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Dierences in the elemental isotope de nition may lead to errors in modern mass-spectrometry-based proteomics Jürgen Claesen,
∗,†
Frederik Lermyte,
‡, ¶
Frank Sobott,
and Dirk Valkenborg
‡, ¶
Tomasz Burzykowski,
§
§,¶,k
†Interuniversity Institute of Biostatistics and statistical Bioinformatics, Hasselt University, Diepenbeek, Belgium ‡Biomolecular and Analytical Mass Spectrometry, University of Antwerp, Antwerp, Belgium ¶Center for Proteomics, University of Antwerp, Belgium §Interuniversity Institute of Biostatistics and Statistical Bioinformatics, Hasselt University, Diepenbeek, Belgium kApplied Bio and Molecular Systems, VITO, Mol, Belgium E-mail:
[email protected] Abstract The elemental isotope definition used to calculate the theoretical masses and isotope distribution of (bio-)molecules is considered to be a fixed, universal standard in mass-spectrometry-based proteomics . However, this is an incorrect assumption. In view of the ongoing advances in mass spectrometry technology, and in particular the ever-increasing mass precision, the elemental isotope definition and its variations should be taken into account.
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2 We illustrate the effect of the elemental isotope uncertainty on the theoretical and experimental masses with theoretical calculations and examples, and a real-life case-study.
Introduction The definition of the elemental isotopes, i.e., their atomic masses and abundance probabilities, is required for the theoretical calculation of the molecular mass and isotope distribution of molecules. In mass-spectrometry-based proteomics the elemental isotope definition is often perceived as generally applicable, and considered as a universal standard. However, this assumption is incorrect and can lead to a bias in the theoretical mass calculation as the assumed elemental isotope abundances most likely deviate from the actual elemental isotope abundances in the measured sample. This bias can play an important role when comparing the theoretical masses to the mass values observed by mass spectrometry and may lead to false positive findings in protein identification. In addition to these “natural" deviations, differences between various standards, such as IUPAC and NIST, and between the elemental isotope definitions used by instrument vendors and software developers 1,2 can be observed. These discrepancies can lead to confusion and can hamper the inter-operability of MS-related software tools. In the early nineties, a number of articles 3–6 illustrated the effect of elemental isotope definition variations on the mono-isotopic and average mass of biomolecules. At that time, the mass precision of most mass spectrometers was insufficient to reflect these minute differences in the average masses when, for example, only considering varying isotope abundances for carbon. In previous analyses, other chemical elements were often ignored, or assumed to have little or no impact ACS Paragon Plus Environment
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3 on the observed mass differences. However, as the mass precision of mass spectrometers has improved over the last two decades 7,8 (see Table 1), and as it will continue to improve in the future, one should carefully consider the influence of the differences in the elemental isotope definition. Table 1: Mass precision and resolution of modern mass spectrometers. 9–11 Mass Spectrometer Mass Precision (ppm) Resolution (FWHM) FTICR
