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Metal Oxide Laser Ionization Mass Spectrometry Imaging (MOLI MSI) using Cerium(IV) Oxide Sankha S Basu, Madison Hailey McMinn, Begona Gimenez-Cassina Lopez, Michael S. Regan, Elizabeth C. Randall, Amanda R. Clark, Christopher R. Cox, and Nathalie Y. R. Agar Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.9b00894 • Publication Date (Web): 26 Apr 2019 Downloaded from http://pubs.acs.org on April 27, 2019
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Analytical Chemistry
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Metal Oxide Laser Ionization Mass Spectrometry Imaging (MOLI
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MSI) using Cerium(IV) Oxide
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Sankha S. Basu1€, Madison H. McMinn2,3,4€, Begoña Giménez-Cassina Lopéz2, Michael S.
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Regan2, Elizabeth C. Randall5, Amanda R. Clark2, Christopher R. Cox6 and Nathalie Y. R.
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Agar2,5,7*
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1 Department
of Pathology, Brigham and Women's Hospital, Harvard Medical School
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2 Department
of Neurosurgery, Brigham and Women's Hospital, Harvard Medical School
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3 Department
of Chemistry and Biochemistry, Southern Illinois University Carbondale
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4
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Harvard Medical School
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5 Department
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6 Cobio
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7 Department
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€
Harvard-Amgen Scholar, Department of Neurosurgery, Brigham and Women's Hospital,
of Radiology, Brigham and Women's Hospital, Harvard Medical School
Diagnostics. Golden, CO. of Cancer Biology, Dana-Farber Cancer Institute, Harvard Medical School
S.S.B. and M.H.M contributed equally to this work
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*Corresponding Author:
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Nathalie Y.R. Agar, Ph.D.
Building for Transformative Medicine
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Department of Neurosurgery
60 Fenwood Road, Suite 8016-J
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Department of Radiology
Boston, Massachusetts 02115
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Brigham and Women’s Hospital
Tel: 617.525.7374, Fax: 617.264.6316
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Harvard Medical School
Email:
[email protected] 1 ACS Paragon Plus Environment
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Abstract
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Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI) is
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a powerful technique for spatially-resolved metabolomics. A variation on MALDI, termed metal
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oxide laser ionization (MOLI), capitalizes on the unique property of cerium(IV) oxide (CeO2) to
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induce laser-catalyzed fatty acyl cleavage from lipids and has been utilized for bacterial
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identification. In this study, we present the development and utilization of CeO2 as an MSI
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catalyst. The method was developed using a MALDI TOF instrument in negative ion mode,
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equipped with a high frequency laser. Instrument parameters for MOLI MS fatty acid catalysis
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with CeO2 were optimized with phospholipid standards and fatty acid catalysis was confirmed
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using lipid extracts from reference bacterial strains, and sample preparation was optimized using
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mouse brain tissue. MOLI MSI was applied to the imaging of normal mouse brain revealing
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differentiable fatty acyl pools in myelinated and non-myelinated regions. Similarly, MOLI MSI
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showed distinct fatty acyl composition in tumor regions of a patient derived xenograft mouse
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model of glioblastoma. To assess the potential of MOLI MSI to detect pathogens directly from
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tissue, a pseudo-infection model was prepared by spotting Escherichia coli lipid extracts on
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mouse brain tissue sections and imaged by MOLI MSI. The spotted regions were molecularly
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resolved from the supporting mouse brain tissue by the diagnostic odd-chained fatty acids and
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reflected control bacterial MOLI MS signatures. We describe MOLI MSI for the first time and
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highlight its potential for spatially resolved fatty acyl analysis, characterization of fatty acyl
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composition in tumors, and its potential for pathogen detection directly from tissue.
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Analytical Chemistry
TOC Graphic
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Fatty acid profiling of tissues and cells has been an extensive area of study, dating back
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nearly a century.1–3 Characterization of the fatty acyl composition of lipids contained within
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biological tissues traditionally involves a number of steps including tissue homogenization,
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followed by lipid extraction, hydrolysis and chemical derivatization, all prior to analysis.4,5
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These additional steps are not only time-consuming but also result in the loss of spatial
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relationships between these molecules, which is of critical importance in the study of diseases
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with heterogenous tissue distribution. Accordingly, an analytical method which not only profiles
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fatty acids, but also preserves their spatial distribution is of considerable value.
