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Charge-Transfer Matrixes as a Tool To Desorb Intact Labile Molecules by Matrix-Assisted Laser Desorption/Ionization. Use of 2,7-Dimethoxynaphthalene in the Ionization of Polymetallic Porphyrins Iolinda Aiello,† Leonardo Di Donna, Mauro Ghedini,† Massimo La Deda,† Anna Napoli, and Giovanni Sindona*
Dipartimento di Chimica, Universita` della Calabria, via P. Bucci, cubo 12/C, I-87030 Arcavacata di Rende (CS)- Italy
2,7-Dimethoxynaphthalene (DMN) is proposed as matrix to investigate the structure of polymetallic porphyrins through matrix-assisted laser desorption/ionization tandem time-of-flight experiments. The peculiarity of DMN is represented by the formation of molecular radical cations and of some diagnostic fragments only. The traditional matrixes do not afford the expected molecular species. The experiments have been performed on extremely labile species such as zinc porphyirinate complexes with aluminum and gallium quinolinate to prove the softness of the methodology.
Matrix-assisted laser desorption/ionization (MALDI) has been widely applied, since its introduction, in a variety of analytical chemistry fields. The effectiveness of the methodology resides in the facility of detecting intact high molecular weight compounds using samples in the low-picomole range. The limiting step of the MALDI procedure is represented by the sample preparation and the choice of appropriate cocrystallizing agents. Different sets of matrixes have been exploited to specifically deal with peptides and proteins,1,2,3 oligo- and polynucleotides,4 carbohydrates,5 and * To whom correspondence should be addressed. Tel: +39-0984-492046. Fax: +39-0984-493307. E-mail:
[email protected]. † Centro di Eccellenza CEMIF.Cal-LASCAMM, Unita ` INSTM della Calabria. (1) Karas, M.; Hillenkamp, F. Anal. Chem. 1988, 60, 2299-2301. (2) Tanaka, K.; Waki, H.; Ido, Y.; Akita, S.; Yoshida, Y.; Yoshida, T. Rapid Commun. Mass Spectrom. 1988, 2, 151-156. (3) Aebersold, R.; Mann, M. Nature 2003, 422, 198-207. (4) Nordhoff, E. Trends Anal. Chem. 1996, 15, 240-250. (5) Harvey, D. J. Mass Spectrom. Rev. 1999, 18, 349-450. 10.1021/ac0490292 CCC: $27.50 Published on Web 09/15/2004
© 2004 American Chemical Society
synthetic polymers.6 The introduction of tandem time-of-flight (TOF) instruments7 has opened new perspectives in the field of both proteomics8 and materials.9 We are not aware of any MALDI application in the structure evaluation of polymetallic porphyrins. The lattersa labile species not amenable to other physicochemical methods of analysisscould benefit by the power of MALDI providing that intact molecular ions and diagnostic fragments could be unambiguously detected. The formation, by MALDI, of gaseous, protonated, and radical molecular cations from nonmetallic oligomeric porphyrins10 represents a well-documented feature of desorption mass spectra of this type of molecule.11 A certain degree of fragmentation is present in the spectra of arrays of covalently bonded porphyrins obtained from R-cyano-4-hydroxycinnamic acid (HCCA) matrix.12 Improvement in the detection of intact molecular ions has been observed when neutral matrixes such as 1,4-benzoquinone derivatives were used.13 (6) Bahr, V.; Deppe, A.; Karas, M.; Hillenkamp, F.; Giessmann, U. Anal. Chem. 1992, 64, 2866-2869. (7) (a) Cotter, R. J.; Cornish, T. J. Anal. Chem. 1993, 65, 1043-1047. (b) Cotter, R. J.; Gardner, B. D.; Iltchenko, S.; English, R. D. Anal. Chem. 2004, 76, 1976-1981. (8) Friedman, D. B.; Hill, S.; Keller, J. W.; Merchant, N. B.; Levy, S. E.; Coffey, R. J.; Caprioli, R. M. Proteomics 2004, 4, 793-811. (9) Cordero, M. M.; Cornish, T. J.; Cotter, R. J. J. Am. Soc. Mass Spectrom. 1996, 7, 590-597. (10) Siegel, M. M.; Tabei, K.; Tsao, R. S.; Pastel, M. J.; Pandey, R. K.; Berkenkamp, S.; Hillenkamp, F.; de Vries, M. S. J. Mass Spectrom. 1999, 34, 661-669. (11) Scha¨fer, M.; Budzikiewicz, H. J. Mass Spectrom. 2001, 36, 1062-1068. (12) Fenyo, D.; Chait, B. T.; Johnson, T. E.; Lindsey, J. S. J. Porphyrins Phthalocyanines 1997, 1, 93-99.
