University of Illinois Urbana-Champaign

Mass spectrometry methods for imaging cellular lipids, organelles, and proteins

Chini, Corryn E. Neumann

Content Files
CHINI-DISSERTATION-2019.pdf

Permalink

https://hdl.handle.net/2142/105924

Description

Title
Mass spectrometry methods for imaging cellular lipids, organelles, and proteins
Author(s)
Chini, Corryn E. Neumann
Issue Date
2019-07-10
Director of Research (if dissertation) or Advisor (if thesis)
Kraft, Mary L
Doctoral Committee Chair(s)
Kraft, Mary L
Committee Member(s)
  • Kong, Hyunjoon
  • Murphy, Catherine J
  • Sweedler, Jonathan V
Department of Study
Chemistry
Discipline
Chemistry
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Date of Ingest
2019-11-26T20:59:42Z
Keyword(s)
  • mass spectrometry
  • lipids
  • spheroids
  • endoplasmic reticulum
  • diglycerides
  • triglycerides
  • Kv2.1
Abstract
Lipids and their secondary metabolites function as energy storage molecules, structural components in cellular membranes, and messengers that can alter cellular function. Imaging the distributions of various lipid species within tissues and cells can yield insight into the mechanism by which lipids influence health and disease. In this thesis, mass spectrometry (MS) is used to identify the structures of various lipid species and image their distributions in parallel with specific organelles and proteins in cell populations and individual cells. Mass spectrometry (MS) is an analytical technique that can detect a wide variety of biomolecules, including lipids and proteins, directly and with chemical specificity. MS employs an ion source, mass analyzer, and detector to identify analytes in a sample based on their mass-to-charge ratio (m/z). In addition to imparting charge upon analytes, ion sources have been designed with imaging modalities for visualizing the spatial distributions of ions with specific m/z in samples, including cells and tissues. The ion source utilized determines whether a distinctive label must be incorporated into the biomolecules of interest to permit identification. For example, ionization sources that impart charges on large biomolecules without fragmentation enable the identification of unlabeled lipid species based on their exact mass. This chemical specificity is essential for the label-free detection of lipids and identification of their precise structures. Desorption electrospray ionization (DESI) and the ion source used for time-of-flight secondary ion mass spectrometry (TOF-SIMS) can ionize lipids and other large biomolecules without fragmentation, so these approaches can be applied to unlabeled samples. In contrast, ionization sources that extensively fragment biomolecules into mono- and di-atomic secondary ions, such as that employed by the Cameca NanoSIMS 50, necessitate labeling the biomolecule of interest with a distinct atom, such as a rare stable isotope, to allow for distinction of its secondary ions from those of other cellular species. Mass analyzers, the second component of mass spectrometers, separate the secondary ions based on their m/z, and may allow for fragmentation and tandem mass spectrometry (MS/MS) experiments that aide in species identification. The last component, the detector, is utilized for the amplification and detection of electrical current. For a particular application of mass spectrometry, the ion source, mass analyzer, and detector are selected based on the efficacy of ionizing the analytes of interest, the mass resolution and degree of fragmentation required for identification, and the sensitivity needed for analyte detection, respectively. Imaging MS instruments are also equipped with a motorized stage or ion source positioning system and corresponding software to automate the collection of individual mass spectra at multiple adjacent locations on the sample. Recording the intensity of the secondary ions detected at each region where a mass spectrum was acquired enables the production of images that show the distributions of each ion on the sample. This direct imaging capability provides location specificity within a sample, such as which lipid species are preferentially enriched in different regions of the plasma membrane versus those present in the bilayer of the endoplasmic reticulum. Here, DESI and TOF-SIMS were used to image lipids, organelles, and proteins in cell populations and individual cells. DESI can image lipids with a lateral resolution on the scale of tens of microns, so it is well suited for elucidating lipid structures and distributions in cell populations and tissues. DESI was used to identify and image the different lipid species at various regions within cell spheroids that serve as simplified models of solid tumors. Various lipid species were detected throughout a spheroid derived from the highly malignant breast cancer cell line, MDA-MB-231. Imaging experiments revealed the localization of ether-linked lipids to the center of the spheroid where nutrients and oxygen are limited. Lipid species were also identified in individual Madin-Darby canine kidney (MDCK) cells using TOF-SIMS, as described in Chapter 3. TOF-SIMS is well suited for these analyses because it is capable of identifying and imaging unlabeled lipids with a lateral resolution of a few microns, which is appropriate for identifying the lipid composition within micron-sized organelles of mammalian cells. Specifically, diglycerides (DAGs) and triglycerides (TAGs) were detected in the lipid droplets within individual MDCK cells, and the specific fatty acid chains within these DAGs and TAGs were elucidated using tandem MS fragmentation patterns. Imaging MS indicated that identified DAG species did not result from the fragmentation of phosphatidylcholine (PC) species, but may have been produced by the fragmentation of TAG species during ionization. In Chapter 4, TOF-SIMS tandem MS was used to detect tubules of the endoplasmic reticulum (ER) within human embryonic kidney (HEK) cells transfected to contain a high number of endoplasmic reticulum-plasma membrane (ER-PM) junctions. ER detection was based on characteristic ions produced from the fragmentation of a commercial dye, ER-Tracker Blue-White DPX®. Identified ions included pentafluorophenyl and fluoride anions that could be detected using TOF-SIMS. Finally, Chapter 5 describes the development of gold nanoparticles conjugated to avidin that can be used to label specific biotinylated proteins on the surface of transfected HEK cells. The presence of a carbon-13-labeled lysine linker in these gold nanoparticle-avidin constructs will allow for the location of the label to be identified by detecting the carbon-13-containing mono- and di-atomic ions it produces using NanoSIMS. Thus, the biotinylated protein will also be localized. These works highlight the versatility of imaging MS techniques as they apply to both cell populations and single cell samples. The characteristic m/z of the detected ions provided the chemical specificity needed to differentiate between specific biomolecules and other cellular components composed of the same basic building blocks. Additionally, imaging modalities with different spatial resolutions provided the location specificity needed to study samples consisting of both cell populations and individual cells.
Graduation Semester
2019-08
Type of Resource
text
Permalink
http://hdl.handle.net/2142/105924
Copyright and License Information
Copyright 2019 Corryn E. Neumann Chini

Owning Collections

Dissertations and Theses - Chemistry
Graduate Dissertations and Theses at Illinois PRIMARY
Graduate Theses and Dissertations at Illinois