ConspectusNative 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.