Monday, May 24, 2004 -- Poster 113

Enzyme-Substrate Characterization by ESSI-MS
Justin M Wiseman; Zoltan Takats; Bogdan Gologan; R. G. Cooks; Purdue University, West Lafayette, IN

Introduction
Both electrospray and MALDI can be used to ionize intact macromolecular complexes. However, under traditional electrospray conditions, i.e. acidified organic solvent, 4-6 kV, protein tertiary and quarternary structures are sometimes denatured leaving it less likely that a protein-substrate interaction will remain intact. As such, there is a need for even "softer" ionization techniques that provide efficient desolvation but reduce the energy load to the ions. Electro-sonic spray ionization (ESSI) couples the advantages of electrospray and sonic spray and can be used to ionize and transfer intact large bio-molecules and their specific complexes into the gas-phase for mass analysis. Utilizing a high velocity nebulizing gas, the technique displays improved performance in resolution and ionization efficiency owing to increased desolvation efficiency.

Methods
All experiments were conducted using a Finnigan LCQ Classic quadrupole ion trap mass spectrometer. The electro-sonic spray device was home-built using a 1/16? Swagelok tee and fused silica capillaries. (inner: 0.05mm i.d.; outer: 0.200mm o.d.) High pressure N2 gas was delivered to the Swagelok tee via ¼ ? PTFE tubing at 120 psi. The enzymes used in these studies were buffer exchanged using diafiltration (centrifuged at 9000 x g) against 0.2M ammonium acetate at pH 7.1. Following five centrifuge cycles, the samples were re-suspended in 10mM ammonium acetate at pH 7.1.

Preliminary Results
Utilizing ESSI we have demonstrated the ability to ionize and transfer the intact phosphorylated cAMP-dependent protein kinase A (catalytic subunit) - adenosine 5' triphosphate (ATP) -magnesium complex. In addition, the yeast hexokinase-glucose complex has been shown to survive ionization and transfer into the mass spectrometer using ESSI. The ability to retain protein substrate complexes of a specific nature in the gas phase unveils a variety of applications involving ESSI-MS. Already demonstrated is the identification of phosphorylating enzymes in solution from a complex mixture of proteins by ESSI-MS. Since these complexes are preserved using electro-sonic spray, specific adduct formation by these enzymes with ATP allows for rapid identification. This technique shows potential for the development of an array of rapid screening methods exploiting the formation of weakly-bound adducts for the identification of specific bio-molecules present in a complex mixture based on their affinities for certain substrates. Also demonstrated are the enzyme- inhibitor interactions of a glutamine amidotransferase, imidazole 3-glycerol phosphate synthase (IGPS), which is competitively inhibited by r-PRFAR (N-1-[(5'-phosphoribulosyl)-formimino]-5-aminoimidazole-4-carboxamide ribonucleotide) and acivicin. The presence of IGPS bound to r-PRFAR and to both r-PRFAR and acivicin were detected in the mass spectrum. These results show preferential binding of the inhibitor rPRFAR over acivicin.

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