Anal. Chem. 1995,67, 1963-1967
Matrix-Assisted Laser Desorption Ionization of Neutral Clusters Composed of Matrix and Analyte Molecules Andrew N. Knitchinsky, Anatoli 1. Dolguine, and Mikhail A. Khodorkovski* LEPTA-Petersbug Ltd., 197198 St. Petersbug, Russia
A new version of the matrix-assisted laser desorption ionization experiment is described. Composed clusters created by laser desorption of a mixture of analyte and matrix material were injected into a supersonic jet and then analyzed by multiphoton ionization time-of-flight mass spectrometry. Selective two-photon ionization of matrix molecules of a cluster component caused their dissociation, followed by intercluster proton or cation that is transparent for the wavelength transfer to an of ionizing radiation. ‘ b e intercluster charge transfer reactions are the only pathway for analyte ion formation under these conditions, as can be concludedfrom analysis of the mass spectra of a variety of substances.
empirical or gas dynamic approaches, the requirements for a matrix material and the rules of sample preparation have been determined.16J7 Among numerous experimentalworks, some have been carried out in a way different from that which became a standard method for MALDI experiments. In these experiments, a “dynamic” rather than a “static”target of the sample layer was laser irradiated to produce the quasimolecular ions of the analytes. Murray and Russells irradiated a beam of particles of dried eluent from the output of a chromatograph. Apparently because of the aerosol character of the target, they called this approach “aerosolWI”. The addition to an eluent of nitroaniline as a matrix strongly absorbing at the wavelength of the ionizing laser caused the protonation of analyte molecules. Thus, it was concluded that the proton transfer from nitroaniline molecules to analyte molWith the introduction of pulsed direct laser desorption O), ecules was the key mechanism of ion formation. In other work, mass spectrometry got a powerful tool for analysis of labile organic Langridge-Smith and cc-~orkers’~ performed two-photon ionizaIt has been shown45 that LD allows transformation tion (2PD of a number of dyes desorbed by laser shots into a of condensed phase molecules into gas phase ions or neutrals. supersonic jet. They have shown that, together with radical However, the widespread application of LD appears to be limited cations, protonated or cationated ions of analytes were formed to the analysis of intact molecules with masses less then a few under the specific conditions of the experiment. However, the thousand Daltons. Either resonant or nonresonant excitation of authors have not proposed any clear explanation of quasimolecular analyte molecules by desorbing laser shots left the possibility of ion formation. uncontrollable energy transfer into dissociative channels of such A common feature of these experiments is the laser irradiation molecules.‘j Many efforts have been made to overcome this of the dynamic target consisting of either pieces of a dried-fromlimitation. It has been shown in recent years that the addition of eluent sample or laser-desorbed neutral species to produce the some organic substrates to create a matrix for analyte molecules allows the introduction of much larger ions into the gas p h a ~ e . ~ , ~ quasimolecular ions of anlaytes. The 2PI of desorbed species such as clusters might not only cause the formation of radical cations The following development of this matrix-assistedlaser desorption of an analyte but also initiate intercluster charge transfer reactions, ionization O D technique, mainly