Isotope Decluttering Reduces Spectral Complexity while Maintaining Protein Structure

Analytical Chemistry, vol. 98, pp. 16063–16072

Abstract

Accurate mass determination is one of the fundamental objectives in mass spectrometry (MS) as it enables confident molecular identification and detection of subtle mass differences. Precise mass determination typically relies on the measurement of the monoisotopic peak. The increasing number of heavier isotopes, as the molecular mass increases, leads to spectral complexity, broadening of the isotopic distribution, and dispersal of signal intensity, ultimately reducing the signal-to-noise ratio (SNR). These effects are enhanced while analyzing larger proteins and protein complexes. In this study, we expressed and purified two Mycobacterium tuberculosis protein complexes, EsxAB and bacterioferritin B (BfrB), under isotope-depleted conditions to reduce the abundances of the heavier isotopes of carbon and nitrogen. We applied isotope depletion to BfrB, which represents the largest mass of the isotope-depleted protein complex studied under native conditions to date, and, for the first time, investigated its structural consequences. Isotope-depleted proteins as well as protein complexes showed simplified mass spectra by reducing the isotopic distribution, with a significant increase in the SNR of the monoisotopic peak followed by improved protein sequence coverage by native top-down MS. Furthermore, our investigation of protein structure, by single-particle analysis using cryo-electron microscopy (cryo-EM), demonstrated that isotope depletion preserves the structural integrity of proteins, even at atomic resolution. Collectively, our findings show that isotope depletion is a suitable method for high-accuracy mass measurement and identification by MS while maintaining the structural integrity of the proteins.

Authors 10

1. Sneha Hakke

   Maastricht University

   Affiliation as printed

   Maastricht Multi-Modal Molecular Imaging Institute (M4i)

   Maastricht University

2. Willem E. M. Noteborn Aachen Netherlands Centre for Electron Nanoscopy (NeCEN)

   Leiden University

   Affiliation as printed

   Leiden University

   Netherlands Centre for Electron Nanoscopy (NeCEN)

3. Birol Cabukusta

   Maastricht University

   Affiliation as printed

   Maastricht Multi-Modal Molecular Imaging Institute (M4i)

   Maastricht University

4. Ye Gao

   Maastricht University

   Affiliation as printed

   Maastricht Multi-Modal Molecular Imaging Institute (M4i)

   Maastricht University

5. Kèvin Knoops

   Maastricht University

   Affiliation as printed

   Maastricht University

   Microscopy CORE Lab

6. Carmen López-Iglesias

   Maastricht University

   Affiliation as printed

   Maastricht University

   Microscopy CORE Lab

7. David P. A. Kilgour
   Affiliation as printed

   Vibrat-Ion Ltd

8. David J. Clarke

   University of Edinburgh

   Affiliation as printed

   EaStCHEM School of Chemistry

   University of Edinburgh

9. Anjusha Mathew

   Maastricht University

   Affiliation as printed

   Maastricht Multi-Modal Molecular Imaging Institute (M4i)

   Maastricht University

10. Ron M. A. Heeren corresponding

    Maastricht University

    Affiliation as printed

    Maastricht Multi-Modal Molecular Imaging Institute (M4i)

    Maastricht University