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Gas-Phase Cationization and Protonation of Neutrals Generated by Matrix-Assisted Laser Desorption

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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/ioniza­ tion (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, Earlier studies have shown that cationized quasi­ matrix-assisted laser desorption/ionization molecular ions produced by laser desorption/ W (MALDI) represents one of the most impor­ ionization (LDI) without matrix are the result of tant developments in ionization technique for large ion-molecule reactions in the gas phase [10-12]. For molecules in recent years [1]. Proteins in excess of 200 the formation of protonated molecular ions by LDI, kDa can now be ionized [2], and other important Parker and Hercules [13] suggested that "pair produc­ classes of compounds, such as polynucleotides [3], tion" is the major mechanism, based on the result of oligosaccharides [4, 5], and synthetic polymers [6], the study of deuterated amino acids. have also been shown to be amenable to this tech­ Previous work from this laboratory has shown that nique. A detection limit of femtomoles has been under certain experimental conditions, LDI produces demonstrated when MALDI is coupled to a time-of­ more radical molecular ions than protonated ones [14]. flight (TOF) mass spectrometer [7, 8]. The utility of Tryptophan, for instance, usually gives abundant MALDI makes a thorough understanding of the mech­ [M + H]+ but little M+; however, when the sample anisms involved in the technique particularly desir­ was cooled to 90 K, the abundances of M + and [M + able. H] + were reversed. Furthermore, when ions were not A key feature of MALDI is the mixing of an analyte extracted promptly, multiple hydrogen attachment to with a small organic compound that absorbs reso­ the molecules was observed. Speir et al. [15] also nantly at the laser wavelength used for desorption. It showed recently that neutral moieties generated by has been observed that although MALDI generates LDI can react with ions trapped in an ion cyclotron predominantly cationized quasimolecular ions for some resonance cell in the gas phase. All of these observa­ peptides, it produces predominantly protonated quasi­ tions seem to suggest that in LDI, [M + H]+ is the molecular ions for other peptides and probably all product of gas-phase reactions between M + and de­ proteins [9]. Currently, it is still unclear how the ab­ sorbed molecules. sorption of photons by the matrix molecules leads to Consistent with the above observations, another re­ the desorption of large molecules. The role of the cent study in this laboratory showed that a host of matrix molecules in the cationization as well as the organic compounds, which can assist the desorption/ protonation process is also poorly understood. 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 prod­ "Dr, Wang's present address is Department of Chemistry, Mas­ uct of gas-phase reactions between matrix ions and sachusetts Institute of Technology, Cambridge, MA 02139. analyte molecules, with photoradical matrix ions initi­ Address reprint requests to Klaus Dreisewerd, Institute of Medical Physics and Biophysics, University of Muenster, Muenster, Germany. ating the reactions. © 1993 American Society for Mass Spectrometry Received Tune 10, 1992 1044-0305/93/$6.00 Revised January 5,1993 Accepted January 5, 1993 394 WANGETAL. J Am Soc Mass Spectrom 1993,4,393-398 To obtain more evidence that ion-molecule reac­ View pod tions 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 Vol'l:°9l! Experimental switCh 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 desorp­ tion. The laser was attenuated and focused to approxi­ Figure 1. Schematic of the experimental setup used in the 6 2 mately 50 ILm to give an irradiance of 3 X 10 Wjcm • study. The system uses two KrF excirner lasers and an ion Under prompt extraction conditions, the sample was reflector. The timing circuit for triggering the lasers and the held at a potential of 3 kV, and the counterelectrode of high-voltage switch are controlled by a master dock and a delay generator. the extraction optics, 5 mm in front of the sample probe, was held at ground potential. For delayed ex­ traction, this counterelectrode is initially floated at 3 kV and switched to ground within 80 ns (90-10% [sar'. val", ala8]-human angiotensin II (HATII) and value) after a variable delay time. In the cross-beam melittin, the concentrations were 1 X 10-4 M. For experiments, in addition to laser I, another KrF ex­ bovine insulin, the concentration was 3 X 10-5 M; this cimer laser (laser II, wavelength 248 run, pulse width solution contained 0.1% trifluoroacetic acid to enhance 20 ns) was used to irradiate a neat NaI sample to solubility. Typically, 1 ILL of the mixture solution was produce a plume of Na+ ions before laser I was fired. transferred to a silver substrate and dried in a stream The NaI sample was placed on a glass surface mounted of cool air. The diameter of the sample deposition on at a right angle to the main sample surface at a the substrate was approximately 2 mm. distance approximately 2 mm from the desorption For the experiment, which required a wide varia­ area. Laser II was attenuated and focused to a spot tion in the molar ratio between DHB and gramicidin S, approximately 200 p..m (horizontal) X400 m (vertical). a 5 X 10-2 M DHB solution was mixed with appropri­ The irradiance was adjusted to approximately 4 X 107 ate amounts of gramicidin S solution in the concentra­ 2 2-7 4 W jcm . This second laser beam propagated parallel to tion range 5 X 10- X 10- M; 0.5 p..L of these mix­ the main sample surface, with the center of the beam tures was transferred to the sample support. As a approximately 250 ILm in front of this surface. The result, the total amount of DHB applied varied only by focusing lens has a focal length of 20 em. The timing of a factor of less than 2 (18-33 nmol), and the full range the two lasers, as well as the delay of the extraction of molar ratios of DHB:gramicidin S varied from 0 (i.e., pulse, was controlled by a master clock and a delay pure compound, no matrix) to 7680. This precaution generator. The jitter in the timing for both laser emis­ was taken to avoid possible influence of the applied sions and the voltage SWitching was approximately 10 DHB amount on the crystallization behavior on solvent ns, Ions were detected in a TOP mass spectrometer evaporation. A 10% ethanol aqueous solution was used equipped with a homebuilt single-stage ion reflector. as solvent for both DHB and gramicidin S, except for The ion detector consists of a conversion dynode and a the two data points with pure gramicidim S and a secondary electron multiplier. The voltage at the con­ DHB to gramicidin S molar ratio of 5:1. Pure ethanol version dynode was -10 kV for detection of peptides was used in these cases to ensure complete solubility with molecular masses below 2000 Da and at -18 kV of gramicidin 5. for detection of peptides with molecular masses above For the cross-beam experiment, approximately 3 ILL 200 Da. The signal from the electron multiplier was of saturated NaI aqueous solution were applied to the amplified and recorded by a transient recorder (LeCroy glass surface and dried in a stream of warm air. 9400); the digitized signal was then transferred to a All peptide samples were purchased from Sigma Personal Computer for summation, storage, and Co. (Deisenhofen, Germany). DHB was purchased from display. Aldrich Chemical Co. (Steinheim, Germany). The com­ With the exception described below, samples were pounds were used as purchased without further pu­ prepared by premixing 2 ILL of a peptide solution of rification. All spectra are summed results of 20 single­ appropriate concentration with 2 ILL of 5 X 10-2 M shot spectra with the exception of HATIl, where the 2,5-dihydroxybenzoic acid (DHB) aqueous solution to summation was performed over 10 single-shot spectra. give a mixture of the desired molar ratio of the peptide All intensities of ion signals were computed from the to DHB. Both solutions contained 10% ethanol. For respective peak areas. J Am Soc Mass Spectrom 1993, 4, 393-398 GAS-PHASE CATIONIZATION / PROTONATION IN MALDI 395 Results and Discussion served.
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