directed toward extending leading to formation of quasimolecular ions. Similar processes the mass range and the list of compounds as well as substances have been studied by Nishi and Shinohara.zOAmmonium clusters applicable for matrix preprati~n,~-’~ gave rise to a number of as the model objects were ionized by UV radiation. It has been models proposed to explain MALDI.I3-l5 Based upon either shown that a competition between intercluster proton transfer and electron-removing reactions generated the protonated ions and (1) Unsold, E.; Hillenkamp, F.; Nitsche, R Analysis 1976, 4, 115. (2) Posthumus, M. A; Kistemaker, P. G.; Meusellaar, H. L. C.; ten Noever de radical cations, respectively. However, it is reasonable to expect Brauw, M. C. Anal. Chem. 1978,50, 985. that this competition may be excluded in the case of photoion(3) Conzemius, R J.; Capellon, J. M. Int.J. Mass Spectrom. Ion Processes 1980, ~~~
~~
34, 197. (4) Cotter, R J. Anal. Chem. 1980,52,1767. (5) Vastova, F. J.; Pirone, A J. Ado. Mass Spectrom. 1968, 4, 107. (6) Antonov, V. S.; Letokhov, V. S.; Shibanov, A N. Appl. Phys. 1981,25, 71. (7) Karas, M.; Bachmann, D.; Bahr, U.; Hillenkamp, F. Int. J. Mass Spectrom. Ion Processes 1987, 78, 53. (8) Tanaka, K.; Waki, H.; Ido, Y.; Akita, S.; Yoshida, Y.; Yoshida, T. Rapid Commun.Mass Spectrom. 1988, 2, 151. (9) Karas, M.; Bahr, U.; Ingendoh, A ; Hillenkamp, F. Angew. Chem., Int. Ed. Engl. 1989,28, 760. (10) Beavis, R C.; Chait, B. T. Org. Mass Spectrom. 1992, 27, 156. (11) Chan, T.-W. D.; Colbum, A W.; Derrick, P. G. Org. Mass Spectrom. 1992, 27, 53. (12) Fitzgerald, M. C.; Parr, G. R; Smith, L. M. Anal. Chem. 1993, 65, 3204.
0003-2700/95/0367-1963$9.00/0 0 1995 American Chemical Society
~
(13) Vertes, A ; Levine, R D.Rapid Commun. Mass Spectrom. 1990,4, 233. (14) Vertes, A. Microbeam. Anal. 1991,25, (special issue, Howitt, D. G., Ed.). (15) Ens, W.; Mao, Y.; Mayer, F.; Standing, K G. Rapid Commun. Mass Spectrom. 1991, 5, 117. (16) Beavis, R C.; Chait, B. T. Proceedings of the 38th ASMS Conference on Mass Spectrometry and Allied Topics, Tuscon, AZ, 1990; Caprioli, R M., Ed.; p 152. (17) Hillekamp, F.; Karas, M.; Beavis, R C.; Chait, B. T. Anal. Chem. 1991, 63, 1993.4. (18) Murray, K. IC; Russel, D. H. Anal. Chem. 1993, 65, 2534. (19) Dale, M. J.;Jones, A C.; LangridgeSmith, P. R R; Costello, K F.; Cummins, P. G.A w l . Chem. 1993, 65, 793. (20) Shinohara, H.; Nishi, N. J. Chem. Phys. 1985, 83 (4), 1939.
Analytical Chemistry, Vol. 67, No. 13, July 1, 1995 1963
ization of clusters composed at least of two components, with the proviso that one of the components absorbs strongly at the wavelength of ionizing radiation and the other one does not. The selective photoionization of an absorbing component may be followed by dissociation, with subsequent charge transfer to the other component transparent for the wavelength of ionizing radiation. Thus, this seems to be it the only probable path for analyte ion formation along charge transfer reaction coordinates. In this paper we present the results of 2PI of neutral clusters composed of matrix and analyte molecules. The clusters were prepared by IR-LD of a matrix material containing analyte molecules. The products of desorption were injected into a supersonic jet. The wavelengths and power density of ionizing radiation were chosen to allow selective 2PI of matrix molecules from neutral clusters. The timeof-flight mass spectra of a number of substances such as synthetic polymers, sugars, and peptides have been obtained under the conditions at which the analytes did not absorb at the wavelengths of ionizing radiation.
