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

Abstract

We demonstrate single-particle charge detection mass spectrometry on an Orbitrap for the analysis of megadalton biomolecular assemblies. We establish that the signal amplitudes of individual ions scale linearly with their charge, which can be used to resolve mixed ion populations, determine charge states and thus also determine the masses of individual ions. This enables the ultrasensitive analysis of heterogeneous protein assemblies including immunoglobulin oligomers, ribosomes, proteinaceous nanocontainers and genome-packed adeno-associated viruses.Native mass spectrometry (MS) is a powerful tool, enabling mass analysis of intact macromolecular assemblies in the megadalton range 1 . The exact mass of the intact macromolecular complex is then used to infer its composition and the stoichiometry of subunits, post-translational modifications and ligands bound to the complex 2 . Various mass analyzers, including quadrupole time-of-flight, Fourier-transform ion cyclotron resonance and, most recently, Orbitraps, have all been adapted for native MS experiments 3 .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 pioneered by Mann and Fenn 4 , 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. This hampers the analysis of larger heterogeneous protein assemblies, such as highly glycosylated proteins, amyloid fibrils, genomepacked viruses and membrane protein complexes decorated with multiple lipid molecules.Even small variabilities in the monomeric building blocks can result in wide distributions of masses in their larger assemblies. In combination with the often poor desolvation, these broadened mass distributions result in overlapping signals between consecutive charge states, leading to inaccurate mass assignments. A possible solution to these problems is to measure one particle (or ion) at a time, thereby avoiding the convolution of signals that stem from insufficient resolving power 5,6 . When such single-particle detection approaches can be combined with an independent measure of the

Higher-order structural characterisation of native proteins and complexes by top-down mass spectrometry

“…For fragile complexes, the result of this is that the achievable mass resolution may be too low for precisely dening the binding of small ligands. Recent publications by the Kelleher 48 and Heck 49 laboratories have demonstrated the use of charge detection MS of noncovalent complexes on Orbitrap instruments. In these experiments, small numbers of ions were allowed in the trap, allowing highly repeatable mass measurement of individual ions.…”

Connecting ‘multi-omics’ approaches to endogenous protein complexes

“…In terms of the development of MS approaches, the introduction of charge detection (CD) [66] is fundamentally changing data processing and improving the assignment of large protein complexes [67][68][69]. The rationale behind this approach is that peaks in nMS, which are often broad and overlap, render charge state assignment ambiguous.…”

Sample preparation and fractionation techniques for intact proteins for mass spectrometric analysis

“…Proteins can be ionized by ESI [27,65,80,81] or MALDI‐MS [75,82–84] and then analyzed by QQQ [27,65,81], Q‐TOF [71–75], linear trap quadrupole (LTQ)‐Orbitrap [74,85–87], or FT‐ICR [83,88–92] mass analyzers. Recently, the ability of Orbitrap MS to assign charge states and masses to proteins and their fragment ions has been demonstrated; this is based on the relationship between the cumulative ion intensity and acquisition time of individual ions [93–95]. Fragmentation can be achieved by collision‐induced dissociation [27,65,74,83,91], higher‐energy C‐trap dissociation [86,96], infrared multiphoton dissociation [83,90], ultraviolet photodissociation [97], electron capture dissociation [83,90,92], electron‐transfer dissociation [84], or in‐source decay [75].…”