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Matrix assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI) is
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an emerging powerful analytical technique, which allows the spatially resolved characterization
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of a wide range of analytes within biological specimens.6 In contrast to commonly used imaging
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techniques, such as immunohistochemistry (IHC), MALDI MSI allows for the untargeted
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analysis of tissue sections.7 Although many of the original applications of MALDI MSI focused
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on protein analyses,8,9 there has been significant progress in utilization of this technique to
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measure drugs10–12 and lipids13 in tissue specimens.
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technology now allow molecular imaging with a spatial resolution of 5 µm, within the range of
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single cell analysis.14
Furthermore, recent advances in the
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Metal oxide laser ionization (MOLI) is a recently described variation on MALDI in
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which a catalytic metal oxide is used instead of a traditional co-ionizing organic acid matrix to
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bring about in situ cleavage of fatty acids ions from surface-associated lipids, .15 Like MALDI,
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MOLI utilizes laser energy to generate ions that are measured by a mass spectrometer, most
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commonly a time of flight (TOF) mass spectrometer.16 Compared with MALDI, one of the
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central advantages of MOLI is the absence of matrix ion peaks, which can obscure ions from the
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Analytical Chemistry
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biological specimen in the small molecule range (m/z < 1000). Most commonly, when MOLI
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MS is used, analyte ionization of lipids occurs by protonation, or sodiation, due to interactions
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with the Lewis acid/base sites on the metal oxide. However, as studies continue, it has been
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found that the ionization mechanism can vary between metal oxides. For certain metal oxides,
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such as nickel (II) oxide (NiO), the method of ionization is thought to involve Lewis acid/base
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interactions between the cation/anion pairs of the metal oxide and the analyte, resulting in the
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protonation from one surface-bound analyte to another without involving solvent or surface-
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absorbed water.17 For others, such as calcium oxide (CaO), analyte ionization occurs due to
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interactions with Lewis acid/base sites on the metal oxide, and results in a calcium adduct of a
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fatty acid carboxylate.15 More recently, cerium(IV) oxide (CeO2), a rare earth lanthanide, has
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also been utilized in MOLI MS. Unlike the other metal oxides, CeO2 demonstrates a unique
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property of laser induced catalysis of fatty acyl cleavage when applied to phospholipids and is
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energized by standard lasers found in commonly used and commercially-available MALDI TOF
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MS instruments.16 This property of laser-induced cleavage catalysis by CeO2 provides a
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considerable opportunity towards various biological and clinical applications in which fatty acid
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profiling may be needed.17,18
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One promising MOLI MS application using CeO2 involves microbial identification using
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fatty acid profiling.19 While conventional MALDI TOF MS has revolutionized the clinical
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laboratory workflow through the identification of bacterial isolates by their proteomic profile,
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differentiation and speciation of certain bacteria, including Acinetobacter, Listeria and
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Enterobacteraciae, have proven challenging due to intra-species similarities.20,21 Fatty acid
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profiling using MOLI MS has been shown to not only improve genus and species level
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identification of these organisms, but has the granularity for even strain level identification22 and 5 ACS Paragon Plus Environment
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differentiation of antimicrobial resistant and susceptible isolates,23 a considerable improvement
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to current approaches utilized in clinical microbiology. In addition to infectious diseases, there
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are also several other potential applications for which this could be used.
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To date, MOLI MS has been limited to target plate spot analysis. We here introduce the
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development and optimization of a new method using CeO2 as a mass spectrometry imaging
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catalyst, providing metal oxide laser ionization mass spectrometry imaging (MOLI MSI) of
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tissue sections. This methodology provides an in situ platform for the spatially resolved specific
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chemical characterization of fatty acyl chains. We demonstrate the translational and clinical
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potential of MOLI MSI with applications to study lipid-rich brain and brain tumor tissue, and the
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detection of diagnostic odd-chain fatty acyls from bacterial extracts directly from tissue.
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Experimental Section
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Chemicals and Materials
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1-palmitoyl-2-oleoyl-glycero-3-phosphocholine
(POPC),
1-palmitoyl-2-oleoyl-sn-
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glycero-3-phosphoethanolamine (POPE), and 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-
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serine (POPS) were obtained from Avanti Polar Lipids (Alabaster, AL).
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(CeO2) (