1
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250 2000
(21) Frey, R.: Holle, A; Weiss, G.; Franzen, J.; Koch, D. Proceedings of the 36th Annual Conference on Mass Spectrometry and Allied Topics, San Francisco, CA, 1988 p 185. (22) Krutchinsky, A N.; Dolgin, A T., Khodorkovski, M. A. J. Mass Spectrom. 1995,30, 375. (23) Beavis, R. C.; Lindner, J.; Grotemeyer, J.; Schlag, E. W. Chem. Phys. Lett. 1988,146, 310. (24) Li. L.; Lubmun, D. M. Appl. Spectrosc. 1989,43, 543. (25) Yu, W.; Vath, J. E.; Huberty, M. C.; Martin, S. A. Anal. Chem. 1993,65, 3015. (26) Walter, K; Boesl, U.: Schlag, E. W. Int.J. Mass Spectrom. Ion Processes 1986, 71, 309. (27) Boesl, V.:Grotemayer, J.; Walter, K; Shlag, E. W. Anal. Instrum. 1987. 16. 151. 1964 Analytical Chemistry, Vol. 67, No. 73,July 7, 7995
I
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EXPERIMENTAL SECTION
Materials and Sample Preparation. The reagents used were poly(ethy1ene glycols) (PEGS), pentaphenylalanine, poly(Lvaline), alanylglysylglysine (AGG) , dalargin CTyr-D-Ala-Gly-Phe Gly-Arg, aminobenzoic acid sodium salt, caffeic acid, 3,4-dyhydroxytruns-cinnamic acid (all from Sigma, St. Louis, MO). All of the sugars used were from Reachim, Russia. Other chemicals and solvents were reagent grade. The solution of an analyte in ethanol/benzene, ethanol/water, or methanol/O.l% acetic acid, typically of 1mg/mL concentration, was mixed with a 1 mg/mL solution of the aminobenzoic acid sodium salt or caffeic acid (3,4dyhydroxy-trans-cinnamicacid) in methanol to prepare the matrix with the desired molar ratio of the components (1:l-1:lO). An aliquot of 10 ,uL of the mixture was applied on the steel surface of a probe tip, dried, and introduced into the desorption chamber of the mass spectrometer. The products of laser desorption injected into a supersonic jet were examined by 2PI/TOF MS. Instrumentation. All experiments were performed using the reflection time-of-flight mass spectrometer TOF-1 (Bruker-Franzen Analytik, Bremen, Germany) with MUPI ion source.21-23 The principal scheme and main parameters of the experimental setup were described e l s e ~ h e r e Briefly, . ~ ~ ~ ~a ~stainless steel pulsed valve (General Valve Co., Fairfield, NJ) of 0.2 mm orifice diameter was used as a supersonic beam source. In the experiments, Ar with a backing pressure of 4-5 bar was the carrier gas; the background pressure remained at 1 x Torr. The jet was skimmed 35 mm downstream by a skimmer with a 1mm orifice. The samples were desorbed by an Altec AL 854 pulse COZlaser (Altec, Luebeck, Germany) directly in front of an expending gas
393
240
/I 1360 1720 2080 2 4 4 A / ; ? 8 0 0
I
Figure 1. Mass spectra of PEG 400 (A), 2000 (B), and 4000 (C) obtained by 2PI (262 nm) of neutral clusters composed of aminobenzoic acid sodium salt and analyte molecules.
jet. The rate of desorption could be controlled by adjusting the COZ laser power density on the sample surface. Based on the known beam profile, the power density of the desorbing beam was held at (1-10) x lo5 W/cm2. The laser ionization was performed 75 mm downstream from the desorption area by a frequency-doubled dye laser, FL 3002 (Lambda Physik, Hottingen, Germany), pumped by a LPX 200 excimer laser (308 nm, 15 ns) (Lambda Physik) . The delay time between desorbing and ionizing laser shots could be changed within 1-1500 ,us. The ionizing laser beam was collimated with a 20 cm focal length lens to produce a laser beam 1-2 mm in diameter. The power density in the ionization region was typically on the order of 105-106 W/cm2. The ion signals were recorded by an RTD-710 200 MHz transient digitizer (Sony-Tektronix, Beaverton, OR) and stored via a GPIB interface on a Motorola 68000 fast computer system. Up to 100 laser shots were averaged to produce a mass spectrum. RESULTS AND DISCUSSION
Synthetic Polymers. The mass spectra of PEGS 400,2000, and 4000 are shown in Figure 1, parts A-C, respectively. All polymers were desorbed from the sodium salt of aminobenzoic acid matrix. 2PI at 262 nm of desorbed products caused the sodiumation of molecules of linear polymers transparent for the wavelength of ionizing radiation. Little formation of the cationated ions occurred by attachment of gas phase sodium ions because of the very low probability of two-body collisions in the ionization region. However, this probability might be increased sufficiently if the process of charge transfer occurred within a cluster. The appearance of charges was promoted via 2PI of a cluster
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Figure 2. LD (10.6 pm)/2PI (262 nm)
124
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spectrum of aminobenzoic
acid sodium salt (m = 159 m/z).
