Anal. Chem. 2007, 79, 9122-9127
Ordered Porous Alumina Geometries and Surface Metals for Surface-Assisted Laser Desorption/ Ionization of Biomolecules: Possible Mechanistic Implications of Metal Surface Melting Yoshinao Wada,*,† Takashi Yanagishita,‡ and Hideki Masuda‡,§
Osaka Medical Center and Research Institute for Maternal and Child Health, 840 Murodo-cho, Izumi, Osaka 594-1101, Japan, Kanagawa Academy of Science and Technology, 5-4-30 Nishi Hashimoto, Sagamihara, Kanagawa 229-1131, Japan, and Department of Applied Chemistry, Tokyo Metropolitan University, 1-1 Minamiosawa, Hachioji, Tokyo 192-0397, Japan
Platinum- or gold-coated porous alumina with submicrometer structures is a potential substrate for surfaceassisted laser desorption/ionization (SALDI) mass spectrometry of biomolecules, not requiring sample matrixes. In this study, a highly ordered porous alumina substrate was fabricated to study the geometric factors allowing good SALDI performance. Evaluation was based on the signalto-noise ratio of protonated angiotensin I ions in the mass spectrum obtained by 337-nm ultraviolet laser irradiation. Varying the geometries, including pore densities and diameters, revealed the laser intensity required to generate ions to be related to surface porosity. Surface platinum was melted upon laser irradiation at the fluence sufficient to generate peptide ions as confirmed by scanning electron microscopy. Moreover, a thin (5-20 nm) platinum coat requires a low intensity of laser light for desorption/ ionization. Considering the size effect on the melting of metals, our findings suggest the surface platinum melting to be involved in ion generation from this SALDI substrate type. Indeed, tantalum, which has a higher melting point, required more laser fluence to generate ions. The porous alumina layer beneath surface metals probably worked as a thermal insulator. This double-layer-type substrate allowed ionization of angiotensin I and verapamil at low femtomole levels. Moreover, small proteins and glycoproteins such as 24-kDa trypsinogen and 15-kDa ribonuclease B could be ionized with sufficient sensitivity on this target. Taking advantage of matrix-free methods, concentrating the sample solution in the target concavity or widening the laser beam focus enhanced the signal-tonoise ratio for analyte ions in the mass spectrum. Activity is maintained for months in air. Matrix-assisted laser desorption/ionization (MALDI) is widely used in mass spectrometry (MS) of biopolymers and synthetic * To whom correspondence should be addressed. Phone: +81-725-56-1220. Fax: +81-725-57-3021. E-mail:
[email protected]. † Osaka Medical Center and Research Institute for Maternal and Child Health. ‡ Kanagawa Academy of Science and Technology. § Tokyo Metropolitan University.
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polymers.1-11 In typical MALDI using an ultraviolet (UV) laser, analytes are mixed and cocrystallized with a UV-absorbing organic molecule, referred to as the sample matrix, or chemical matrix on the MALDI sample target. The matrix transfers energy from a pulsed laser beam to the analyte molecules, but using a matrix has certain disadvantages; The matrix-derived ions complicate the mass spectrum of low molecular weight analytes. The matrix/ analyte ratio, which is critical for efficient ionization, is not uniformly distributed in the sample spot, causing wide signal intensity variations among laser irradiation points. Moreover, the suitability of different matrixes for analyte compounds is mostly an empirical issue, such that matrix selection and optimization of the sample preparation procedure were the issues in utilizing the MALDI technique.12,13 The technique of laser desorption/ionization of biomolecules without the aid of a chemical matrix was originally developed by Tanaka et al.,4 who suspended fine cobalt particles (diameters ∼30 nm) in glycerol and obtained spectra of proteins and synthetic polymers by using a pulsed ultraviolet laser. Sunner et al. replaced the metal powder with micrometer-sized graphite particles and named this method graphite surface-assisted laser desorption/ ionization (graphite SALDI).14 Subsequently, a remarkable advance was made with desorption/ionization on (porous) silicon (DIOS), in which high-quality UV laser desorption/ionization mass spectra (1) Karas, M.; Bachmann, D.; Hillenkamp, F. Anal. Chem. 1985, 57, 29352939. (2) Karas, M.; Bachmann, D.; Hillenkamp, F. Int. J. Mass Spectrom. 1987, 78, 53-68. (3) Karas, M.; Hillenkamp, F. Anal. Chem. 1988, 60, 2299-2301. (4) Tanaka, K.; Waki, H.; Ido, Y.; Akita, S.; Yoshida, Y.; Yoshida, T. Rapid Commun. Mass Spectrom. 1988, 2, 151-153. (5) Chance, D. H. Chem. Rev. 2001, 101, 445-477. (6) Aebersold, R.; Goodlett, D. R. Chem. Rev. 2001, 101, 269-295. (7) Rappsilber, J.; Moniatte, M.; Nielsen, M. I. L.; Podtelejnikov, A. V.; Mann, M. Int. J. Mass Spectrom 2003, 226, 223-237. (8) Montaud, G.; Lattimer, R. P. (eds). Mass Spectrometry of Polymers; CRC Press: Boca Raton, FL, 2001. (9) Hanton, S. D. Chem. Rev. 2001, 101, 527-69. (10) Macha, S. F.; Limbach, P. A. Curr. Opin. Solid State Mater. Sci. 2002, 6, 213-220. (11) Murgasova, R. M.; Hercules, D. M. Int. J. Mass Spectrom. 2003; 226, 151162. (12) Vorm, O.; Roepstorff, P.; Mann, M. Anal. Chem. 1994, 66, 3281-3287. (13) Dai, Y.; Whittal, R. M.; Li, L. Anal. Chem. 1996, 68, 2494-2500. (14) Sunner, J.; Dratz, E.; Chen, Y.-C. Anal. Chem. 1995, 67, 4335-4342. 10.1021/ac071414e CCC: $37.00
