Direct Observation of Biomolecular Complexes by Cold-Spray Ionization Time-of-Flight Mass Spectrometry

Many biological processes are regulated by the specific and noncovalent intermolecular recognition between enzyme–substrate, antigen–antibody, receptor–ligand, carbohydrate–protein, and carbohydrate–carbohydrate. The direct observation of these biomolecular interactions is important for understanding the mechanisms behind these biological processes. Electrospray ionization mass spectrometry (ESI MS) is an important tool for the investigation of noncovalent complex formation [16]. The advantages of MS over traditional methods such as UV/Vis spectroscopy, fluorescence spectroscopy, surface plasmon resonance (SPR), isothermal titration microcalorimetry (ITC), and nuclear magnetic resonance (NMR) spectroscopy include the accuracy of mass measurement, speed of analysis and small sample quantities. However, the harsh conditions of the ionization process in MS are often detrimental to the survival of noncovalent and unstable biomolecular interactions between sugar-protein, sugar-sugar and sugarwater complexes. Recently, Yamaguchi and co-workers developed cold-spray ionization mass spectrometry (CSI MS) [7, 8] which does not require heat or an electric field and provides a snapshot of the state of compounds in solution. It is suggested that a cooled ion spray promotes stable solvation-ionization processes through increased compound polarizability by higher dielectric constants at low temperature. Using the CSI system, facile and precise characterization of labile self-assembling nanostructures and unstable organometalic complexes in solution was achieved [9, 10]. CSI MS may become one of the most promising and versatile tools for characterization of a variety of weak but specific biomolecular interactions, as ionization at a low temperature (–20°C) allows direct observation of unstable largescale aggregates of amino acids or nucleosides in organic solvents with a magnetic-sectorequipped instrument [11-13]. Moreover, CSI MS combined with an orthogonal acceleration time-of-flight (oa-TOF) mass analyzer extends the applicability of this method to the characterization of the dynamic interactions of biomacromolecules. This is possible through a number of attractive features of TOF MS analyzers, such as their theoretically unlimited mass range, very high spectrum acquisition rates, high ion transmission, high sensitivity, multiplex detection capacity, reasonable mass resolution and simplicity in instrument design. Our interest is focused on the potential of CSI-TOF MS, with particular attention to specific and weak biomolecular interactions in water at a low temperature. We have previously tried to monitor directly the formation of noncovalent protein-carbohydrate, carbohydrate-carbohydrate and carbohydrate-water complexes in an aqueous solution [14].