Cryo-LESA Mass Spectrometry—a Step Towards Truly Native Surface Sampling of Proteins

Probing Folded Proteins and Intact Protein Complexes by Desorption Electrospray Ionization Mass Spectrometry

“…3a). Compared with typical denaturing ubiquitin mass spectra, 29,30 here high charge states 10+ to 12+ were not detected, suggesting that very few ions of completely unfolded protein were detected. This may be attributed to the very brief, transient, interaction time between the harsh solvent and the protein within DESI analysis as well as very stable folded structure of ubiquitin 31 leading to an incomplete unfolding in this experiment.…”

“…Results presented in figures 3 and 4 showed that folded protein structures and intact protein complexes can be detected using DESI with a standard inlet capillary. In previous LESA studies, 29,33 structure collapse was observed during room temperature drying of droplets of proteins and protein complexes prepared in native conditions. Furthermore, refolding of unfolded proteins and protein complexes has been shown to occur when using native sampling solvent conditions for analysis of denatured samples.…”

“…Furthermore, refolding of unfolded proteins and protein complexes has been shown to occur when using native sampling solvent conditions for analysis of denatured samples. 29,33 As a result, during native protein surface analysis, the sampling solvent seems to play a vital role in determining the observed spectra features. In these experiments we investigate whether refolding can also be observed in native DESI-MS. Ubiquitin, cytochrome c and the protein complex myoglobin were analyzed.…”

Probing Folded Proteins and Intact Protein Complexes by Desorption Electrospray Ionization Mass Spectrometry

“…Our next study plans will investigate methods to improve the detection sensitivity of native DESI analysis and explore strategies to overcome the protein refolding phenomenon therein. The solutions for the latter may include 1) narrowing the spray coverage area on surface or employing faster DESI stage moving speed to reduce the protein analytes extraction time into solvent before they are ejected 2) employing a cryo sampling strategy like we used in our native LESA studies 29 to frozen the structures of surface proteins to be analyzed.…”

Rapid structural discrimination of IgG antibodies by multicharge-state collision-induced unfolding

“…24 As a highly complementary method, native MS has recently emerged as a promising method for producing intact gas-phase protein complexes and even membrane proteins, providing valuable stoichiometry and topology information. 25,26 Gratifyingly, native MS can be hyphenated with ion mobility-mass spectrometry (IM-MS), which has been widely acknowledged in studying intact protein assemblies, such as dynamics, 13,27 thermal stability, 28,29 protein refolding, 26,30 and antibody-drug conjugates. 31,32 Despite these successful applications, great challenges remain because the available IM-MS normally has limited spectral resolution, and therefore, fails to produce sufficient information for dening macromolecular structures in detail.…”