Are Native Conformations of Proteins Retained Throughout Laser Ablation Capture?

Abstract

Combining ion mobility-mass spectrometry (IM-MS) with laser ablation (LA) is an attractive approach for characterizing protein higher-order structure in native ambient MS (NAMS). Recently, we demonstrated that proteins ablated from native deposits are detected in their native higher-order structure. However, it remains unclear whether the proteins are ablated and captured in their native form or denatured by ablation and refolded after capture in the solvent. We used refolding properties of bovine carbonic anhydrase II (CA) and bovine serum albumin (BSA) to investigate the stabilities of these proteins during the LA and solvent capture. The denatured BSA refolded to a native structure when captured in a native solvent, yielding ESI mass spectra and collision-induced unfolding (CIU) curves comparable to those from native protein deposits. In contrast, denatured CA sample deposits dissolved in native solvent did not produce ESI mass spectra. However, matrix-assisted laser desorption ionization (MALDI) MS and bottom-up proteomics analysis of the same denatured deposit extracted samples confirmed the presence of CA, suggesting aggregate formation rather than refolding. The inability of denatured CA to refold upon redissolving in the ammonium acetate solvent was leveraged to investigate protein stability in LA NAMS. Observed charge-state distributions (CSDs) from native MS of intact and surface-deposited laser-ablated CA were comparable. However, minor variations in their CIU plots were observed. These findings, along with structural characterization of laser-ablated bovine hemoglobin, as well as weakly associated concanavalin A-mannose carbohydrate and CA-sulfanilamide complexes highlight the potential of LA as a promising surface sampling method for investigating protein structures.