Probing the Fundamentals of Native Liquid Extraction Surface Analysis Mass Spectrometry of Proteins: Can Proteins Refold during Extraction?
journal contributionposted on 19.09.2019, 17:39 by Eva Illes-Toth, Helen J. Cooper
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.
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Native ambient mass spectrometryNative Liquid Extraction Surface Analysis Mass Spectrometrynative-like LESA solventsZn-binding protein carbonic anhydraseprotein structuresdrift time profilesMSextraction surface analysisextraction surface analysis representativeextraction surface analysis ion mobility mass spectrometrycharge state distributionsrefold denatured proteinsambient mass spectrometry