Stabilizing protein-ligand complexes in ESI-MS using solution additives: Comparing the effects of amino acids and imidazole

- Results of comparative study of the stabilizing effects of amino acids and imidazole on ESI-MS protein-ligand affinity measurements reported.
- Imidazole provided some stabilization at low source cone voltages.
- Amino acids tested provided little or no stabilization of the protein-ligand complexes.
- The stabilizing effects of solution additives are highly dependent on the ESI source configuration and instrumental parameters used.

The results of a comparative study of the stabilizing effects of amino acids and imidazole on electrospray ionization mass spectrometry (ESI-MS) measurements of protein-ligand complexes are described. Affinity measurements were carried out, using a range of source cone voltages, for two protein-ligand complexes, trypsin (Tr) with benzamidine (L1) and lysozyme (Lyz) with β-d-GlcNAc-(1 → 4)-β-d-GlcNAc-(1 → 4)-d-GlcNAc (L2), in the presence of individual amino acids (Gly, Ser, Pro or His) or imidazole at mM concentrations. Consistent with earlier reports, imidazole was found to protect the (Tr + L1) complex against in-source dissociation, although only partial stabilization was observed at higher cone voltages. No stabilization by imidazole was observed at cone voltages that promoted in-source dissociation of the (Lyz + L2) complex. It is proposed that, under these conditions, the influence of imidazole on the internal energy of the (Lyz + L2) ions is negligible compared to that due to collisional activation. The addition of amino acids to solution led to extensive adduct formation, which complicated data analysis. However, and in contrast to the results of a recently reported study, the amino acids tested provided little or no stabilization of the two protein-ligand complexes. While the reasons for this discrepancy are not known, this finding suggest that the stabilizing effects of amino acids and, presumably, other stabilizing additives, is highly dependent on the ESI source configuration and instrumental parameters used.

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