We are using matrix-assisted laser desorption ionization (MALDI) of peptides as a model system to study peptide ion fragmentation. Ion energetics relationships between laser fluence and peptide ion fragmentation. This type of study is fundamental to optimizing MALDI TOF/TOF experiments for the purpose of peptide sequencing. In these studies we obtain peptide fragmentation spectra, typically 5000 laser shots, in both the unimolecular decomposition and collision induced dissociation (CID) modes. We have the ability to easily follow two time points for each peptide decomposition, i.e., the in-source fragmentation consisting of ions formed within 1 usec after the laser firing and the longer, mass dependent fragmentation occurring within the instruments collision cell. To date we have used the fragmentation of a model peptide, leucine enkephalin, YGGFL, (LeuEnk) over the full range of laser fluence. While not a peptide of the type normally encountered in protein characterizations, LeuEnk is an excellent model to enable studies of short lived processes in the laser plume and been studied extensively by other mass spectrometric approaches. Spectra are acquired as a function of laser fluence beginning at the onset of ionization and extending to the maximum fluence available in the instrument. We have shown earlier that the MS mode spectra show a region of extensive fragmentation occurring in what must be a very short time frame following the onset of ionization. These rapid fragmentations, leading to the observation only of immonium ions, are associated with low laser plume densities. A second set of process takes place within the first several hundred nanoseconds following the laser pulse and occur in the realm of much higher laser plume density. These ions formed in these processes undergo a large number of collisions with the high temperature gases present in the laser plume, and begin to fragment; these fragmentations proceed in a series of consecutive reactions in which the amide backbone bonds are ruptured. Finally, the MS-MS mode spectra of LeuEnk exhibit little fragmentation.? ? Our initial studies were conducted using a laser pulse length of 5 nsec duration. as summarized above, these studies showed that the extent of fragmentation varied with laser fluence. That is, the total extent of fragmentation increased as fluence increased and the fragments appeared in an order consistent with the sequence of amino acids in the peptide. The sequential fragmentation occurred from both the amino and carboxy terminals of the peptide. In more recent studies, we have investigated these same phenomena using a laser pulse length of 0.6 nsec duration, and have determined the fragmentation in each of three different common MALDI matrices: alpha cyano-4-hydroxy cinamic acid (ACHA), 3,5-dimethoxy-4-hydroxy cinamic acid (Sinapinic acid, SA) and 2,5-dihydroxy benzoic acid (DHB). In both of the latter matrix materials, very little fragmentation was observed, as might be expected from matrices that are widely considered to be cooler than ACHA, but the DHB consistently showed more extensive fragments than the SA. What was appreciably more surprising however is that the onset of extensive fragmentation in ACHA occurred at laser fluences about 50% lower than than observed using the 5 nsec pulse length laser. These observations imply that the concentration of energy on the solid surface undergoing MALDI, in terms of photons/unit area/unit time, has a dominant influence on the extent the fragmentation that is observed in spectra.
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