Anal. Chem. 1998, 70, 2360-2365
Surface Plasmon Resonance-Laser Desorption/ Ionization-Time-of-Flight Mass Spectrometry Sandy Owega, Edward P. C. Lai,* and A. D. O. Bawagan
Ottawa-Carleton Chemistry Institute, Department of Chemistry, Carleton University, Ottawa, Ontario K1S 5B6, Canada
The laser desorption/ionization (LDI) process is investigated under surface plasmon resonance (SPR) conditions using time-of-flight mass spectrometry (TOFMS). We demonstrate that LDI-TOFMS at the SPR angle requires a lower minimum laser fluence for the production of silver monomer and cluster cations from ablation of a thin silver film substrate. In the LDI of gramicidin S deposited on a thin silver film substrate, the largest intensity for the molecular cation peak occurs when the laser light is incident on the substrate at a specific SPR angle. These results fully confirm SPR enhancement of the LDI process. The capability to perform SPR-LDI on a larger molecular weight analyte (1141 amu for gramicidin S) represents a new milestone beyond the previous achievement with rhodamine B (479 amu). A better understanding of the SPR mechanism is gained with respect to the substrate metals (silver vs aluminum), desorption (microscopic vs mesoscopic), and ionization (chemical vs multiphoton). These findings may be useful in the future design of SPR-LDI techniques for better TOFMS analysis of higher mass biomolecules. The analysis of peptides and proteins by time-of-flight mass spectrometry (TOFMS) using various ionization techniques is an important area of current research activity.1,2 Since the early introduction of laser desorption of neutral peptide molecules on an organic substrate,3 particular attention has been focused on various analytical benefits of substrate-assisted laser desorption. Substrates ranging from glass, graphite, stainless steel, to other metals can be used in laser desorption to decrease the internal energy of analyte molecules, thereby reducing their fragmentation by an order of magnitude.4 Depending on the electronic nature of metal substrates, different types of reactions are promoted in the laser desorption/ionization (LDI) of different analytes.5-10 It (1) Amado, F. M. L.; Santana-Marques, M. G.; Ferrer-Correia, A. J.; Tomer, K. B. Anal. Chem. 1997, 69, 1102-1106. (2) Kleinekofort, W.; Avdiev, J.; Brutschy, B. Int. J. Mass Spectrom. Ion Processes 1996, 152, 135-142. (3) Speir, J. P.; Amster, I. J. Anal. Chem. 1992, 64, 1041-1046. (4) Zhan, Q.; Wright, S. J.; Zenobi, R. J. Am. Soc. Mass Spectrom. 1997, 8, 525-531. (5) Bjarnason, A. Anal. Chem. 1996, 68, 3882-3883. (6) Ge´ribaldi, S.; Breton, S.; Decouzon, M.; Azzaro, M. J. Am. Soc. Mass Spectrom. 1996, 7, 1151-1160. (7) Lei, Q. P.; Amster, I. J. J. Am. Soc. Mass Spectrom. 1996, 7, 722-730. (8) Wu, H. F.; Brodbelt, J. S. J. Am. Chem. Soc. 1994, 116, 6418-6426. (9) Cromwell, E. F.; Reihs, K.; de Vries, M. S.; Ghaderi, S.; Wendt, H. R.; Hunziker, H. E. J. Phys. Chem. 1993, 97, 4720-4728.
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has recently been reported that noble transition metal ions produced in the gas phase by laser ablation of a metal probe tip or a metal-substituted zeolite can act as chemical ionization (CI) reagents for analyte molecules, with minimal fragmentation.11,12 Surface plasmon resonance (SPR) was first reported by Lee et al. to improve laser desorption of intact rhodamine B dye molecules from a thin aluminum substrate.13 SPR is a light absorption process in which surface plasmon waves are excited along the interface between a thin metal film substrate and a sample overlayer.14,15 When the ablation laser was tuned to a resonant angle of incidence specific for the thin metal film substrate and sample overlayer, a lower laser fluence could be utilized to minimize unwanted fragmentation of the desorbed organic dye molecules. Dawson and Cairns also confirmed that the fluence required for laser ablation of thin aluminum films under SPR conditions is 3-5 times less than that needed when direct laser irradiation is used.16 In this work, we couple SPR with LDI-TOFMS to investigate laser ablation of thin silver film substrates under SPR and nonSPR conditions. Our purpose is to attempt development of SPR for the LDI-TOFMS of biomolecules, even though these analytes have no appreciable light absorption at the 532-nm laser wavelength. The potential benefit of minimal fragmentation is evaluated using a cyclic decapeptide, gramicidin S. The deformation, desorption, and ionization processes, as well as the kinetic energy distributions, are examined to elucidate a stepwise mechanism by which silver cluster cations are produced during the laser ablation. EXPERIMENTAL SECTION Thin Silver Film Substrates. Thin silver film substrates were prepared at Lumonics Optics Group (Nepean, ON, Canada) by thermal evaporation of pure silver metal at a pressure of 10-7 Torr and deposition on one face of clean microscope slides. Their nominal thickness, as given by the quartz crystal oscillator in the evaporation system, was 40 nm. These thin silver film substrates were wrapped in a special oven-dried paper to prevent contamina(10) Wang, B. H.; Dreisewerd, K.; Bahr, U.; Karas, M.; Hillenkamp, F. J. Am. Soc. Mass Spectrom. 