Tuesday, May 25, 2004 -- 4:40 pm -- Oral Presentation

Solution vs gas phase protein structure as studied by sonic spray and ion soft landing
Zoltan Takats; Justin M. Wiseman; R. Graham Cooks; Purdue University, West Lafayette, IN

Introduction
Preparative mass spectrometry of proteins by ion soft-landing represents a new separation technology. One of the greatest challenges to this type of application is to retain the solution phase structure and biological activity of protein molecules throughout the entire process. A complete solution for this problem involves development of novel types of ion sources and atmospheric interface as well as special surfaces onto which to perform the ion landing. The retention of bioactivity also restricts various instrument parameters including the voltage profile along the ion path, vacuum conditions, and the overall time ions spend in the gas phase.

Methods
A modified LTQ instrument (Thermo Finnigan, San Jose, CA) was used for the soft-landing of mass-selected multiply charged ions of various proteins. The instrument was equipped with a home built electro-sonic spray ion source and an axially positioned moving stage for holding the soft-landing substrate. In some of the experiments, the commercial heated capillary inlet was replaced by a home built multi-capillary interface. Enzymes including hexokinase, protein kinase A, lysozyme, IGPS, trypsin, and chymotrypsin were used as model systems. The proteins were dissolved in aqueous ammonium-acetate buffer and ionized using micro-electrospray and electro-sonic spray ionization. Appropriate protein ions were accumulated and mass selected in the linear trap and ejected axially to land gently on the surface.

Preliminary Results
Three steps in the preparative mass spectrometry process were identified as danger points for loss of protein biological activity. The first is gas-phase ion formation; the second is ion transfer from atmospheric pressure to high vacuum, while the third is soft-landing itself. Electro-sonic spray ionization (ESSI) was developed as a variant on electrospray utilizing a micro-electrospray emitter with a supersonic nebulizing gas. This ionization technique is able to ionize proteins dissolved in aqueous buffers and yields considerably higher ion currents than nanospray. Preliminary data shows, that ESSI normally generates a single charge state of the ionized protein, and a single chemical species is responsible for more than 50% of the observed ion current. This single species can be either a completely desolvated protein ion or one with a specific adduct. The observed single charge state indicates that the protein is folded when the ionization takes place. Another important feature of ESSI is the lack of dependence of spectral characteristics on atmospheric interface settings. In this way, mild conditions can be implemented when ions are transferred to the high vacuum. Unfolding processes were investigated either by determining the activity of soft-landed proteins or performing gas-phase H/D exchange on protein ions. Both studies clearly showed that by using ESSI the unfolding can be kept in the reversible range. Two major factors were identified as being responsible for unfolding occurring upon landing. One is the landing energy of the ions, which was optimized for several systems in the 1-5 eV/charge range. The other factor is the type of surface on which the landing takes place. Preliminary investigations revealed that the immediate re-solvation of proteins is essential for preserving the activity. This can be carried out by providing a low vapor pressure, H-bonded liquid (e.g. glycerol) as a landing medium.

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