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Isotope Depletion Mass Spectrometry (ID-MS) for Enhanced Top-Down Protein Fragmentation

submitted on 01.10.2018, 14:34 and posted on 02.10.2018, 15:24 by Kelly J. Gallagher, Michael Palasser, Sam Hughes, C. Logan Mackay, David P. A. Kilgour, David Clarke
Top-down mass spectrometry has become an important technique for the identification of proteins and characterisation of chemical and posttranslational modifications. However, as the molecular mass of proteins increases intact mass determination and top-down fragmentation efficiency become more challenging due to the partitioning of the mass spectral signal into many isotopic peaks. In large proteins, this results in reduced sensitivity and increased spectral complexity and signal overlap. This phenomenon is a consequence of the natural isotopic heterogeneity of the elements which comprise proteins (notably 13C). Here we present a bacterial recombinant expression system for the production of proteins depleted in 13C and 15N and use this strategy to prepare a range of isotopically depleted proteins. High resolution MS of isotope depleted proteins reveal dramatically reduced isotope distributions, which results in increases in sensitivity and deceased spectral complexity. We demonstrate that the monoisotopic signal is observed in mass spectra of proteins up to ~50 kDa. This allows confident assignment of accurate molecular mass, and facile detection of low mass modifications (such as deamidation). We outline the benefits of this isotope depletion strategy for top-down fragmentation. The reduced spectral complexity alleviates problems of signal overlap; the presence of monoisotopic signals allow more accurate assignment of fragment ions; and the dramatic increase in single-to-noise ratio (up to 7-fold increases) permits vastly reduced data acquisition times. Together, these compounding benefits allow the assignment of ca. 3-fold more fragment ions than analysis of proteins with natural isotopic abundances. Thus, more comprehensive sequence coverage can be achieved; we demonstrate near single amino-acid resolution of the 29 kDa protein carbonic anhydrase from a single top-down MS experiment. Finally, we demonstrate that the ID-MS strategy allows far greater sequence coverage to be obtained in time limited top-down data acquisitions – highlighting potential advantages for top-down LC-MS/MS workflows and top-down proteomics.


This work was funded by the University of Edinburgh and a Royal Society Research Grant awarded to D.J.C. K. J. G. is supported by an Engineering and Physical Sciences Research Council (EPSRC) PhD studentship. M.P. was supported by the Erasmus programme.


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University of Edinburgh



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Declaration of Conflict of Interest

no conflict of interest