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Are Native Conformations of Proteins Retained Throughout Laser Ablation Capture?
Neda Feizi1, Blessing C Egbejiogu2, Kelcey B Hines2
1Department of Chemistry and Biochemistry, Baylor University, Waco, Texas76798, United States.
Analytical Chemistry
|August 11, 2026
Summary
Laser ablation with native ambient mass spectrometry (NAMS) can preserve protein structure. While bovine serum albumin refolded after ablation, carbonic anhydrase II aggregated, revealing protein-specific stability during this surface sampling method.
Area of Science:
- Analytical Chemistry
- Biophysical Chemistry
- Structural Biology
Background:
- Combining ion mobility-mass spectrometry (IM-MS) with laser ablation (LA) is a promising technique for analyzing protein higher-order structures in native ambient mass spectrometry (NAMS).
- Previous work showed proteins ablated from native deposits retain their higher-order structure.
- Uncertainty exists whether proteins denature during ablation and refold post-capture or remain native throughout the process.
Purpose of the Study:
- To investigate the stability of proteins during laser ablation (LA) and solvent capture in native ambient mass spectrometry (NAMS).
- To determine if proteins denature during LA and subsequently refold in solution.
- To assess the suitability of LA as a surface sampling method for protein structure analysis.
Main Methods:
- Utilized refolding properties of bovine carbonic anhydrase II (CA) and bovine serum albumin (BSA) to assess protein stability.
- Employed electrospray ionization mass spectrometry (ESI-MS) and collision-induced unfolding (CIU) to analyze refolded proteins.
- Applied matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) and bottom-up proteomics for denatured sample analysis.
- Compared charge-state distributions (CSDs) and CIU plots of intact and laser-ablated carbonic anhydrase II.
Main Results:
- Denatured bovine serum albumin (BSA) successfully refolded to its native structure upon capture in a native solvent, showing comparable ESI mass spectra and CIU curves to native deposits.
- Denatured carbonic anhydrase II (CA) did not refold in the native solvent, instead forming aggregates confirmed by MALDI-MS and proteomics.
- Laser-ablated CA exhibited comparable charge-state distributions to intact CA, with minor differences in collision-induced unfolding plots, suggesting structural integrity.
- Structural characterization of laser-ablated hemoglobin and protein-carbohydrate/ligand complexes further supported LA's potential.
Conclusions:
- Laser ablation (LA) combined with native ambient mass spectrometry (NAMS) shows potential for analyzing protein higher-order structures.
- Protein refolding post-ablation is protein-dependent, with BSA refolding but CA forming aggregates.
- LA is a viable surface sampling technique for investigating protein structures, including complexes, with varying protein stabilities.
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