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Dec 27, 2017 - Analyzer Using Isotope Ratio Mass Spectrometry. Sitora Khodjaniyazova,. †,#. Milad Nazari,. †,#. Kenneth P. Garrard,. †,‡. Maya...
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Characterization of the Spectral Accuracy of an Orbitrap Mass Analyzer using Isotope Ratio Mass Spectrometry Sitora Khodjaniyazova, Milad Nazari, Kenneth P Garrard, Mayara P.V. Matos, Glen P. Jackson, and David C. Muddiman Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b03983 • Publication Date (Web): 27 Dec 2017 Downloaded from http://pubs.acs.org on December 29, 2017

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Analytical Chemistry

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Characterization of the Spectral Accuracy of an

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Orbitrap Mass Analyzer using Isotope Ratio Mass Spectrometry

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Sitora Khodjaniyazovaa‡, Milad Nazaria‡, Kenneth P. Garrarda,b, Mayara P. V. Matosc,d, Glen P.

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Jackson , and David C. Muddiman

c

a

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a

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(METRIC), North Carolina State University, Raleigh, NC 27695

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b

Department of Chemistry and Molecular Education, Technology, and Research Innovation Center

Precision Engineering Consortium, Department of Mechanical and Aerospace Engineering, North

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Carolina State University, Raleigh, NC 27606

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c

Department of Forensic and Investigative Science, West Virginia University, Morgantown, WV 26506

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d

Department of Biology, West Virginia University, Morgantown, WV 26506

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Authors contributed equally to this work

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Submitted to: Analytical Chemistry

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Submitted: 09/23/2017 Revised: 12/22/2017

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Manuscript: 42 Pages / 6 Figures / 2 Tables / 7 Supplemental Figures

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Keywords: Orbitrap, Spectral Accuracy, Isotopic distribution, Mass Measurement Accuracy, Q

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Exactive Plus, Isotope Ratio Mass Spectrometry, Mass Spectrometry Imaging

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*Author for Correspondence

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David C. Muddiman, Ph.D.

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Department of Chemistry

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Molecular Education, Technology, and Research Innovation Center (METRIC)

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North Carolina State University

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Raleigh, North Carolina 27695

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Phone: 919-513-0084

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Email: [email protected]

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Abstract Infrared

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matrix-assisted

laser

desorption

electrospray

ionization

(IR-

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MALDESI) source coupled to the Q Exactive PlusTM has been extensively used in

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untargeted mass spectrometry imaging (MSI) analyses of biological tissue sections.

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Although the Orbitrap is a high-resolution and accurate-mass (HRAM) mass

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analyzer, these attributes alone cannot be used for the reliable identification of

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unknown analytes observed in complex biological matrices. Spectral accuracy (SA)

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is the ability of the mass spectrometer to accurately measure the isotopic

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distributions which, when used with high mass measurement accuracy (MMA), can

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facilitate the elucidation of a single elemental composition. To investigate the effects

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of different ion populations on an Orbitrap’s SA and MMA, a solution of caffeine, the

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tetrapeptide MRFA, and ultramark was analyzed using a Q Exactive Plus across

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eight distinct automatic gain control (AGC) targets. The same compounds from the

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same lot numbers were also individually analyzed using isotope ratio mass

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spectrometry (IRMS) to accurately determine the isotopic abundance of 13C, 15N, and

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S. We demonstrated that at optimum absolute ion abundances the Orbitrap can be

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used to accurately count carbons, nitrogens, and sulfurs in samples with varying

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masses. Additionally, absolute monoisotopic ion abundances required for high SA

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were empirically determined by using the expected (IRMS) and experimental

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(Orbitrap) isotopic distributions to calculate the Pearson chi-square test. These

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thresholds for absolute ion abundances can be used in untargeted MSI studies to

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shorten an identification list by rapidly screening for isotopic distributions whose

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absolute ion abundances are high enough to accurately estimate the number of

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atoms.

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Analytical Chemistry

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Introduction

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Accurate determination of elemental compositions is one of the most

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challenging aspects in untargeted metabolomics analyses.1 High resolving power

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coupled with high mass measurement accuracy (MMA) alone cannot be used for

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confident identification of unknowns2-5 because, even at high MMA (95% of false candidates.6 Spectral accuracy is the ability of the mass

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analyzer to accurately measure isotopic distributions (including isotopic fine

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structures) which, when coupled with high MMA, can be used for estimating the

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number of specific elements in an unknown.4,5,7-14 For example, to estimate the

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number of carbon atoms in a molecule, the relative abundance of A+1 (13C1) peak is

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divided by the natural abundance of

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abundance values reported by the National Institute of Standards and Technology

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(NIST) or International Union of Pure and Applied Chemistry (IUPAC).15 However,

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the values in these libraries are presented as “best-measurement” values, whereas

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the true isotopic abundances fall somewhere within the observed range of natural

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variations.15 For instance, the relative abundance of

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ranges from 0.96% to 1.15%, which provides sufficient variance to cause incorrect

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assessments of the number of carbons in a molecule.15

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C on Earth (~1.11%) based on natural

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C in terrestrial matter actually

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The motivation of the present work is to characterize SA of the Orbitrap mass

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analyzer2,16-18 based on the absolute ion abundances to allow its use in untargeted

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mass spectrometry imaging (MSI) studies with infrared matrix-assisted laser

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desorption electrospray ionization (IR-MALDESI)19,20 coupled to the Thermo Fisher

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Scientific Q Exactive PlusTM mass spectrometer.21 IR-MALDESI is an ambient

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ionization source where a mid-IR laser is used to desorb neutral species from a

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sample. The desorbed materials partition into the charged droplets of an orthogonal

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electrospray, where ions are formed in an ESI-like fashion.19 These ions are next

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stored in the C-trap for a pre-determined amount of time, denoted by the maximum

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injection time (IT),22 and the automatic gain control (AGC) function in the Q Exactive

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Plus mass spectrometer is disabled due to the pulsed nature of the IR-MALDESI

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source. While MS/MS analyses can be performed on peaks of interest to confirm the

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structure of putative identifications,21 acquiring MS/MS spectra for every analyte in

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every voxel is virtually impossible in untargeted MSI analyses. Therefore,

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identification of unknowns in untargeted IR-MALDESI studies relies solely on MMA

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and SA. Nazari et al. have previously demonstrated the utility of SA and sulfur

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counting in the identification of metabolites in an untargeted polarity switching MSI

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analysis,7 where analyte was confidently identified from four potential candidates

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generated in MELTIN database.23

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One critically important and limiting factor in accurately characterizing the SA

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of a mass analyzer is the fact that there is a variation in the relative abundance of

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different isotopes of each element found in nature. Thus, to investigate the effects of

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absolute ion abundance on the Orbitrap’s ability to recover expected isotopic

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distributions, we characterized known compounds spanning a wide mass range

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using

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characterized standards permits accurate abundance measurements of stable

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isotopes since the values obtained from NIST or IUPAC15 database have a relatively

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large variance. These analyses allowed us to establish two experimentally

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deteremined optimal conditions for each of three arbitrarily-defined mass windows of

isotope ratio

mass

spectrometry

(IRMS).

Analyzing

such

precisely

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Analytical Chemistry

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small (100 < MW (Da) < 400), medium (400 < MW (Da) < 900), and large (1000