© 2007 American Chemical Society Published on Web 11/03/2007
of organic compounds directly deposited and dried on porous silicon were obtained, with neither a chemical matrix nor glycerol being required.15 DIOS inspired a line of SALDI studies using various materials and structures (see review by Peterson16), mostly of submicrometer or nanometer size, including etched silicon and carbon surfaces,17 carbon nanotubes,18 zinc oxide nanowires,19 titanium dioxide nanostructure film,20 ordered silicon nanocavity arrays,21 silicon nanoparticles,22 silicon nanowires,23 gold nanoparticles,24 gold nanodots,25 and germanium nanodots.26 Porous silicon is a UV-absorbing semiconductor produced by electrochemical anodization or chemical etching of crystalline silicon providing a large surface area. Although the DIOS mechanism is largely unknown, electron transfer between the silicon and analytes participates in the ionization process as inferred from our studies on analyte reduction and oxidation.27-29 DIOS applications cover a wide range of compounds including peptides, natural products, small organic molecules, and synthetic polymers.15,27-36 DIOS sensitivity for peptides has been improved by pentafluorophenyl derivatization or functionalization of the porous silicon surface.33 Most DIOS MS applications involve small molecules, less than 3 kDa. DIOS MS of large biomolecules has not been thoroughly investigated, though a signal (at m/z 18 000) derived from β-lactoglobulin was reported.31 In our previous study, surface electroconductivity was one of the requirements for porous or submicrometer structures to be effective for SALDI, and porous alumina yielded molecular ion signals of angiotensin and insulin with UV irradiation when the surface was coated with gold or platinum.37 This study has been (15) Wei, J.; Buriak, J. M.; Siuzdak, G. Nature 1999, 399, 243-246. (16) Peterson, D. S. Mass Spectrom. Rev. 2007, 26, 19-34. (17) Alimpiev, S.; Nikiforov, V.; Minton, T.; Sunner, J. J. Chem. Phys. 2001, 115, 1891-1901. (18) Ren, S. F.; Zhang, L.; Cheng, Z. H.; Guo, Y. L. J. Am. Soc. Mass Spectrom 2005, 16, 333-339. (19) Kang, M. J.; Lee, J. C.; Choi, Y. J.; Park, J. H.; Park, J. G.; Lee, J. G.; Choi, H. J. Rapid Commun. Mass Spectrom 2005, 19, 3166-3170. (20) Chen, C. T.; Chen, Y. C. Anal. Chem. 2004, 76, 1453-1457. (21) Finkel, N. H.; Prevo, B. G.; Velev, O. D.; He, L. Anal. Chem. 2005, 77, 1088-1095. (22) Go, E. P.; Apon, J. V.; Luo, G.; Saghatelian, A.; Daniels, R. H.; Sahi, V.; Dubrow, R.; Cravatt, B. F.; Vertes, A.; Siuzdak, G. Anal. Chem. 2005, 77, 1641-1646. (23) Wen, X.; Dagan, S.; Wysocki, V. H. Anal. Chem. 2007, 79, 434-444. (24) Su, C. L.; Tseng, W. L. Anal. Chem. 2007, 79, 1626-1633. (25) Chen, L. C.; Yonehama, J.; Ueda, T.; Hori, H.; Hiraoka, K. J. Mass Spectrom 2007, 42, 346-353. (26) Seino, T.; Sato, H.; Yamamoto, A.; Nemoto, A.; Torimura, M.; Tao, H. Anal. Chem. 2007, 79; 4827-4832. (27) Okuno, S.; Arakawa, R.; Wada, Y. J. Mass Spectrom. Soc. Jpn. 2004, 52, 13-20. (28) Okuno, S.; Nakano, M.; Matsubayashi, G.; Arakawa, R.; Wada, Y. Rapid Commun. Mass Spectrom. 2004, 18, 2811-2817. (29) Okuno, S.; Oka, K.; Arakawa, R. Anal. Sci. 2005, 21, 1449-1451. (30) Shen, Z.; Thomas, J. J.; Averbuj, C.; Broo, K. M.; Engelhard, M.; Crowell, J. E.; Finn, M. G.; Siuzdak, G. Anal. Chem. 2001, 73, 612-619. (31) Thomas, J. J.; Shen, Z.; Crowell, J. E.; Finn, M. G.; Siuzdak, G. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 4932-4937. (32) Kruse, R. A.; Li, X.; Bohn, P. W.; Sweedler, J. V. Anal. Chem. 2001; 73, 36393645. (33) Lewis, W. G.; Shen, Z.; Finn, M. G.; Siuzdak, G. Int. J. Mass Spectrom 2003, 226, 107-116. (34) Trauger, S.; Go E. P.; Shen, Z.; Apon, J. V.; Compton, B.; Bouvier, E. S. P.; Finn, M. G.; Siuzdak, G. Anal. Chem. 2004; 76, 4484-4489. (35) Arakawa, R.; Shimomae, Y.; Morikawa, H.; Ohara, K.; Okuno, S. J. Mass Spectrom. 2004; 18, 961-965. (36) Okuno, S.; Wada, Y.; Arakawa, R. Int. J. Mass Spectrom. 2005, 241, 43-48. (37) Okuno, S.; Arakawa, R.; Okamoto, K.; Matsui, Y.; Seki, S.; Kozawa, T.; Tagawa, S.; Wada, Y. Anal. Chem. 2005, 77, 5364-5369.
