High-throughput matrix-assisted laser desorption/ionization mass spectrometry for single-cell and single-organelle measurements

Little is known regarding the molecular heterogeneity of specific classes of individual cells and organelles. However, clear phenotypical differences are seen between cells of same type, which may be attributed to organelle size, shape and composition differences. Organelle heterogeneity is ubiquitous and an indispensable property in biology that shapes cellular architecture and allows for specialized function by influencing the biochemical output of the cell. To understand how emergent properties of networks arise from an ensemble of cells, understanding their chemical differences can be important. Here, we present high-throughput mass spectrometry-based protocols to chemically profile large populations of single cells and individual organelles. Therefore, to address the fundamental heterogeneity present in these different cellular systems, we take on a single-cell approach where we enzymatically digest tissue to remove the extracellular matrix and connective components, releasing the individual cells from the tissue matrix. Single-cell staining and scattered deposition of the single cells onto the microscopy slide allows the relative spatial locations to be mapped across the microscopy slide for translation of pixel coordinates to physical coordinates, such as millimeters or microns. Performing point-based image registration between the optical microscopy image and a matrix-assisted laser desorption/ionization mass spectrometer allows the locations of the single cells to be mapped to physical stage coordinates on the mass spectrometer’s stage. With the single-cell deposition leaving space between the individual cells, the individual cells can be chemically characterized using the matrix-assisted laser desorption/ionization laser for analyte ionization and subsequent detection by mass spectrometry. In a similar manner, this single-cell workflow can be adapted to individual organelles through distinct modifications of the approach to allow targeting of objects that are tenfold smaller in diameter than the single cells. Specifically, modification of the single-cell approach can allow the targeting and chemical analysis of individual dense-core vesicles and mitochondria. Due to the high-throughput nature of the work, the downstream data analysis and interpretation requires the use and development of informatic based data analysis tools for processing and handling the highly dimensional datasets generated. Together, this work allows the high-throughput investigation of single cells and single organelles with a granularity of detail that was no previously possible.

Name thesis:degree_name

Ph.D.

Level thesis:degree_level

Dissertation

Discipline thesis:degree_discipline

Molecular & Integrative Physi

Grantor

University of Illinois at Urbana-Champaign

Year dc:date

2023

Author dc:creator

• Castro, Daniel Cruz

Contributors dc:contributor

• Sweedler, Jonathan
• Grosman, Claudio
• Boppart, Marni
• Raetzman, Lori

• Maldi
• Mass Spectrometry
• High-throughput
• Single-cell
• Single-organelle
• Cell
• Organelle
• Fourier-transform Ion Cyclotron Resonance.

Statement dc:rights

• Copyright 2023 Daniel Castro

Language dc:language

en, eng

Handle dc:identifier

https://hdl.handle.net/2142/121413

OAI record oai:identifier

https://www.ideals.illinois.edu/oai-pmh?verb=GetRecord&metadataPrefix=etdms&identifier=oai%3Awww.ideals.illinois.edu%3A2142%2F121413