T
140
192
244
296
34a,,z400
Figure 4. Mass spectrum of a mixture of fructose and lactose obtained by 2PI (262 nm) of neutral clusters composed of aminobenzoic acid sodium salt and analyte molecules. MFNa+, sodiumated fructose (MF= 180 "2); MLNa+,sodiumated lactose (ML = 342 Wz).
240
1
350
550
750
950 115R,2350
Flgure 5. Mass spectrum of /3-cyclodextrin obtained by 2PI (262 nm) of neutral clusters composed of aminobenzoic acid sodium salt and analyte molecules. McDNa+,sodiumated/3-cyclodextrin(MCD = 1134 m/z); McDm+, ions of composed clusters.
Molar
ratio
Dependence of the intensity of the mass peak (Wz2021) in mass spectra of PEG 2000 on the molar ratio of the matrix of the analyte. Figure 3.
components. The ion signal of two-photon ionization of pure sodium, which might be presented as an impurity, was not as abundant as in the 2PI mass spectrum of sodium salt of aminobenzoic acid (see Figure 2). Thus, the most probable process occumng within a neutral composed cluster after ionization of the matrix molecules was Na+ detachment and transfer to an analyte molecule with subsequent reorganization and dissociation of the clusters. The investigation was carried out to determine the optimal matrix to analyte molar ratio. For this, a series of mass spectra were obtained for PEG 2000 desorbed from the matrices with different relative concentrations of aminobenzoic acid sodium salt and the polymer. The normalized amplitude values of the mass peak at m/z 2021 near the maximum of the polymer distribution as a function of the matrix to analyte molar ratio by constant amount of the polymer applied onto a probe tip (-2 pg, -1 nmol) are shown in Figure 3. It can be seen in this figure that the signals were increased by increasing the molar ratio until the ratio was approximately equal to 10. We did not use any system to deflect low-weight ions, so the drop of the signal was not due to the deviation from the optimal ratio but was caused by charging of the microchannel plates because of a large number of matrix ions incident upon its surface. However, from these results, it may be concluded that the greater the molar ratio, the more effectively the matrix assisted desorption, apparently both as a heat buffer for excessive desorption energy and as a cation donor.
This approach was successfully applied for analysis of a number of synthesized polymers, such as poly(ethy1ene glycols), poly(propy1ene glycols), and polyfurans, not only to determine the final products but also to optimiie the conditions of polymerization. Sugars. The process of cation transfer appears to be effective also for a number of sugars. Sodium salt of aminobenzoic acid was appropriated as matrix material to obtain the mass spectra of fructose, lactose, glucose, and sucrose. Figure 4 shows the mass spectrum of fructose (m/z 180) and lactose (m/z 342). Although in the mass spectrum there are many peaks that can be identified with the ions of clusters composed of the fragments of matrix molecules, the sodiumated ions are clearly recognizable. Analysis of the mass spectrum of j3-cyclodexb-h (m/z 1134) in Figure 5 shows that the mechanism described above was involved in the processes of cationated ion formation. Peptides. The mass spectra of a number of peptides containing no aromatic amino acid residues were obtained by 2PI of caffeic acid molecules of a composed cluster component. The dissociative ionization at 262 nm of the matrix molecules was followed by proton transfer to the analyte molecules. The mass spectra of the AGG (m/z 203) and caffeic acid matrix shown in parts A and B of Figure 6 were obtained at -5 x 105 and -108 W/cm2 power densities of the ionizing laser radiation, respectively. The ultimate high-power density of W radiation used for ionization of clusters did not cause any fragmentation of the peptide transparent at 262 nm. The dissipation of W radiation energy occurred along dissociative ionization reaction coordinates of the matrix molecules which acted as a buffer for excessive energy. Analytical Chemistry, Vol. 67, No. 13, July 1, 1995
1965
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500
400
600
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Figure 8. Mass spectrum of acetyldalargin (CH3-CO-YAGFGR) obtained by 2PI (308 nm) of neutral clusters composed of caffeic acid and analyte molecules. MH+, protonated analyte (M = 71 1 m/z); (M - 42)H+ and (M - 42 - 18)H+, protonated pyrolytic fragments of the original peptide.
