Hyphenation of analytical techniques to single cell capillary electrophoresis-mass spectrometry

Philip, Marina Catherine

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Description

Title

Hyphenation of analytical techniques to single cell capillary electrophoresis-mass spectrometry

Author(s)

Philip, Marina Catherine

Issue Date

2020-05-04

Director of Research (if dissertation) or Advisor (if thesis)

Sweedler, Jonathan V

Doctoral Committee Chair(s)

Sweedler, Jonathan V

Committee Member(s)

• Gewirth, Andrew A
• Han, Hee-Sun
• Yau, Peter M

Department of Study

Chemistry

Discipline

Chemistry

Degree Granting Institution

University of Illinois at Urbana-Champaign

Degree Name

Ph.D.

Degree Level

Dissertation

Keyword(s)

• analytical chemistry
• single cell
• capillary electrophoresis
• mass spectrometry
• neurochemistry
• metabolomics

Abstract

Single cell chemical analysis pushes the limits of analytical measurements. Small volumes, large dynamic range, and chemical complexity all contribute to the analytical challenge of investigating single cell biology. To comprehensively study single cell chemistry, technologies require low limits of detection, accurate quantitation, confident identity assignment, and ability to process complex chemical mixtures. Heterogeneity in nucleic acids, proteins, peptides, metabolites, lipids, and more result in functionally significant differences between cells within the same tissue. Mass spectrometry (MS) has emerged as an effective tool for single cell chemical analysis because of its versatility, sensitivity, and ability to distinguish analytes in complex samples. Different MS approaches have various advantages and disadvantages for chemical analysis; for instance, matrix-assisted laser desorption ionization (MALDI)-MS enables rapid analysis but suffers in quantitation, whereas capillary electrophoresis–electrospray ionization (CE–ESI)-MS has low limits of detection and quantitative capabilities, but is comparatively low-throughput. Additionally, each method tends to be better suited for different chemical classes. Improvements in technology are constantly evolving to address the limitations of individual MS analyses, yet they tend to be limited in at least one dimension. This thesis describes a strategy to bridge the gap between the advantages and disadvantages of orthogonal analytical techniques by performing multiple measurements on the same single cell. By taking advantage of material left behind after a sample isolation or analysis, we can expand chemical coverage of single cell analysis, confidently classify cell types, and correlate different chemical classes within the same cell. Our approach uses a liquid microjunction (LMJ) extraction probe which extracts chemical content from a single cell off a microscope slide. We describe the assembly, operation, and validation of the LMJ probe as a strategy for performing optically guided single cell MALDI-MS profiling followed by CE–ESI-MS metabolite profiling. The first demonstrated application of the device is the analysis of single cells from rat pancreatic islets of Langerhans, which perform the canonical glucose-regulating function of the pancreas. Each cell type within islets of Langerhans are defined by the peptide hormone complement contained therein (e.g. α cells contain glucagon, β cells contain insulin), and cells can be rapidly profiled and classified by their peptide content with MALDI-MS. By screening cells prior to extraction, we can specifically target only the cells of interest for lower-throughput CE–ESI-MS analysis. After MALDI-MS profiling, we use the LMJ probe to extract metabolites directly from the microscope slide for follow-up CE–ESI-MS metabolite profiling. After validating the LMJ probe, we further apply the technology to different chemical measurements and biological systems. First, we describe application of the probe to CE–ESI-MS metabolite profiling of single rat cerebral neurons and astrocytes. The gold standard in biological differentiation and classification of neurons and astrocytes is immunocytochemistry (ICC), where cells must be fixed and crosslinked for antibody incubation, which precludes follow-up MS analysis. To circumvent this limitation, we extract metabolite content from the cell with the LMJ probe prior to ICC. The neuron and astrocyte markers we selected are a transmembrane protein and structural protein, respectively, which remain adhered to the microscope slide after extraction. Thus, we can profile cells with CE–ESI-MS and later classify them as neurons or astrocytes after ICC to correlate chemical content with cell type. Second, we applied LMJ extraction to RNA, which has potential applications in both MS and transcriptomics, a powerful tool for gene extrusion profiling. We designed a biphasic extraction addition to the LMJ probe to enable RNA extraction from buccal ganglia from Aplysia californica with simultaneous delivery of an aqueous and organic phase. The biphasic extraction reduces the number of preprocessing steps, preventing sample loss, and successfully extracts suitable amounts of RNA for MS analysis. Finally, we applied field-amplified sample injection (FASI) to our CE–ESI-MS system to improve limits of detection by 100-fold for single cell analysis. FASI is typically of limited use in MS applications because it favors the injection of ionic salts into the capillary, which causes ion suppression at the electrospray interface. We addressed this by incorporating a desalting step into the sample preparation, which preserved metabolite material while precipitating interfering salts. We expect this addition to our CE–ESI-MS toolkit to enable greater metabolite detection and identifications in our single cell analyses.

Graduation Semester

2020-05

Type of Resource

Thesis

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Copyright and License Information

Copyright 2020 Marina Philip

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