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Down to single-cell metabolomics: Developments of direct infusion tools for electrospray ionization mass spectrometry
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Analytical Chemistry. (Lanekoff)ORCID iD: 0000-0002-8433-1725
2024 (English)Doctoral thesis, comprehensive summary (Other academic)
Description
Abstract [en]

Metabolomics provides a snapshot of the metabolic status of biological samples. This knowledge can give insights into several biological processes, including understanding the etiology and development of diseases such as cancer or type 2 diabetes. Mass spectrometry is the golden standard technique for metabolite profiling due to its high sensitivity and comprehensive analyte coverage. Although several sampling techniques are available for routine metabolomics studies, these usually require sample sizes of around 1 million cells. The work entailed in this thesis focuses on developing sampling strategies to perform metabolomic studies on challenging samples, namely, low cell number and minute volume samples, down to single cells. 

The direct infusion probe (DIP) that I developed enables the analysis of metabolites and lipids in minute sample volumes with low cell numbers. Specifically, it enables analysis of samples containing as low as 20 cells/µL in 5 µL volumes. The DIP has a user-friendly and simple design that allows for rapidly switching samples. Furthermore, it provides minimal carryover between samples by simply washing with the next sample for 8 seconds, which enhances the analysis throughput by eliminating extra washing steps. I utilized the DIP to conduct metabolite and lipid profiling of insulin-releasing cells (INS-1) exposed to high glucose. I observed that high glucose exposure causes changes in the abundance of amino acids, lipids, and other small molecules. Our results are linked with pathways, such as glycolysis, fatty acid biosynthesis, and glutamate and sphingolipid metabolisms that are altered in type 2 diabetes. 

Another analytically challenging task that I address in this thesis is the chemical analysis of individual cells. Single-cell metabolomics is crucial to decoding cellular heterogeneity and identifying phenotype variations within a cell population. I redesigned the pneumatically assisted nanospray desorption electrospray ionization (PA nano-DESI) probe to enable the analysis of metabolites and lipids from single cells. This was accomplished by reducing the inner diameter of capillaries to enhance ionization and tapering the capillaries of the probe to establish a miniaturized liquid bridge. Then, I utilized the tapered PA nano-DESI probe to profile the metabolome and lipidome of single INS-1 cells and senescent IMR-90 cells. The results show alterations in glutamate and arachidonic acid levels, characteristic of insulin release and aging processes, respectively. 

Overall, the DIP and the tapered PA nano-DESI probes represent significant analytical advances in conducting metabolomic studies of samples containing low cell numbers down to single cells.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2024. , p. 85
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 2406
Keywords [en]
mass spectrometry, single-cell, metabolomics, lipidomics, DIP-MS, tapered PA-nano-DESI
National Category
Analytical Chemistry
Research subject
Chemistry with specialization in Analytical Chemistry
Identifiers
URN: urn:nbn:se:uu:diva-527116ISBN: 978-91-513-2142-4 (print)OAI: oai:DiVA.org:uu-527116DiVA, id: diva2:1853953
Public defence
2024-06-13, A1:107a, BMC, Husargatan 3, Uppsala, 09:15 (English)
Opponent
Supervisors
Available from: 2024-05-22 Created: 2024-04-24 Last updated: 2024-05-22
List of papers
1. A Direct Infusion Probe for Rapid Metabolomics of Low-Volume Samples
Open this publication in new window or tab >>A Direct Infusion Probe for Rapid Metabolomics of Low-Volume Samples
2022 (English)In: Analytical Chemistry, ISSN 0003-2700, E-ISSN 1520-6882, Vol. 94, no 37, p. 12875-12883Article in journal (Refereed) Published
Abstract [en]

Targeted and nontargeted metabolomics has the potential to evaluate and detect global metabolite changes in biological systems. Direct infusion mass spectrometric analysis enables detection of all ionizable small molecules, thus simultaneously providing information on both metabolites and lipids in chemically complex samples. However, to unravel the heterogeneity of the metabolic status of cells in culture and tissue a low number of cells per sample should be analyzed with high sensitivity, which requires low sample volumes. Here, we present the design and characterization of the direct infusion probe, DIP. The DIP is simple to build and position directly in front of a mass spectrometer for rapid metabolomics of chemically complex biological samples using pneumatically assisted electrospray ionization at 1 mu L/min flow rate. The resulting data is acquired in a square wave profile with minimal carryover between samples that enhances throughput and enables several minutes of uniform MS signal from 5 mu L sample volumes. The DIP was applied to study the intracellular metabolism of insulin secreting INS-1 cells and the results show that exposure to 20 mM glucose for 15 min significantly alters the abundance of several small metabolites, amino acids, and lipids.