extended by optimization of the porous alumina structures.38 Porous alumina is a substrate allowing regulated fabrication of a highly ordered structure by a finely tuned anodization process.39,40 In the present study, geometric factors including pore density and porosity as well as different coating metals were investigated to explore factors allowing SALDI MS of biomolecules. Melting of surface metals was suggested to underlie the desorption/ionization mechanism of this SALDI substrate type, and the porous alumina layer was assumed to work as a thermal insulator. This doublelayer-type substrate allowed ionization of proteins over 20 kDa. EXPERIMENTAL SECTION Chemicals. Glucose, glycine, verapamil, and aluminum sheet were purchased from Wako (Osaka, Japan), angiotensin I (human) was from Bachem (Bubendorf, Switzerland). pancreatic ribonuclease (bovine), trypsinogen (bovine), and O-acetyl-L-carnitine from Sigma-Aldrich (Milwaukee, WI), heptadecafluorooctanesulfonic acid (PFOS) from TokyoKasei (Tokyo, Japan), and methyl methacrylate monomer from Kanto Kagaku (Tokyo, Japan). Fabrication of Ordered Porous Alumina and Metal Nanohole Array. Highly ordered porous alumina was prepared by anodizing an Al sheet as reported previously.40 Briefly, an Al sheet (99.99%) was annealed at 400 °C for 1 h and then polished electrochemically in a solution of perchloric acid and ethanol at 20 kDa.16 In this study, 500 fmol of 24-kDa trypsinogen could be ionized with good sensitivity as shown in Figure 8a, in which a 72-kDa trimer was detectable as well. Interestingly, the charge state of observed ions was the same as that found in the MALDI mass spectrum (Figure 8a and b), suggesting a similar mechanism underlying the porous alumina SALDI with MALDI or room for an argument whether (44) Chen, C. T.; Chen, Y. C. Anal. Chem. 2005, 77, 5912-5919.
thermal desorption at the substrate-analyte interface is the only basis for the SALDI process on metal-coated porous alumina. In Figure 8c, a heterogeneous mixture of ribonucleases could be analyzed without any additives, and the glycan heterogeneity of ribonuclease B was delineated in this mass spectrum. These measurements demonstrated that the double-layer substrate composed of porous alumina and surface Pt is indeed a sample target allowing soft laser desorption/ionization or good desorption efficiency for biomolecules. The laser fluence required for protein ionization was quite similar to that needed for small molecules, being consistent with the concept that metal melting allows SALDI activity. The SALDI activity with 337-nm laser irradiation was dependent on porosity but was not sensitive to a specific geometry, indicating that the absorption of laser light, which was actually demonstrated by ellipsometry in some wide-pore structures examined herein, was not a major determinant of activity. The independence of the UV laser wavelength was also seen with DIOS.32 Other parameters including the laser repetition rate and duration, beam profile homogeneity, and possible advantages of infrared wavelength should be considered with the aim of improving performance. In addition, the properties of generated ions such as internal energy and initial velocity, as well as their relations to the surface temperature upon laser irradiation, remain to be determined. CONCLUSIONS Porous alumina substrate with a 50-100-nm surface Pt coat allowed soft SALDI MS of biomolecules including small proteins. Melting of surface metals corresponded to ion generation and might be involved in the mechanism of this double-layer type of SALDI sample target, which has potential to be a practical SALDI platform for biomolecular analysis. ACKNOWLEDGMENT We thank Prof. F. Hillenkamp for helpful comments. The late Dr. S. Okuno participated in the early stage of this work. This study was supported by a Grant-in-Aid for Exploratory Research (18657037) from MEXT (Ministry of Education, Culture, Sports, Science and Technology) of Japan. SUPPORTING INFORMATION AVAILABLE Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.
Received for review July 3, 2007. Accepted September 25, 2007. AC071414E
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