Gas-Phase cationization and protonation of neutrals generated by matrix-assisted laser desorption

Gas-Phase Cationization and Protonation of Neutrals Generated by Matrix-Assisted Laser Desorption Bing H. Wang,* Klaus Dreisewerd, Ute Bam, Michael Karas, and Franz Hillenkamp Institute of Medical Physics and Biophysics, University of Muenster, Muenster, Germany The ionization mechanisms involved in matrix-assisted ultraviolet laser desorption/ionization (MALDI) were studied with a time-of-flight mass spectrometer. When protonated or cationized quasimolecular ions generated by MALDI are not extracted promptly, their abundance is a function of the delay time between laser irradiation and ion extraction, maximizing at an optimum delay time (DTM) of a few hundred nanoseconds. The ion abundance at DTM exceeds that of prompt extraction by a factor of 2 or more. Increasing the cation density near the sample surface reduces the DTM, whereas increasing the desorption laser irradiance has the opposite effect. The enhancement suggests extensive gas-phase ion-molecule reactions after irradiation by the desorption laser has ceased. (J Am Soc Mass Spectrom 1993, 4, 393-398) ith its versatility and high sensitivity, matrix-assisted laser desorption/ionization (MALDI) represents one of the most important developments in ionization technique for large molecules in recent years [1]. Proteins in excess of 200 kDa can now be ionized [2], and other important classes of compounds, such as polynucleotides [3], oligosaccharides [4, 5], and synthetic polymers [6], have also been shown to be amenable to this technique. A detection limit of femtomoles has been demonstrated when MALDI is coupled to a time-offlight (TOF) mass spectrometer [7, 8]. The utility of MALDI makes a thorough understanding of the mechanisms involved in the technique particularly desirable. A key feature of MALDI is the mixing of an analyte with a small organic compound that absorbs resonantly at the laser wavelength used for desorption. It has been observed that although MALDI generates predominantly cationized quasimolecular ions for some peptides, it produces predominantly protonated quasimolecular ions for other peptides and probably all proteins [9]. Currently, it is still unclear how the absorption of photons by the matrix molecules leads to the desorption of large molecules. The role of the matrix molecules in the cationization as well as the protonation process is also poorly understood. W "Dr, Wang's present address is Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA 02139. Address reprint requests to Klaus Dreisewerd, Institute of Medical Physics and Biophysics, University of Muenster, Muenster, Germany. © 1993 American Society for Mass Spectrometry 1044-0305/93/$6.00 Earlier studies have shown that cationized quasimolecular ions produced by laser desorption/ ionization (LDI) without matrix are the result of ion-molecule reactions in the gas phase [10-12]. For the formation of protonated molecular ions by LDI, Parker and Hercules [13] suggested that "pair production" is the major mechanism, based on the result of the study of deuterated amino acids. Previous work from this laboratory has shown that under certain experimental conditions, LDI produces more radical molecular ions than protonated ones [14]. Tryptophan, for instance, usually gives abundant [M + H]+ but little M+; however, when the sample was cooled to 90 K, the abundances of M + and [M + H] + were reversed. Furthermore, when ions were not extracted promptly, multiple hydrogen attachment to the molecules was observed. Speir et al. [15] also showed recently that neutral moieties generated by LDI can react with ions trapped in an ion cyclotron resonance cell in the gas phase. All of these observations seem to suggest that in LDI, [M + H]+ is the product of gas-phase reactions between M + and desorbed molecules. Consistent with the above observations, another recent study in this laboratory showed that a host of organic compounds, which can assist the desorption/ ionization of proteins, form odd-electron molecular ions [16]. The study consequently suggests that in MALDI, the [M + H] + ion of the analyte is the product of gas-phase reactions between matrix ions and analyte molecules, with photoradical matrix ions initiating the reactions. Received Tune 10, 1992 Revised January 5,1993 Accepted January 5, 1993 394 J Am Soc Mass Spectrom 1993,4,393-398 WANGETAL. View pod To obtain more evidence that ion-molecule reactions in the gas phase may account for the ionization of analyte molecules in MALDI, the effect of delaying the extraction of ions was studied. Delaying the extraction presumably gives more time for bimolecular reactions or other processes to take place in the ion source [17]. High Experimental Figure 1 shows the experimental setup used in this study. A KrF laser (laser I) with a wavelength of 248 run and a pulse width of 15 ns was used for desorption. The laser was attenuated and focused to approximately 50 ILm to give an irradiance of 3 X 10 6 W jcm 2 • Under prompt extraction conditions, the sample was held at a potential of 3 kV, and the counterelectrode of the extraction optics, 5 mm in front of the sample probe, was held at ground potential. For delayed extraction, this counterelectrode is initially floated at 3 kV and switched to ground within 80 ns (90-10% value) after a variable delay time. In the cross-beam experiments, in addition to laser I, another KrF excimer laser (laser II, wavelength 248 run, pulse width 20 ns) was used to irradiate a neat NaI sample to produce a plume of Na + ions before laser I was fired. The NaI sample was placed on a glass surface mounted at a right angle to the main sample surface at a distance approximately 2 mm from the desorption area. Laser II was attenuated and focused to a spot approximately 200 p..m (horizontal) X400 m (vertical). The irradiance was adjusted to approximately 4 X 10 7 W jcm 2 . This second laser beam propagated parallel to the main sample surface, with the center of the beam approximately 250 ILm in front of this surface. The focusing lens has a focal length of 20 em. The timing of the two lasers, as well as the delay of the extraction pulse, was controlled by a master clock and a delay generator. The jitter in the timing for both laser emissions and the voltage SWitching was approximately 10 ns, Ions were detected in a TOP mass spectrometer equipped with a homebuilt single-stage ion reflector. The ion detector consists of a conversion dynode and a secondary electron multiplier. The voltage at the conversion dynode was -10 kV for detection of peptides with molecular masses below 2000 Da and at -18 kV for detection of peptides with molecular masses above 200 Da. The signal from the electron multiplier was amplified and recorded by a transient recorder (LeCroy 9400); the digitized signal was then transferred to a Personal Computer for summation, storage, and display. With the exception described below, samples were prepared by premixing 2 ILL of a (...truncated)