, allow sequences of events
to be evaluated and new mechansims to be uncovered.
SPEAKER ABSTRACTS
228 JOURNAL OF BIOMOLECULAR TECHNIQUES, VOLUME 11, ISSUE 4, DECEMBER 2000
S9
Profiling signal transduction networks in mammalian cells.
N.L. Allbritton, C.E. Sims, G. Meredith; Univ. of California, Irvine,
Medical Sciences I, Rm D380, Irvine, CA 92697-4560
A central goal of genomics and proteomics is to catalog the biological molecules
present in different organisms and cell types under various conditions.
A greater challenge for accurate and comprehensive characterization, however,
lies in determining the activities and functional relationships of the biological
molecules, particularly the enzymes, as they occur within the complex
cellular networks that comprise biological systems. To accomplish this task,
new technologies must be developed to measure multiple chemical species
within intact intracellular networks. We have demonstrated a new method,
the laser micropipet system, for the simultaneous measurement of the activation
of key regulatory enzymes in small groups of cells, a single cell, or
portions of a cell. This assay strategy should be broadly applicable to measurements
of a broad range of enzymes, including kinases, phosphatases,
proteases, and nucleases.
S11
Genetic analysis by mass spectrometry.
L.M. Smith; Univ. of Wisconsin–Madison, 1101 University Ave.,
Madison, WI 53706-1396
In the last decade two powerful new tools for the mass spectrometric analysis
of biomolecules have been developed, Matrix-Assisted Laser Desorption
Mass Spectrometry (MALDI-MS), and Electrospray Ionization Mass Spectrometry
(ESI-MS). The power of these methods lies in their ability to produce
and mass analyze intact gas phase ions from very large molecules such as
proteins and nucleic acids. The speed, accuracy, and sensitivity of the technologies
make them well-suited to address a number of problems in genetic
analysis, including the analysis of DNA sequence, genetic variations, and
gene expression. Results in these areas will be presented, including recent
work in which single nucleotide polymorphisms (SNPs) in genomic DNA
may be analyzed without need for a prior PCR amplification step.