Collision cross‐section values, which can be determined using ion mobility experiments, are sensitive to the structures of protein ions and useful for applications to structural biology and biophysics. Protein ions with different charge states can exhibit very different collision cross‐section values, but a comprehensive understanding of this relationship remains elusive. Here, we review cation‐to‐anion, proton‐transfer reactions (CAPTR), a method for generating a series of charge‐reduced protein cations by reacting quadrupole‐selected cations with even‐electron monoanions. The resulting CAPTR products are analyzed using a combination of ion mobility, mass spectrometry, and collisional activation. We compare CAPTR to other charge‐manipulation strategies and review the results of various CAPTR‐based experiments, exploring their contribution to a deeper understanding of the relationship between protein ion structure and charge state.

The ability of nanoelectrospray ionization (nanoESI) to generate a continuous flow of charged droplets relies on the electrolytic nature of the process. This electrochemistry can lead to the accumulation of redox products in the sample solution. This consequence can have significant implications for native mass spectrometry (MS), which aims to probe the structures and interactions of biomolecules in solution. Here, ratiometric fluorescence imaging and a pH-sensitive, fluorescent probe are used to quantify changes in solution pH during nanoESI under conditions relevant to native MS. Results show that the extent and rate of change in sample pH depends on several experimental parameters. There is a strong correlation between the extent and rate of change in solution pH and the magnitude of both the nanoESI current and electrolyte concentration. Smaller changes in solution pH are observed during experiments when a negative potential is applied than for those when a positive potential is applied. Finally, we make specific recommendations for designing native MS experiments that control for these effects.

Recent developments in ion mobility (IM) technology have expanded the capability to separate and characterize gas-phase ions of biomolecules, especially when paired with mass spectrometry. This next generation of IM technology has been ushered in by creative innovation focused on both instrument architectures and how electric fields are applied. In this review, we focus on the application of high-resolution and multidimensional IM to biomolecular analyses, encompassing the fields of glycomics, lipidomics, peptidomics, and proteomics. We highlight selected research that demonstrates the application of the new IM toolkit to challenging biomolecular systems. Through our review of recently published literature, we outline the current strengths of respective technologies and perspectives for future applications. Expected final online publication date for the Annual Review of Analytical Chemistry, Volume 16 is June 2023. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.