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Title:Mass spectrometry-based investigations to characterize specific cell types within the brain
Author(s):Knolhoff, Ann M.
Director of Research:Sweedler, Jonathan V.
Doctoral Committee Chair(s):Sweedler, Jonathan V.
Doctoral Committee Member(s):Bailey, Ryan C.; Gillette, Martha L.; Scheeline, Alexander
Department / Program:Chemistry
Discipline:Chemistry
Degree Granting Institution:University of Illinois at Urbana-Champaign
Degree:Ph.D.
Genre:Dissertation
Subject(s):mass spectrometry
single-cell analysis
glia
astrocytes
Aplysia californica
mast cells
metabolomics
metabolite profiling
transcriptomics
peptides
cell culture
Abstract:Investigations of the chemical content of the brain and its many constitutive cell types yield information regarding normal and abnormal brain function. Frequently, proteomics, peptidomics, and metabolomics experiments survey brain regions and attribute detected species to neurons. However, many other cell types are present, including astrocytes, oligodendrocytes, and microglia. It is also known that different cell types can vary greatly in their analyte content and concentration; therefore, to obtain an adequate representation of brain function, specific cell types require further characterization. Cell types of particular interest are astrocytes, which are involved in neuronal communication, and mast cells, which are involved in allergic response. Moreover, morphologically similar cells can exhibit chemical heterogeneity; to characterize these differences, single-cell analyses are necessary. In this case, single cells from the model organism, Aplysia californica, are isolated for analysis. Mass spectrometry (MS) is well-suited for such applications because it has limits of detection in the attomole range for both small molecule metabolites and peptides, has a wide dynamic concentration range, and can detect and identify molecules of interest without a priori knowledge of sample content. To characterize metabolite profiles in single cells and tissue homogenates, a laboratory-built capillary electrophoresis (CE) system coupled to electrospray ionization (ESI) MS is implemented. This is a useful technique for such measurements because it only requires a small amount of sample (injection of nanoliter volumes), it can efficiently separate and detect hundreds of analytes within a single CE separation, and it has detection limits in the low nanomolar (attomole) range for several neurotransmitters. This technique has been successfully applied to characterize and chemically distinguish single cells of various neuron types from Aplysia californica. Here, hundreds of ions are detected within single-cell samples and 36 ions are identified. Furthermore, neuron-specific analytes are revealed, chemical classification of neurons is achieved via principal component analysis, and cellular concentrations of serine and glutamic acid are determined for the different neuron types. CE-ESI-MS has also been effective in determining relative chemical differences between different sample types. To determine the chemical contribution that mast cells make to the brain, tissue homogenates from mast cell-deficient mice are compared to their heterozygous littermates. In this comparison, a number of metabolites, including amino acids and choline, are statistically different between the two sample types; furthermore, this data agrees with differences observed in gene expression data. The combined metabolite and transcriptomics data reveal global chemical differences that affect a number of metabolic pathways, which may be related to behavioral and developmental traits observed in mast cell-deficient mice. The characterization of peptide content in astrocytes is also achieved by employing several instrumental platforms, such as CE-ESI-MS and matrix-assisted laser desorption/ionization MS. By carefully selecting defined samples, the identification of a large number of peptides from purified astrocyte samples is accomplished.
Issue Date:2011-08-26
URI:http://hdl.handle.net/2142/26414
Rights Information:Copyright 2011 Ann M. Knolhoff
Date Available in IDEALS:2013-08-27
Date Deposited:2011-08


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