Inaccuracies in selected ion monitoring determination of isotope ratios obviated by profile acquisition: nucleotide 18O/16O measurements

Abstract

Precise and accurate measurements of isotopologue distributions (IDs) in biological molecules are needed for the determination of isotope effects, quantitation by isotope dilution and quantifying isotope tracers employed in both metabolic and biophysical studies. While single ion monitoring (SIM) yields significantly greater sensitivity and signal/noise than profile mode acquisitions, we show that small changes in the SIM window width and/or center can alter experimentally determined isotope ratios by up to 5%, resulting in significant inaccuracies. This inaccuracy is attributed to mass granularity, the differential distribution of digital data points across the m/z ranges sampled by SIM. Acquiring data in the profile mode and fitting the data to an equation describing a series of equally spaced and identically shaped peaks eliminates the inaccuracies associated with mass granularity with minimal loss of precision. Additionally a method of using the complete ID profile data that inherently corrects for "spillover" and for the natural abundance ID has been used to determine 18 Keywords isotope ratio; isotopologue distribution; mass isotopomer distribution; tandem mass spectrometry; heavy atom isotope effect; oxygen-18; nucleotide; mass granularity; whole molecule mass spectrometry 1 This work supported by NIH grants GM56740 (M.E.H.), R33 DK07029 (VEA & SP) and a HHMI predoctoral fellowship (A.G.C.). *Corresponding Authors: Vernon E. Anderson and Michael E. Harris, Department of Biochemistry, Case Western Reserve University School of Medicine, Cleveland, OH 44106-4935, Phone: 216-368-2511 (VEA); 216-368-4779 (MEH), Fax: 216-368-3419, Email: vernon.anderson@case.edu; michael.harris@cwru.edu. 3 Current address: Department of Chemistry, Drew University, Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. NIH Public Access Author ManuscriptAnal Biochem. Author manuscript; available in PMC 2008 August 1. Published in final edited form as:Anal Biochem. 2007 August 1; 367(1): 28-39. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author ManuscriptHeavy atom isotope effects generated by substituting 13 C, 15 N or 18 O are important tools for probing the mechanisms and transition states of chemical reactions [1], including enzymecatalyzed reactions [2][3][4][5][6][7]. Because the observed effects for isotopic substitution of heavy atoms are extremely small, typically a few percent, they cannot be determined by direct comparison of experimentally determined rate constants. Instead, heavy atom isotope effects are generally measured by internal competition methods that...

Isotopologue distributions of peptide product ions by tandem mass spectrometry: Quantitation of low levels of deuterium incorporation

“…In these results, to our surprise, the ion trap instrument performed better than the SIM quadrupole determination which had greater precision than the MALDI-TOF analyses. The performance of the quadrupole analysis contributed to our more thorough evaluation of the potential effects of mass granularity reported in the accompanying paper [15]. Based on the results reported there, the accuracy of the quadrupole could have been improved if the data had been acquired and analyzed in profile scan rather than SIM mode.…”

“…The areas of the successive ions in the IIC comprise the ID and are denoted M n . The isotopologues contributing to the intensity of the M 1 peak will have a single heavy atom substitution, exactly one 2 H, 13 C, 15 N or 17 O atom substituted for the more common isotope (with the higher resolution of ion cyclotron resonance mass spectrometry it is possible to separate the different isotopologues, but not with the typical resolution of commercial TOF instruments).…”

“…The reflectron mode was used with a laser power attenuation range from 70-80 and an average of 500 scans acquired so that the base peak had an intensity in excess of 20,000 counts. MALDI data were converted to ASCII files using the m over z [13] export window function and the data fit in Origin [14] as described in the accompanying paper [15].…”

“…These approximations place constraints on the use of equation 14 to cases where the number of isotopically substituted atoms is greater than the number of isotopologue ions analyzed 4 , and where the mol fractions x e1 and x e2 < 0.25. To determine the isotopic composition from the ID in cases with a small number of highly enriched sites, the method of the accompanying paper may be used [15].The approximations of equation 11 result in a small overestimation of M n /M 0 for n ≥ 2 which increases with n. For molecules at or near natural abundance, this overestimation can be empirically corrected by a fudge factor, f (≤ 1), as shown in equation 15. Using n-1 as the exponential power of f accounts both for the fact that M 1 /M 0 is exactly correct (so the correction is unity for n=1) and subsequently increases with n. is less than the spread in calculated values from "exact" calculations.…”

“…The reflectron mode was used with a laser power attenuation range from 70-80 and an average of 500 scans acquired so that the base peak had an intensity in excess of 20,000 counts. MALDI data were converted to ASCII files using the m over z [13] export window function and the data fit in Origin [14] as described in the accompanying paper [15].ESI-quadruple mass spectrometry was carried out on either a Finnigan TSQ 7000 or Micromass Quattro II in the SIM mode, while tandem mass spectrometry was performed with a ThermoFinnigan LTQ linear ion trap or an Applied Biosystems Q-STAR XL hybrid quadrupole-TOF mass spectrometer. All IICs detected by ion trap were acquired in the Zoom Scan mode.…”