Resolving heterogeneous high-mass macromolecular machineries by Orbitrap-based single particle charge detection mass spectrometry

Abstract

Here we show that single particle charge-detection mass spectrometry (CD-MS) can be performed on a ubiquitous Orbitrap mass analyser and applied to the analysis of highmass (megadalton) heterogeneous biomolecular assemblies. We demonstrate that single particle high-mass ions can survive in the Orbitrap for seconds, whereby their measured signal amplitudes scale linearly with charge state over the entire m/z range. Orbitrap based single particle CD-MS can be used to resolve mixed ion populations, accurately predict charge states, and consequently also the mass of the ions. We successfully applied CD-MS to challenging natural and biotherapeutic protein assemblies, such as IgM oligomers, designed protein nano-cages, ribosome particles and intact, empty-and genome-loaded Adeno-associated virus particles. Single particle CD-MS combined with native MS on existing Orbitrap platforms will greatly expand its application, especially in the mass analysis of megadalton heterogeneous biomolecular assemblies.3 Main text:Over the past two decades, native mass spectrometry (MS) has developed into a powerful tool for the characterization of biological macromolecular complexes 1 . These complexes are analysed from solvents that preserve the noncovalent interactions, enabling mass analysis of intact macromolecular assemblies into the megadalton (MDa) range 2 . The exact mass of the intact macromolecular complex is then used to infer its composition, the subunit stoichiometry, and even the number and chemical nature of post-translational modifications and small molecule ligands bound to the complex 3-6 . Various mass analysers, including Quadrupole Time-of-Flight, Fourier-Transform Ion CyclotronResonance, and most recently Orbitrap™-based mass analysers, have all been adapted for native MS experiments 7-12 . With the successful modifications of the Orbitrap for use in native MS, an unprecedented mass resolving power in the high mass range could be achieved, opening the avenue for high resolution analysis on ever more heterogeneous protein assemblies like therapeutic and plasma glycoproteins, designed protein cages, membrane protein assemblies, intact viruses, and ribosomes 12,13 .Notably, masses are not measured directly in most MS approaches, but need to be inferred from the mass-to-charge (m/z) ratios of the detected ions. As first shown in the pioneering work of Mann and Fenn 14 , the charge states from a population of multiply charged ions generated by electrospray ionization (ESI) can be determined from the m/z values by matching consecutive peaks in the charge state distribution to calculate accurate masses. A general limitation in native MS studies then stems from the fact that the charge state, and thus also the mass, can only be accurately measured when multiple charge states of the same molecular species can be resolved and assigned in the m/z for the trimer and 56 for the tetramer precisely coincide at 10,649 m/z (see Fig. 4B).However, the single ion intensities of the IgG oligomers scale with charge state (see Fig. 4C...

Native top‐down mass spectrometry for higher‐order structural characterization of proteins and complexes

“…CDMS is particularly suitable to the analysis of high‐mass, heterogeneous analytes. Its eye‐catching performance has recently been demonstrated in several exciting studies, including characterizing highly complicated mixtures of proteoforms and their complexes (Kafader, Melani, et al, 2020), ribosomes, and genome‐packed adeno‐associated viruses (Wörner et al, 2020).…”

Characterizing Adeno-Associated Virus Capsids with Both Denaturing and Intact Analysis Methods

“…Intensity-Based CD-MS. Intensity-based CD-MS, pioneered by the Heck lab and Alexander Makarov, 36,37 was performed on a UHMR Orbitrap (Bremen, Germany) as described previously. 28,29,31,37 Intensity-based CD-MS uses the signal intensity or the signal-to-noise (S/N) ratio of single ions to determine their charge. 31 For our intensity-based CD-MS measurements, we used the S/N ratio to determine the charge of single ions.…”

Individual Ion Mass Spectrometry Enhances the Sensitivity and Sequence Coverage of Top-Down Mass Spectrometry

“…1,2 Traditionally, these experiments have been performed on specialized, custom-built instruments specifically designed for the charge detection of individual ions in an electrostatic linear ion trap analyzer. 3−5 Recent developments have revealed that the charge of an ion can be accurately assigned using the commercially available Orbitrap analyzer; two recent reports showed the detection of individual ions, with one having a charge assignment with an uncertainty of ±3 to 4 unit charges 6 and relying on prior m/z knowledge for species separation, whereas the other approach performs direct charge assignment on 6+ to 70+ ions with >96% reliability. 7 The latter was accomplished utilizing the summation of the ion signal as a function of time (Selective Temporal Overview of Resonant Ions; STORI plot analysis) 8 coupled to the Orbitrap analyzer's ability to measure ion frequencies with high accuracy.…”