The preferred ligands for the Hck Src homology 2 domain among a combinatorial library containing 324 different peptides were determined in a single experiment involving Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry (MS), electrospray ionization (ESI), stored-waveform inverse Fourier transformation (SWIFT), and infrared multiphoton laser disassociation (IRMPD). These were compared with the results obtained by conventional screening of the peptide library in solution using affinity chromatography. The results reported here show that by combining ESI, FT-ICR MS, SWIFT, and IRMPD, ligands likely to bind under physiological conditions are rapidly and efficiently identified, even from complex library mixtures. In the gas phase some discrimination against hydrophobic ligands could be observed. However, the illustrated feasibility of identifying high affinity ligand via gas-phase screening of complex library mixtures should lead to broad applications in the development of ligands for proteins with interesting biological activity, the first step that must be taken to develop a therapeutic agent. Important in the application of FT-ICR methods to biological macromolecules are "soft" ionization techniques, which generate macromolecular ions without fragmentation. Electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI) are two of these [4,5]. ESI involves spraying an aqueous solution of a biological macromolecule under "quasiphysiological" conditions directly into a mass spectrometer. The solvent evaporates in the vacuum from the droplets, gently "elevating" a biological ion from its native environment in solution. This process frequently leaves non-covalent macromolecular complexes intact and allows the mass spectrometric observation of enzyme-substrate, receptor-ligand, peptide-peptide, protein-subunit, oligonucleotide-toxin/drug, and DNA duplex binding in the gas phase [6].In many cases, binding constants estimated from mass spectrometric studies match the binding constants obtained in solution to within an order of magnitude [7]. The gentleness of ESI allows H/D exchange experiments in the gas phase to give information about protein conformation and protein/protein interactions based on measurements of the exchange rates of protein backbone amide protons [8,9]. This combination of

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