Thursday, May 27, 2004 -- Poster 453

Soft-landing on active liquid chips: a new approach to protein purification and identification
Bogdan Gologan; Zoltan Takats; Justin M. Wiseman; Justin Oliver; V. Jo Davisson; R. Graham Cooks; Purdue University, West Lafayette, IN

Introduction
Ion soft-landing can be applied to separate and purify compounds from complex mixtures. In the case of proteins, the mixtures are ionized by ESI, followed by separation of the components according to their mass/charge ratios, and then landed on a surface. The deposited proteins can be made to form an array of spots on the "chip" surface. In this study, enzymatically active functionalized liquid surfaces are used for ion soft-landing, and MALDI is used for product analysis. The objective is to determine the feasibility of using vacuum compatible liquid substrates as a media for protein capture as well as for identification of the soft-landed species through enzyme assisted fragmentation and peptide recognition using automated search processes.

Methods
The instrument consisted of single-stage quadrupole SSQ c770 mass spectrometer (ThermoFinnigan, San Jose, CA) and was modified to allow positioning of surface on a moving stage between the quadrupole and the detector. Mixtures of chicken egg lysozyme and cytochrome c, among other proteins, were ionized by ESI and particular charge states from the protein envelope were mass-selected. By an appropriate choice of deposition energies, the ionic projectiles were soft-landed into various liquid surfaces containing active enzymes. Subsequent to soft-landing, buffer was added and the enzymatic digestion was initiated. The resulting digestion mixtures were analyzed by UV-MALDI-MS on a Bruker Reflex III Instrument and the identified peptides were compared by a peptide search algorithm (MS-FIT) to identify the originating protein.

Preliminary Results
Ion/surface interactions were explored through soft-landing of various mass selected projectile ions such as cytochrome c (M+15H)+15 into a range of functionalized liquid surfaces, which contained enzymatically active components, such as trypsin. The collision energy used for the landing process was varied between 5 and 20 eV and the deposition time varied between 1 and 2 hours. Following to the soft-landing process, the enzymatic digestions were initiated by addition of NH4HCO3 buffer to the liquid chips and by incubation at constant temperature. Upon MALDI analysis of the digestion medium, peptides corresponding to the soft-landed compound were identified and recorded. Through a computerized search using MS-FIT (Protein Prospector) the identities of the soft-landed proteins were determined. A detailed investigation of the spectral characteristics for various samples, established the feasibility of these liquid surfaces as MALDI targets for protein identification and analysis. Preliminary results revealed that in the case less stable enzymes, such as the catalytic subunit of protein kinase A, the use of liquid surfaces as landing substrates during the purification by ion soft-landing considerably increases the chances of recovering a biologically active purified protein. This work was supported by InProteo, Inc. (Indianapolis, IN).

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