MATRIX-ASSISTED LASER DESORPTION-IONIZATION TIME-OF-FLIGHT MASS-SPECTROMETRY IN THE SUBUNIT STOICHIOMETRY STUDY OF HIGH-MASS NONCOVALENT COMPLEXES

This study explores the potential of MALDI-TOF MS for the mass measure ment of large non-covalent protein complexes. The following non-covale nt complexes have been investigated: aerolysin from Aeromonas hydrophi la (335 kDa) and cr-haemolysin from Staphylococcus aureus (233 kDa) wh ich are both cytolytic toxins, three enzymes known to be homotetramers in solution: bovine liver catalase (235 kDa), rabbit muscle pyruvate kinase (232 kDa), yeast alcohol dehydrogenase (147 kDa) and finally a lectin, concanavalin A (102 kDa). Three different matrix preparations were systematically tested under various conditions: ferulic acid diss olved in THF, 2,6-dihydroxyacetophenone in 20 mM aqueous ammonium citr ate and a two-step sample preparation with sinapinic acid. It was poss ible to find a suitable combination of matrix and preparation type whi ch allowed the molecularity of all complexes tested to be deduced from the MALDI mass spectrum. Trimeric and tetrameric intermediates accumu lating during the formation of the active heptameric aerolysin complex were also identified, this allowing a formation mechanism to be propo sed, The observation of large specific non-covalent complexes has been found to be dependent on the choice of matrix, the type of sample pre paration used, the solvent evaporation speed, the pH of the resulting matrix-sample mixture and the number of shots acquired on a given area . From this set of experiments, some useful guidelines for the observa tion of large complexes by MALDI could therefore be deduced. Fast evap oration of the solvent is particularly necessary in the case of pH sen sitive complexes. An ESMS study on the same non-covalent complexes ind icated that, rather surprisingly, reliable results could be obtained b y MALDI-TOF MS on several very large complexes (above 200 kDa) for whi ch ESMS yielded no clear spectra. (C) 1997 Elsevier Science B.V.