The propensities of a series of peptide ions produced by both electrospray and atmospheric pressure matrix assisted laser desorption ionization (AP-MALDI) to fragment in an ion trap mass spectrometer under various conditions were studied in detail by measuring the extent of fragmentation of precursor ions by collision induced dissociation (CID) as a function of applied resonance excitation RF voltage. For the most basic peptides, the energy required to fragment MH ϩ ions generated by electrospray exceeded that required to fragment equivalent AP-MALDI ions under identical instrumental conditions; the reverse was observed for a peptide incorporating no basic residues, while peptides of intermediate basicity showed little difference between the ionization methods. This correlation between peptide basicity and the difference in the energy required to induce fragmentation of MH ϩ ions generated by AP-MALDI and electrospray is attributed primarily to a trend in the internal energies of the ions generated by AP-MALDI (the greater the difference in gas-phase basicities between the matrix and the analyte the greater the internal energy of the analyte ions produced). Furthermore the internal energies of ions produced by AP-MALDI, but not the equivalent ions formed by electrospray, were observed to decrease with decreasing analyte concentration. We attribute this finding to the cooling effect of endothermic dissociation of analyte ion/matrix molecule clusters following the matrix assisted laser desorption step. Time-resolved analyses (measurement of extent of fragmentation of precursor ions by CID as a function of pre-CID "cool times") revealed that cooling periods in excess of 250 ms were required to achieve internal energy equilibrium through cooling collisions with the helium buffer gas. Furthermore, these analyses demonstrated that, even after these extended cooling times, equivalent ions formed by the two ionization techniques showed different propensities to fragment. We conclude that the two different ionization techniques produce ion populations that may differ in their three-dimensional structure. T he initial internal energy of an ion is determined by the internal energy of the analyte molecule before ionization and the energy deposited during ionization. The additional internal energy that must be acquired during ion activation in order for dissociation to occur is defined by the initial internal energy of the ion. Thus, for a fixed activation energy the extent of ion decomposition is a measure of the initial internal energy of that ion. Therefore, in order to compare the initial internal energies of peptide ions produced by electrospray and AP-MALDI, a series of energyresolved tandem mass spectrometry experiments were performed, in which the extent of decomposition of precursor ions by CID was measured as a function of applied resonance excitation RF voltage.Danell and Glish [1] and Wysocki and coworkers [2] investigated the differences between the internal ener-

In an initial investigation into the electrospray ionisation ion trap mass spectrometry (ESI/ITMS) of simple organophosphate esters we reported that in one of the collision induced fragmentation steps of protonated dimethyl methylphosphonate (DMMP), formaldehyde was eliminated with a concomitant partial scrambling of the methyl group attached to the phosphorus and a methoxy group. The present paper describes a further investigation of this reaction. Three novel isotopomers of DMMP were used for this study and their synthesis and properties are reported. A mechanism for the formaldehyde elimination and scrambling of the methyl groups is proposed and supported by a kinetic analysis of a limiting case. During this study ' black holes ' were found to occur in one of the ITMS instruments used and it is shown that these could lead to severe distortions in the amounts of product ions observed. A brief analysis of this observation is presented.

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