The structure and folding of a protein in solution depends on noncovalent interactions within the protein and those with surrounding ions and molecules. Decoupling these interactions in solution is challenging, which has hindered the development of accurate physics-based models for structure prediction. Investigations of proteins in the gas phase can be used to selectively decouple factors affecting the structures of proteins. Here, we use Cation to Anion Proton Transfer Reactions (CAPTR) to reduce the charge states of denatured ubiquitin ions in the gas phase, and ion mobility to probe their structures. In CAPTR, a precursor charge state is selected (P) and reacted with monoanions to generate charge-reduced product ions (C). Following each CAPTR event, denatured ubiquitin ions (13+ to 6+) yield products that rapidly isomerize to structures that have smaller collision cross sections (Ω). The Ω of CAPTR product ions depend strongly on C and very weakly on P. Pre- and post-CAPTR activation was then used to probe the potential-energy surfaces of the precursor and product ions, respectively. Post-CAPTR activation showed that ions of different P fold differently and populate different regions of the potential-energy surface of that ion. Finally, pre-CAPTR activation showed that the structures of protein ions can be indirectly investigated using ion mobility of their CAPTR product ions, even for subtle structural differences that are not apparent from ion mobility characterization of the activated precursor ions. More generally, these results show that CAPTR strongly complements existing techniques for characterizing the structures and dynamics of biological molecules in the gas phase.

The relationship between structures of protein ions, their charge states, and their original structures prior to ionization remains challenging to decouple. Here, we use cation-to-anion proton-transfer reactions (CAPTR) to reduce the charge states of cytochrome c ions in the gas phase and ion mobility to probe their structures. Ions were formed using a new temperature-controlled, nanoelectrospray ionization source at 25 °C. Characterization of this source demonstrates that the temperature of the liquid sample is decoupled from that of the atmospheric-pressure interface, which is heated during CAPTR experiments. Ionization from denaturing conditions yields 18+ to 8+ ions, which were each isolated and reacted with monoanions to generate all CAPTR products with charge states of at least 3+. The highest, intermediate, and lowest charge-state products exhibit collision cross section distributions that are unimodal, multimodal, and unimodal, respectively. These distributions depend strongly on the charge state of the product, although those for the intermediate charge-state products also depend on that of the precursor. The distributions of the 3+ products are all similar, with averages that are less than half that of the 18+ precursor ions. Ionization of cytochrome c from native-like conditions yields 7+ and 6+ ions. The 3+ CAPTR products from these precursors have slightly more compact collision cross section distributions that are indistinguishable from those for the 3+ CAPTR products from denaturing conditions. More broadly, these results indicate that the collision cross sections of ions of this single domain protein depend strongly on charge state for charge states greater than ~4.

The fidelity between the structures of proteins in solution and protein ions in the gas phase is critical to experiments that use gas-phase measurements to infer structures in solution. Here we generate ions of lysozyme, a 129-residue protein whose native tertiary structure contains four internal disulfide bonds, from three solutions that preserve varying extents of the original native structure. We then use cation-to-anion proton-transfer reactions (CAPTR) to reduce the charge states of those ions in the gas phase and ion mobility to probe their structures. The collision cross section (Ω) distributions of each CAPTR product depends to varying extents on the original solution, the charge state of the product, and the charge state of the precursor. For example, the Ω distributions of the 6+ ions depend strongly on the original solutions conditions and to a lesser extent on the charge state of the precursor. Energy-dependent experiments suggest that very different structures are accessible to disulfide-reduced and disulfide-intact ions, but similar Ω distributions are formed at high energy for disulfide-intact ions from denaturing and from aqueous conditions. The Ω distributions of the 3+ ions are all similar but exhibit subtle differences that depend more strongly on the original solutions conditions than other factors. More generally, these results suggest that specific CAPTR products may be especially sensitive to specific elements of structure in solution.

Stimulated Raman scattering (SRS) microscopy is a powerful method for imaging molecular distributions based on their intrinsic vibrational contrast. However, despite a growing list of biological applications, SRS is frequently hindered by a parasitic background signal which both overpowers the signal in low-signal applications and makes the extraction of quantitative information from images challenging. Frequency modulation (FM) has been used to suppress this parasitic background. However, many FM-SRS methods require either the acquisition of multiple images or the addition of multiple optomechanical components and an extensive realignment procedure. Herein, we report a new procedure for alignment-free FM-SRS utilizing polarization encoding. We demonstrate the efficacy of this approach, along with parabolic amplification of the Stokes pulse, at removing parasitic background signals in SRS microscopy applications. We further highlight how this technique can be used to suppress Raman signals from major molecular species to unveil spectral signatures from nucleic acids in both murine brain tissue and whole blood. Due to its ease of use and demonstrated experimental capabilities, we expect this technique to see broad use in the SRS microscopy community.

In the quest to decipher the chain of life from molecules to cells, the biological and biophysical questions being asked increasingly demand techniques that are capable of identifying specific biomolecules in their native environment, and can measure biomolecular interactions quantitatively, at the smallest possible scale in space and time, without perturbing the system under observation. The interaction of light with biomolecules offers a wealth of phenomena and tools that can be exploited to drive this progress. This Roadmap is written collectively by prominent researchers and encompasses selected aspects of bio-nano-photonics, spanning from