Place, publisher, year, edition, pages
American Chemical Society (ACS), 2022
National Category
Analytical Chemistry
Identifiers
urn:nbn:se:uu:diva-489972 (URN)10.1021/acs.analchem.2c02918 (DOI)000853252300001 ()36070505 (PubMedID)
Funder
Swedish Foundation for Strategic Research, ITM17-0014Swedish Research Council, 2017-04125
Available from: 2022-12-06 Created: 2022-12-06 Last updated: 2024-04-24Bibliographically approved
2. Online direct infusion mass spectrometry of LLE phases for metabolite and lipid profiling with the direct infusion probe
Open this publication in new window or tab >>Online direct infusion mass spectrometry of LLE phases for metabolite and lipid profiling with the direct infusion probe
(English)Manuscript (preprint) (Other academic)
Keywords
DIP-MS, lipidomics, low cell number, LLE, BUME
National Category
Analytical Chemistry
Research subject
Chemistry with specialization in Analytical Chemistry
Identifiers
urn:nbn:se:uu:diva-527113 (URN)
Available from: 2024-04-24 Created: 2024-04-24 Last updated: 2024-04-24
3. Global and Spatial Metabolomics of Individual Cells Using a Tapered Pneumatically Assisted nano-DESI Probe
Open this publication in new window or tab >>Global and Spatial Metabolomics of Individual Cells Using a Tapered Pneumatically Assisted nano-DESI Probe
Show others...
2023 (English)In: Journal of the American Society for Mass Spectrometry, ISSN 1044-0305, E-ISSN 1879-1123, Vol. 34, no 11, p. 2518-2524Article in journal (Refereed) Published
Abstract [en]

Single-cell metabolomics has the potential to reveal unique insights into intracellular mechanisms and biological processes. However, the detection of metabolites from individual cells is challenging due to their versatile chemical properties and concentrations. Here, we demonstrate a tapered probe for pneumatically assisted nanospray desorption electrospray ionization (PA nano-DESI) mass spectrometry that enables both chemical imaging of larger cells and global metabolomics of smaller 15 mu m cells. Additionally, by depositing cells in predefined arrays, we show successful metabolomics from three individual INS-1 cells per minute, which enabled the acquisition of data from 479 individual cells. Several cells were used to optimize analytical conditions, and 93 or 97 cells were used to monitor metabolome alterations in INS-1 cells after exposure to a low or high glucose concentration, respectively. Our analytical approach offers insights into cellular heterogeneity and provides valuable information about cellular processes and responses in individual cells.

Place, publisher, year, edition, pages
American Chemical Society (ACS), 2023
National Category
Analytical Chemistry Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-516884 (URN)10.1021/jasms.3c00239 (DOI)001097001800001 ()37830184 (PubMedID)
Funder
Swedish Research Council, 2017-04125EU, European Research Council, 101041224 - X CELLSwedish Research Council, VR MH 2019-02050Novo Nordisk, NNF21OC0070086Novo Nordisk, NNF22OC0078239Swedish Society for Medical Research (SSMF)
Available from: 2023-12-07 Created: 2023-12-07 Last updated: 2024-04-24Bibliographically approved
4. Enhanced lipidomics from single-cells using the tapered PA nano-DESI-MS
Open this publication in new window or tab >>Enhanced lipidomics from single-cells using the tapered PA nano-DESI-MS
Show others...
(English)Manuscript (preprint) (Other academic)
Keywords
single-cell, lipidomics, mass spectrometry, lipid online separation, lipids, senescence, tapered PA nano-DESI
National Category
Analytical Chemistry
Identifiers
urn:nbn:se:uu:diva-527114 (URN)
Available from: 2024-04-24 Created: 2024-04-24 Last updated: 2024-04-24

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