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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] ACS Paragon Plus Environment
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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
