Probing the Fundamentals of Native Liquid Extraction Surface Analysis Mass Spectrometry of Proteins: Can Proteins Refold during Extraction?

Abstract

Native ambient mass spectrometry has the potential for simultaneous analysis of native protein structure and spatial distribution within thin tissue sections. Notwithstanding sensitivity, this information can, in principle, be obtained for any protein present with no requirement for a priori knowledge of protein identity. To date, native ambient mass spectrometry has primarily made use of the liquid extraction surface analysis (LESA) sampling technique. Here, we address a fundamental question: Are the protein structures observed following native liquid extraction surface analysis representative of the protein structures within the substrate, or does the extraction process facilitate refolding (or unfolding)? Specifically, our aim was to determine whether protein–ligand complexes observed following LESA are indicative of complexes present in the substrate, or an artifact of the sampling process. The systems investigated were myoglobin and its noncovalently bound heme cofactor, and the Zn-binding protein carbonic anhydrase and its binding with ethoxzolamide. Charge state distributions, drift time profiles, and collision cross sections were determined by liquid extraction surface analysis ion mobility mass spectrometry of native and denatured proteins and compared with those obtained by direct infusion electrospray. The results show that it was not possible to refold denatured proteins with concomitant ligand binding (neither heme, zinc, nor ethoxzolamide) simply by use of native-like LESA solvents. That is, protein–ligand complexes were only observed by LESA MS when present in the substrate.

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

“…34,35 However, refolding of holo-myoglobin was not observed in native LESA studies of surface dried sample, although adjusting native sampling solvent of 25mM ammonium acetate by 2.5% ammonium hydroxide led to ~ 3% of myoglobin detected as holo-protein. 33 Surprisingly, an extraordinary amount of refolding was observed during our native DESI studies of denatured myoglobin. As seen in Fig.…”

“…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.…”

Integrating ion mobility and imaging mass spectrometry for comprehensive analysis of biological tissues: A brief review and perspective

“…60,74,75 For example, TWIMS has been coupled to many ion sources including MALDI, 36,[76][77][78][79][80] DESI, 60,74,80,81 LAESI, 82,83 and LESA (Figure 2A-D). 48,61,84,85 is a point of much discussion in the scientific community. [85][86][87] In another example, Hale and colleagues showed that mobility information provided by TWIMS enabled filtering of spectral noise that contributed to improved ion image quality.…”

“…48,61,84,85 is a point of much discussion in the scientific community. [85][86][87] In another example, Hale and colleagues showed that mobility information provided by TWIMS enabled filtering of spectral noise that contributed to improved ion image quality. 61 For example, the authors…”