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280
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,
Figure 6. Mass spectra of AGG peptide (M = 203 m/z) obtained by 2PI (262 nm) of neutral clusters composed from caffeic acid (m = 180 m/z) and analyte molecules. Power density of the ionizing radiation: (A) 5 x 105 and (E) -lo8 W/cmz.
+
1000
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0 200
520
840 1160 148@,/2800
Figure 9. Mass spectrum of SPPTSTPDPKPKNNT peptide (m/z = 1579) obtained by 2PI (262 nm) of neutral clusters composed from caffeic acid and analyte molecules. The mass peak at m/z 797 is identified with the (-DPKPKNNT)H+ fragment ion formed due to the breaking of the labile -P. .D- bond of the peptide. 9
0
250
400
550
700
850,,,2000
Figure 7. Mass spectrum of pentaphenylalanine obtained by 2PI (308 nm) of neutral clusters composed of caffeic acid and analyte molecules. MH+, protonated analyte (M = 754 m/z); Mm+, ions of composed clusters.
However, no clear, recognizable effects of intercluster charge transfer have been observed upon study of the compounds having aromatic groups strongly absorbing at 262 nm. This can be explained by a low dilution factor of an analyte in the matrix (obviously, the numbers of analyte and matrix molecules in a composed cluster were comparable) and by the competition between the intercluster charge transfer reactions and the electron-removing reactions which generate the protonated (or cationated) ions and radical cations, respectively.2O To avoid competition between two possible reaction channels for those compounds that have absorption maxima in the region of 260290 nm, the wavelength of ionizing laser radiation should be above 300 nm. On this assumption, the mass spectrum of pentaphenylalanine (m/z 754) was obtained by 2PI of caffeic acid molecules of a cluster component. The 2PI of the matrix was performed at 322 nm, where the weak absorbency band of the gas phase molecules had been found. The resonance 2PI of the protondonated matrix molecules can be also induced at 308 nm, where the absorbency of the matrix molecules was stronger (Figure 7). Thus, the caffeic acid matrix appeared to be very convenient for the selective photoionization of matrix molecules of a cluster component using the 308 nm wavelength of an XeCl excimer laser. 1966 Analytical Chemistry, Vol. 67, No. 73, July 7, 7995
It should be pointed out that, whereas the processes of direct deposition of the ionizing radiation energy can be excluded by causing matrix-assisted ionization, the processes of molecule excitation during laser desorption still might occur. The processes of thermopyrolyses were clearly discerned in the mass spectrum of acetyldalargin (CH3-CO-YAGFGR, m/z 711, Figure 8). The intensive protonated fragment ions correspond to the loss of the guanidine group of the arginine residue, reduction (m/z 669), and loss of water (m/z 651), the typical pyrolytic fragment^.?^,?^ Probably, the excitation of the SPF'TSTPDPKPKNNT (m/z 1579) molecules during laser desorption caused the gas phase cleavage of the -P..-D- labile bond of the peptide, which was clearly observed in the mass spectrum (see Figure 9). This evidence is in good agreement with the recent results of a study of some peptides having the -P.--D- (-Pro-Asp) bond.25 It has been proposed that the gas phase cleavage of the peptide is due to the rather short lifetime of the excited quasimolecular ions. For some polypeptides, the process of cationation seemed to be preferable over protonation. The poly @-valine)mass spectrum obtained by using aminobenzoic acid sodium salt as a matrix is shown in Figure 10; the yield of the protonated ions appeared to be very low when the proton-donated matrix was used. All mass spectra were obtained at a time delay between IR and W laser shots of 250-260 ps. The measured time delay spectra for almost all of the compounds showed that the maximum of the distribution was within mentioned values and the fwhm was less than 50 ps. Figure 11 shows the normalized time delay spectra for a number of AGG protonated clusters. The spectra
1000
500
300
540
780
1020 126@,,2500
Figure 10. Mass spectrum of poly(L-valine) obtained by 2PI (262 nm) of neutral clusters composed of aminobenzoic acid sodium salt and analyte molecules. The main set of mass peaks corresponds to (H(CsH~NO),,OH)2Na+; peaks labeled with asterisk correspond to (H(CsH9NO),,0H)Na+.