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Chart 1
The structure determination of an interesting class of polymetallic porphyrin complexes is currently under investigation as materials for applications in energy-transfer devices.14 EXPERIMENTAL SECTION Mass Spectrometry. The MALDI mass spectra were acquired on a 4700 proteomics analyzer mass spectrometer from Applied Biosystems (Foster City, CA) equipped with a 200-Hz Nd:YAG laser at 355-nm wavelength. The MS spectra, internally calibrated, were acquired in reflectron mode (20-keV accelerating voltage), with 400-ns delayed extraction, averaging 2000 laser shots with a mass accuracy of 10 ppm. 2,7-Dimethoxynaphthalene (DMN; Sigma-Aldrich Corp., Saint Louis, MO) was used as matrix. The DMN was recrystallized from CH3OH to remove the impurities. A 0.45-µL aliquot of a premixed solution of DMN and sample (400: 1) dissolved in MeOH/CH3Cl (1:1) was spotted on the matrix target, dried at room temperature, and analyzed into the mass spectrometer. The MS/MS experiments were acquired at the collision energy of 1 kV, defined by the potential difference between the source acceleration voltage (8 kV) and the floating collision cell (7 kV); 3400 laser shots were averaged, while the pressure inside the collision cell was 8 × 10-7 Torr. Chemicals. Commercially available 5,10,15,20-tetrakis(4-hydroxyphenyl)-21H,23H-porphyrin (H2TPP(OH)4; Chart 1) and stoichiometric amounts of zinc acetate provide smoothly the zinc porphyrinate complex (Zn-TPP(OH)4), whose molecular structure is featured by four peripheral phenolic groups that can further bind other metal ions. Their structure and purity were determined by NMR, IR, and melting point and confirmed by MALDI. Safety Considerations. 2,7-Dimethoxynaphthalene requires no specific safety precaution aside from universal safety precautions for handling chemical samples; there is no information about the toxicity of polymetallic porphyrins; therefore, caution is recommended when these compounds are handled. RESULT AND DISCUSSION The present investigation deals with the structure determination by MALDI of the insoluble materials that separate on standing from an ethanolic solution of 1:4:8 H2TPP(OH)4- (or Zn-TPP(OH)4)-GaNO3-HQ′ (HQ′ ) 2-methyl-8-hydroxyquinoline) mixture, 1(or 2), respectively, and of the toluene-insoluble material 3 obtained when [(CH3)2CHO)]3Al has replaced the gallium salt (Chart 2). (13) Srinivasan, N.; Haney, C. A.; Lindsey, J. S.; Zhang, W.; Chait, B. T. J. Porphyrins Phthalocyanines 1999, 3, 283-291. (14) (a) Baldo, M. A.; O’Brien, D. F.; You, Y.; Shoustikov, A.; Sibley, S.; Thompson, M. E.; Forrest, S. R. Nature 1998, 395, 151-154. (b) Wieb Van der Meer, B.; Coker, G.; Simon Chen, S. Y. Resonance Energy Transfer. Theory and Data; VCH Publisher: New York, 1994. (c) Haustein, E.; Jahnz, M.; Schwille, P. Chem. Phys. Chem. 2003, 4, 745-748.
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When 1 and 2 are submitted to MALDI experiments in traditional matrixes, no molecular ion peaks are detected. In the presence of HCCA matrix, for instance, the spectrum displayed species in which one or more matrix molecules have replaced ligands in the coordination sphere of the sampled analyte. DMN has been involved in the formation of charge-transfer complexes either in the condensed15 or in the gas phase.16 Moreover, it has been observed that the mechanism of ion formation in classic matrixes such as sinapinic acid (SA) depends on the production of the SA•+ radical cations.17 Taking into account the structure of the analytes, it was considered that DMN could act as a tool to promote intracluster single electron transfer (SET) between matrix and analytes. Remarkably, the MS spectrum of the species of 1 shows the formation of intact molecular species at m/z 2214.3862 and of the fragment ion at m/z 1830.3345. These peaks are accompanied by satellite ions at 69 mass units higher, which might correspond to gallium adducts. The base peak of the spectrum is the fragment ion at m/z 385.0426, which represents the stable species GaQ′2+ (Figure 1). The spectrum of the primary ions indicates that the fully substituted species of mixture 1 was formed, but it does not allow us to exclude the presence in the mixture of a trisubstituted isomer. The two ions were identified by matching the experimental with the theoretical isotopic pattern with a 98-99% score and by the evaluation of the elemental composition, which was in the 10 ppm range. The molecular ion species, isolated with the first TOF and allowed to decompose in the field-free region in front of the second TOF, spontaneously decays by forming and releasing the GaQ′2 moiety and displaying the GaQ′2+ species at m/z 385 as the base peak. The trisubstituted isomer, [(M - GaQ′2) + H]+ at m/z 1830 (Figure 2) is now clearly formed by a gas-phase reaction path taken by the fully substituted species, which requires a hydrogen transfer from the leaving neutral moiety. The mobile hydrogen should reasonably originate from the methyl groups of the hydroxyquinoline moieties, by analogy with the gas-phase chemistry of methylated pyridine and quinoline radical cations.18 The evidence for the proton transfer from the gallium moiety to the porphyrin system suggests that the trisubstituted species might not be present in the insoluble mixture indicated as product 1. When zinc is in the inner core of the porphyrin, the feature of the TOF spectrum of species 2 is more compatible with that of a mixture of compounds. In fact, it shows, besides the base peak at m/z 385, the species at m/z 740 that corresponds to the unreacted porphyrin (Zn-TPP(OH)4) and minor peaks at m/z 1124, 1508, 1892 and 2276 corresponding to ionized Zn-TPP(OGaQ′2)1, Zn(15) (a) Cottrell, P. T.; Mann, C. K. J. Am. Chem. Soc. 1971, 93, 3579-3583. (b) Hamad, T.; Nishida, A.; Matsumoto, Y.; Yonemitsu, O. J. Am. Chem. Soc. 1980, 102, 3978-3980. (16) (a) De Pauw, E. Anal. Chem. 1983, 55, 2195-2196. (b) Athanassopoulos, C.; Papaioannou, D.; Napoli, A.; Siciliano, C.; Sindona, G. J. Mass Spectrom. 1995, 30, 1284-1290. (c) Liguori, A.; Napoli, A.; Sindona, G. J. Am. Soc. Mass Spectrom. 2001, 12, 176-179. (17) Land, C. M.; Kinsel, G. R. J. Am. Soc. Mass Spectrom. 1998, 9, 10601067. (18) Sample, S. D.; Lightner, D. A.; Buchardt, O.; Djerassi, C. J. Org. Chem. 1967, 32, 997-1005.
