THE USE OF BIOREACTIVE PROBES IN PROTEIN CHARACTERIZATION

Matrix-assisted laser desorption/ionization time-of-flight mass spectr ometry (MALDI-TOF) has in the past decade found routine use in the bio logical sciences. With this use has evolved several mass spectrometric -based methods directed at the intricate investigation of biomolecular structure and function. One such methodology involves the enzymatic m odification of a protein prior to the mass spectrometric readout of th e resulting products. The enzyme-modification/mass spectrometric appro ach has a definite use in a number of applications, including: the ver ification/identification of protein sequence, elucidation of post-tran slational modifications, the investigation of protein higher-order str ucture, and even the characterization of the modifying enzyme. To avoi d the potentials of sample loss and autolytic interferences in the mas s spectrum, mass spectrometer targets can be covalently derivatized wi th enzymes for use in the characterization procedures. The enzymatical ly active, or bioreactive, probes are used by application of the analy te to the activated surface, followed by application of a suitable MAL DI matrix and mass analysis from the surface of the probe. Limited tra nsfer and handling steps eliminate sample losses, and surface-tethered enzymes (and autolytic fragments) are prohibited from interfering wit h analytical signals in the mass spectra. In addition, the probes are rapid and easy to use. Reviewed here are Issues of concern during the manufacture and use of the bioreactive probes,and application of the p robes to investigate protein structure and function. (C) 1998 John Wil ey & Sons, Inc.