Orbitrap-Based Mass and Charge Analysis of Single Molecules

Abstract

Native mass spectrometry is nowadays widely used for determining the mass of intact proteins and their noncovalent biomolecular assemblies. While this technology performs well in the mass determination of monodisperse protein assemblies, more real-life heterogeneous protein complexes can pose a significant challenge. Factors such as co-occurring stoichiometries, subcomplexes, and/or post-translational modifications, may especially hamper mass analysis by obfuscating the charge state inferencing that is fundamental to the technique. Moreover, these mass analyses typically require measurement of several million molecules to generate an analyzable mass spectrum, limiting its sensitivity. In 2012, we introduced an Orbitrap-based mass analyzer with extended mass range (EMR) and demonstrated that it could be used to obtain not only high-resolution mass spectra of large protein macromolecular assemblies, but we also showed that single ions generated from these assemblies provided sufficient image current to induce a measurable charge-related signal. Based on these observations, we and others further optimized the experimental conditions necessary for single ion measurements, which led in 2020 to the introduction of single-molecule Orbitrap-based charge detection mass spectrometry (Orbitrap-based CDMS). The introduction of these single molecule approaches has led to the fruition of various innovative lines of research. For example, tracking the behavior of individual macromolecular ions inside the Orbitrap mass analyzer provides unique, fundamental insights into mechanisms of ion dephasing and demonstrated the (astonishingly high) stability of high mass ions. Such fundamental information will help to further optimize the Orbitrap mass analyzer. As another example, the circumvention of traditional charge state inferencing enables Orbitrap-based CDMS to extract mass information from even extremely heterogeneous proteins and protein assemblies (e.g., glycoprotein assemblies, cargo-containing nanoparticles) via single molecule detection, reaching beyond the capabilities of earlier approaches. We so far demonstrated the power of Orbitrap-based CDMS applied to a variety of fascinating systems, assessing for instance the cargo load of recombinant AAV-based gene delivery vectors, the buildup of immune-complexes involved in complement activation, and quite accurate masses of highly glycosylated proteins, such as the SARS-CoV-2 spike trimer proteins. With such widespread applications, the next objective is to make Orbitrap-based CDMS more mainstream, whereby we still will seek to further advance the boundaries in sensitivity and mass resolving power.

Figure 1

Transmission of macromolecular ions into the Orbitrap and different methods of mass measurement. (A) In standard, ensemble native MS experiments, millions of ions are simultaneously introduced into the Orbitrap mass analyzer, and the recorded image current is converted to an m/z spectrum. Signal accumulates for ions that bear the same mass and charge, resulting in a distribution of charge states. (B) In Orbitrap-based CDMS the number of ions entering the Orbitrap mass analyzer is reduced to 10⁰–10¹ to avoid detecting multiple ions of identical mass and charge and the associated signal accumulation. Mass spectra recorded in the single-ion regime thus exhibit a “spike” for each ion detected, where the intensity relates to the ion’s induced image current (charge). Because charge is quantized, coincidental measurement of two ions of identical m/z in the same acquisition is distinguishable by the appearance of signals at ∼double intensity.

Figure 2

Processing of single-particle data in Orbitrap-based CDMS. (A) The single ion peaks are obtained in the frequency domain (=m/z) after FT of the image current collected in the time domain (the transient). For every peak both an m/z and intensity are recorded. During acquisition, some ions can drift in frequency, causing signal splitting and tailing to lower values in the intensity domain. Implementing a filter step in which split peaks are detected and eliminated, improving the determination of charge and thus also mass. This approach requires only the standard (frequency-domain) mass spectrum. (B) In the frequency-chasing approach, transients are segmented into time intervals, allowing ion drifts to be monitored over the detection period. Ions that would have previously been filtered can thus be addressed, enabling longer acquisitions and in turn improving the accuracy and precision of Orbitrap-based CDMS measurements. This approach requires access to the time-domain Orbitrap data. (C) The image current amplitude of an ion orbiting in the Orbitrap scales linearly with the ion charge. To determine this relationship quantitatively, single ion intensities of well-characterized biomolecules are plotted against their a priori determined charge values (i.e., by standard inferencing) to generate an intensity-to-charge calibration curve. Once calibrated, single ion intensities can be directly converted to a corresponding charge value. Adapted with permission from ref (1). Copyright 2020 Springer Nature. (D) Calibrated assignment of charge together with their m/z values allows the mass of each individual ions to be calculated. These individual ion masses can then be plotted together in a zero-charge mass histogram.

Figure 3

Mass determination of antibody–antigen complexes comparing ensemble native MS and Orbitrap-based CDMS. (A) Although ensemble native MS can resolve individual glycoforms of IgG1 (upper panel), the extensive glycosylation of the antigen sEGFR prohibits resolving features of sEGFR alone (middle) and of IgG1-sEGFR complexes (lower). (B) Orbitrap-based CDMS enables accurate mass determination of both sEGFR (upper) and of all co-occurring species of IgG1 bound to sEGFR (lower). Insets depict the two-dimensional separation in CDMS. Masses determined for each species, shown across the top of the panel, correspond to the mean (dotted line) of each fitted normal distribution. Adapted with permission from ref (48). Copyright 2022 American Chemical Society.

Figure 4

Characterization of the gene-delivery adeno-associated viral vectors (AAVs) using ensemble native MS and Orbitrap-based CDMS. (A) Ensemble native mass spectrum of AAV8 recorded on a UHMR Orbitrap (32 ms transient). Two populations, corresponding to filled and overfilled capsids, can be discerned, but precise mass determination is hampered by sample heterogeneity. (B) Two-dimensional CDMS histogram of an AAV6a sample (left) and its corresponding mass histogram (right). Adapted with permission from ref (3). Copyright 2022 Elsevier. (C) Mass histograms of genome-packed AAVs containing a CMV-GFP transgene measured by Orbitrap-based CDMS. Distinct mass distributions are observed across AAV2 samples produced either from human-cell or insect-cell based platforms and/or obtained from separate providers. Adapted with permission from ref (28). Copyright 2022 Elsevier.