Issue 8, 2024

Overcoming aggregation with laser heated nanoelectrospray mass spectrometry: thermal stability and pathways for loss of bicarbonate from carbonic anhydrase II

Abstract

Variable temperature electrospray mass spectrometry is useful for multiplexed measurements of the thermal stabilities of biomolecules, but the ionization process can be disrupted by aggregation-prone proteins/complexes that have irreversible unfolding transitions. Resistively heating solutions containing a mixture of bovine carbonic anhydrase II (BCAII), a CO2 fixing enzyme involved in many biochemical pathways, and cytochrome c leads to complete loss of carbonic anhydrase signal and a significant reduction in cytochrome c signal above ∼72 °C due to aggregation. In contrast, when the tips of borosilicate glass nanoelectrospray emitters are heated with a laser, complete thermal denaturation curves for both proteins are obtained in <1 minute. The simultaneous measurements of the melting temperature of BCAII and BCAII bound to bicarbonate reveal that the bicarbonate stabilizes the folded form of this protein by ∼6.4 °C. Moreover, the temperature dependences of different bicarbonate loss pathways are obtained. Although protein analytes are directly heated by the laser for only 140 ms, heat conduction further up the emitter leads to a total analyte heating time of ∼41 s. Pulsed laser heating experiments could reduce this time to ∼0.5 s for protein aggregation that occurs on a faster time scale. Laser heating provides a powerful method for studying the detailed mechanisms of cofactor/ligand loss with increasing temperature and promises a new tool for studying the effect of ligands, drugs, growth conditions, buffer additives, or other treatments on the stabilities of aggregation-prone biomolecules.

Graphical abstract: Overcoming aggregation with laser heated nanoelectrospray mass spectrometry: thermal stability and pathways for loss of bicarbonate from carbonic anhydrase II

Supplementary files

Article information

Article type
Paper
Submitted
09 févr. 2024
Accepted
12 mars 2024
First published
13 mars 2024

Analyst, 2024,149, 2281-2290

Overcoming aggregation with laser heated nanoelectrospray mass spectrometry: thermal stability and pathways for loss of bicarbonate from carbonic anhydrase II

J. S. Jordan, K. J. Lee and E. R. Williams, Analyst, 2024, 149, 2281 DOI: 10.1039/D4AN00229F

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