Approaches to Heterogeneity in Native Mass Spectrometry

Abstract

Native mass spectrometry (MS) is aimed at preserving and determining the native structure, composition, and stoichiometry of biomolecules and their complexes from solution after they are transferred into the gas phase. Major improvements in native MS instrumentation and experimental methods over the past few decades have led to a concomitant increase in the complexity and heterogeneity of samples that can be analyzed, including protein–ligand complexes, protein complexes with multiple coexisting stoichiometries, and membrane protein–lipid assemblies. Heterogeneous features of these biomolecular samples can be important for understanding structure and function. However, sample heterogeneity can make assignment of ion mass, charge, composition, and structure very challenging due to the overlap of tens or even hundreds of peaks in the mass spectrum. In this review, we cover data analysis, experimental, and instrumental advances and strategies aimed at solving this problem, with an in-depth discussion of theoretical and practical aspects of the use of available deconvolution algorithms and tools. We also reflect upon current challenges and provide a view of the future of this exciting field.

UniDecCD: Deconvolution of Charge Detection-Mass Spectrometry Data

“…Since the introduction of electrospray ionization-mass spectrometry (ESI-MS), deconvolution of the charge and mass dimensions of mass spectra has been a critical step in their analysis. 1 Because ESI-MS of peptides and proteins produces ions with multiple charges, measuring the mass requires first assigning the charge states. The most direct way to assign charge states in ESI-MS is from the isotope distribution, which leverages the known mass spacing between isotopologues.…”

Spectral Interferences Impede the High-Resolution Mass Analysis of Recombinant Adeno-Associated Viruses

“…35,46 In a high-resolution native MS experiment, standard charge state inference strategies are utilized to indirectly assign charge state values to each species. 47,48 The primary advantage of this approach is that a substantially higher mass resolution to CD-MS can be obtained as there is essentially no uncertainty in charge determination - with the caveat that the mass spectra must be sufficiently well-resolved for charge states to be accurately inferred. In cases where this requirement cannot be fulfilled, CD-MS out-performs standard native MS in mass determination ability.…”

Fundamentals: How Do We Calculate Mass, Error, and Uncertainty in Native Mass Spectrometry?

“…In homogeneous spectra where charge states are clearly assignable we will assume essentially zero uncertainty in the charge state. Heterogeneous spectra , with uncertainty in the charge-state assignments will be partially captured by the metrics described below but may require additional considerations not explored here.…”

“…Finally, we will primarily consider homogeneous spectra with easily assignable charge states to simplify our discussion. Heterogeneous spectra can present additional challenges of intrinsic ambiguity and are nicely reviewed by Roland and Prell 19 and discussed previously. 20 Precise definitions of mass, error, and uncertainty specific to native MS will help ensure reproducibility of results, standardize methods, and guide software development for data analysis.…”

“…Finally, we will primarily consider homogeneous spectra with easily assignable charge states to simplify our discussion. Heterogeneous spectra can present additional challenges of intrinsic ambiguity and are nicely reviewed by Roland and Prell and discussed previously …”