I
210
230
250
Time
delay
290
270 ( JIS
310
)
Figure 11. Time delay spectra of (AGG),, peptide clusters. n = 1 (a), 2 (b), 3 (c), and 4 (d).
were obtained by integrating the ion signals of corresponding masses, scanning the delay time between desorbing and ionizing laser shots. We suppose that the emission of clusters occurred within a short period of time, comparable with the duration of desorbing laser pulse (z = 4 ps). If this is true, then the full time for clusters to reach the ionization area was mainly determined by the velocity of the jet, not the time of cluster emission. Thus, the time delay distribution can be interpreted as the velocity distribution of the clusters in the ionization area. The mass resolution of 1000-1500 obtained for almost all of the compounds studied (see,for example, Figure 7) was typical of the resolution
achieved in our experiments with 2PI of neutral molecules. It may imply that the initial velocity distribution of ions resulting from charge transfer with subsequent cluster reorganization is simiiar to the measured velocity distribution of neutral clusters. Thus, it seems that the maximum and width of the initial velocity distribution of quasimolecularions in the ionization area are much lower and narrower than were determined by irradiation of “static” matrix 1a~ers.l~ We did not make efforts to improve the mass resolution, but it is believed that the resolution might be improved sufficiently due to the properties inherent in the experiments by separately performing desorption into a supersonic jet and multiphoton ionizationF6 CONCLUSIONS The LD of various analyte compounds embedded in a matrix of aminobenzoic acid sodium salt or caffeic acid and 2PI of desorbed products showed that the ionization of analyte molecules had occurred via selective photodissociativeionization of a matrix component of the neutral clusters, followed by charge transfer reactions with subsequent cluster reorganization and fragmentation. The wavelengths of ionizing radiation, chosen on the basis of knowledge of the absorption bands of the matrix molecules in the gas phase, allowed the selective deposition of laser energy into these molecules but not into the analytes that are transparent for these wavelengths. Under this condition, the only possible route for analyte ion formation was along charge transfer coordinates. The ionization of composed cluster may be viewed as a simplest model of an irradiated target. Because of the small number of the components involved in photochemical reactions, all possible paths of deposition and dissipation of the energy of laser radiation can be easily traced. Obviously, there is a direct analogy between the processes occurring upon irradiation of the composed clusters and in the “static” matrix layers. One can consider this analogy to elucidate the basis principles underyling the physical processes involved in MALDI. ACKNOWLEDGMENT We gratefully acknowledge the support of this work by BrukerFranzen Analytik GmbH (Bremen) and the helpful discussions with Drs. R Frey, C. Koster, A. Holle, and J. Franzen. Received for review August 29, 1994. Accepted January 4, 1995.@ AC940855S e Abstract published in Advance ACS Abstracts, February 1, 1995.
Analytical Chemistry, Vol. 67, No. 13, July 1, 1995
1967
