Top-down mass spectrometry of supercharged native protein–ligand complexes

Tandem mass spectrometry (MS/MS) of intact, noncovalently bound protein–ligand complexes can yield structural information on the site of ligand binding. Fourier transform ion cyclotron resonance (FT-ICR) top-down MS of the 29 kDa carbonic anhydrase-zinc complex and adenylate kinase bound to adenosine triphosphate (ATP) with collisionally activated dissociation (CAD) and/or electron capture dissociation (ECD) generates product ions that retain the ligand and their identities are consistent with the solution phase structure. Increasing gas phase protein charging from electrospray ionization (ESI) by the addition of supercharging reagents, such as m-nitrobenzyl alcohol and sulfolane, to the protein analyte solution improves the capability of MS/MS to generate holo-product ions. Top-down proteomics for protein sequencing can be enhanced by increasing analyte charging.

Increasing ESI-charging of protein complexes improves the ability to measure ligand-bound product ions and to determine ligand-binding sites by tandem mass spectrometry.Figure optionsDownload high-quality image (142 K)Download as PowerPoint slide