Figure 1. MALDI/TOF spectrum of species 1.
Figure 2. TOF/TOF spectrum of the m/z 2214-2224 cluster. The precursor ion is out of scale.
Chart 2. Expected Products of Ethanolic Insoluble Materials from 1:4:8 (Zn)H2TPP(OH)4-GaNO3-HQ′ Mixture (1 and 2), and of Toluene-Insoluble Materials from H2TPP(OH)4-[(CH3)2CHO)]3Al-HQ′ Mixture (3)
TPP(OGaQ′2)2, Zn-TPP(OGaQ′2)3, and Zn-TPP(OGaQ′2)4, respectively. All these species behave similarly when allowed to dissociate spontaneously, as shown by the corresponding MS/MS spectra.
The ions at m/z 1892 and elemental composition [Zn-TPP(OGaQ′2)3] afford, in fact, the species [Zn-TPP(OGaQ′2)2 - H]+ at m/z 1507 (Figure 3), by homolysis of the O-Ga bond. Comparison of the MS/MS spectra of species 1 and 2 suggests Analytical Chemistry, Vol. 76, No. 20, October 15, 2004
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Figure 3. TOF/TOF spectrum of the m/z 1892-1902 cluster. The isotopic pattern of the [M - GaQ′2]+ fragment is shown in the expanded view.
Figure 4. MS/MS spectrum of Zn-TPPO-(GaQ′M) species. Taken from HCCA matrix.
that in the presence of a metal ion in the core of the porphyrin a single bond cleavage process prevails over the rearrangement process taken by species 1. The other peaks present in the spectrum correspond to GaQ′2+ (m/z 385) and [M - Q]+ (m/z 1734). This methodology has been extended to similar compounds that possess aluminum quinolinate moieties [(H2TPP(OAlQ′2)n (n ) 1, 4) (3)]. The TOF spectra shows the presence of only two major species: the first one at m/z 2046.6801, which corresponds to the tetrakis complex with AlQ′2, and the second one at m/z 1704.5576, which is the compound bearing three aluminated moieties. All the peaks are due to radical cation species whose isotopic pattern has been recognized with 96% accuracy. The MS/ MS experiments taken on the H2TPP(OAlQ′2)4 precursor ions show, by analogy with 1, the formation of the AlQ′2+ cation at m/z 343 and the formation of [(M - AlQ′2) + H]+ at m/z 1704. 5988
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This confirms that, in the absence of the metal in the porphyrin core, the dissociation of the bond between the phenolic oxygen and the outer-shell metal requires a rearrangement processes. When an acidic matrix like HCCA is used many peaks are present, likely due to in-source and plume reactions. For example, the spectrum of the mixture of compounds 2 in HCCA shows only two peaks in the higher mass region: the peak at m/z 740, corresponding to the unsubstituted zinc-porphyrin, and the minor peak at m/z 1154, which corresponds to the species Zn-TPP(OGaQ′M), which has lost a molecule of quinolinate (Q′) and has included in the complex a molecule of HCCA (M). This picture is confirmed by the MS/MS spectrum of the latter species showed in Figure 4. The gallium moiety is represented now by the peak at m/z 415, which corresponds to the species GaQ′M+, and the major peak at m/z 371 due to the loss of CO2 from the former.
CONCLUSIONS
ACKNOWLEDGMENT
DMN, besides its known properties to promote SET processes within interacting molecules, offers the advantage of providing a single background peak, due to its molecular radical cation, in the low-mass region of the MALDI spectrum. This feature should allow the use of this new matrix even for low molecular weight complexes. Moreover, no aggregates have been detected between the analytes and the matrix that could hamper the interpretation of spectra of extremely labile species, such as already happened when HCCA was used in the examination of 2.
Funds from European Community and PON-avviso 68 are acknowledged.
Received for review July 2, 2004. Accepted September 2, 2004. AC0490292
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