Metal Oxide Laser Ionization Mass Spectrometry Imaging (MOLI MSI

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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]

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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) (