MALDI TOF mass spectrometry of noncovalent biocomplexes

Research project P 14181 MALDI TOF mass spectrometry of noncovalent biocomplexes Günter ALLMAIER 24.01.2000 During the last two decades the developments of so-called soft desorption/ionization techniques have revolutionized the mass spectrometric characterization of high-mass biomolecules, especially of proteins and carbohydrates. The generation of intact, charged molecules as biopolymers in the gas phase was a big achievement. Techniques based on highenergy fission fragments as Californium-252 (Plasmadesorption, PD) or fast, neutral gas atoms (Fast Atom Bombardment, FAB) as primary beam or on an electrospray process (Electrosprayionization, ESI) to desorb and ionize biomolecules exhibit still limits in the molecular mass, -which can be determined. Matrix-assisted laser desorption/ionization (MALDI) - applying photons as primary beam - combined with a time- of-flight mass spectrometer allowed for the first time to analyze macromolecules above a molecular mass of I million Dalton. The remarkable capabilities of MALDI and ESI mass spectrometry (MS) were used with considerable success to determine the exact molecular mass and the primary structure of biopolymers as well as to identify vanishingly small quantities of peptides from complex matrices. The possibility of direct characterization of high-mass non-covalent biocomplexes by means of MALDI MS was recognized but received much less attention than ESI MS. The ability to probe non-covalent interactions and complexes in the high mass range by MALDI time-of-flight MS opens up new avenues in analytical biochemistry (e.g. studies of the non-covalent interaction of HIV proteins with inhibitors or of multi heteroprotein complexes) and supramolecular chemistry. In contrast to other mass spectrometric desorption/ionization techniques UV MALDI desorbs the intact analyte mainly from the solid phase. Therefore sample preparation, that means the incorporation of intact, mostly labile, non-covalent complexes into a solid matrix lattice, is a difficult task. Nevertheless this procedure is feasible. The investigation of the important parameters during this sample preparation step and the subsequent optimization as well as standardization will allow new areas of applications (e.g. heteroprotein complexes) and to tackle new problems related to supramolecular complexes. Liquid matrix systems with UV MALDI for non-covalent complexes will be evaluated and can reduce or avoid some obstacles related to solid matrices. The influence of ion source related parameters on the desorption/ionization behavior of noncovalent biocomplexes will be studied, too. The importance of data as the exact molecular mass of non-covalent, mostly very large, homo- or heteroprotein complexes, obtained by MALDI MS lies in the determination of the complex stoichiometry and in the theoretical unlimited mass range of a linear time-of-flight analyzer with a very low sample consumption (in the picomole - 10-1 2 - range and below). Systematic investigations, and optimizations of the MALDI sample preparation process as well as new sample preparation techniques and ion source designs for the analysis of noncovalent biocomplexes will generate a quantum leap in the MALDI MS application to biochemical and biotechnological important, high- mass non-covalent complexes. The detailed characterization of supramolecular structures and molecular recognition processes as antigenantibody complexes, enzyme-substrate complexes or receptor-ligand interaction, beyond a molecular mass of 200000 Dalton will be possible by MALDI time-of-flight MS.