1993, 4, 393-398. (11) Gill, C. G.; Garrett, A. W.; Hemberger, P. H.; Nogar, N. S. J. Am. Soc. Mass Spectrom. 1996, 7, 664-667. (12) Gill, C. G.; Garrett, A. W., Earl, W. L.; Nogar, N. S.; Hemberger, P. H. J. Am. Soc. Mass Spectrom. 1997, 8, 718-723. (13) Lee, I.; Callcott, T. A.; Arakawa, E. T. Anal. Chem. 1992, 64, 476-478. (14) Lee, I.; Callcott, T. A.; Arakawa, E. T. Phys. Rev. B 1993, 47, 6661-6666. (15) Johnston, K. S.; Karlsen, S. R.; Jung, C. C.; Yee, S. S. Mater. Chem. Phys. 1995, 42, 242-246. (16) Dawson, P.; Cairns, G. J. Modern Opt. 1994, 41, 1287-1294. S0003-2700(97)01166-9 CCC: $15.00
© 1998 American Chemical Society Published on Web 04/26/1998
Figure 1. Schematic diagram of the SPR-LDI-TOFMS system.
tion of their surfaces and stored under vacuum in a desiccator until needed. Chemicals. Gramicidin S was obtained from Sigma (St. Louis, MO), and methanol was an HPLC grade solvent purchased from Caledon (Georgetown, ON). All chemicals were used without further purification or treatment. Preparation of Gramicidin S on Thin Silver Film Substrates. A 10-µL aliquot of 4 mM gramicidin S solution in methanol was applied to a 2.5 cm × 2.5 cm thin silver film substrate. After air-drying, the thin silver film substrate was placed inside the ion source chamber of a TOFMS instrument for analysis. SPR Characterization. The SPR activity of thin silver film substrates was tested on a SPR sensing system using a red lightemitting diode source, a polarizer, a glass prism in the Kretschman geometry, and a photodiode array detector. SPR-LDI-TOFMS System. SPR-LDI experiments were performed on a linear TOFMS instrument constructed in our laboratory, as illustrated in Figure 1. The polarized 532-nm beam of a Nd-HyperYAG laser (Lumonics, Kanata, ON, Canada), operating at a repetition rate of 1 Hz, was directed toward a prism mounted on a rotation/translation stage. The laser beam was next sent into a glass prism which was coupled to a Plexiglas window of the ion source chamber with diffusion pump oil, to accommodate a Kretschmann geometry for SPR-LDI. A thin silver film substrate was oil-coupled to the other side of the Plexiglas window, directing the silver film surface toward the field-free drift tube of the TOFMS. A +20-kV high-voltage connection was made to the silver film which acted as the repeller grid a. A ground potential was held at a distance of 2 cm away on grid b. The TOFMS was
pumped down to a base pressure of 5 × 10-7 Torr using a vapor diffusion pump (Edwards EO4 Speedivac, Manor Royal Crawley, Sussex, U.K.). Using laser fluences ranging from 50 to 70 mJ/ cm2, the laser beam was incident at a variety of angles (θ) on the thin silver film substrate. The ions produced by LDI were accelerated toward grid b and flew through a 55-cm field-free drift tube to strike a dual-microchannel plate detector with a front-face voltage of -1.6 kV. The detector signal was fed into 50-Ω coupling of a 300-MHz LeCroy digital storage oscilloscope (DSO), which was triggered by a fast photodiode signal generated by a branch of the laser beam. Deformation on the backside of the silver film was visually observed while the laser fluence was increased, to the point where laser irradiation of the silver film substrate induced melting. This laser fluence was then measured and used to record all subsequent mass spectra. Each mass spectrum was recorded as the sum-average of over 50 shots on a single spot for a given angle of incidence, θ. This mass spectrum was stored onto a 3.5-in. floppy disk for data processing. After the experiment, the SPR-LDI-induced damage on the thin silver film substrates was examined by a change in the He-Ne laser light absorption within the deformed spot. RESULTS AND DISCUSSION One requirement for SPR-LDI-TOFMS experiments is SPR activity of the thin silver film substrate. A dip in the angular profile of reflected light intensity at a specific SPR angle, as shown in Figure 2, lessens SPR activity in the thin silver film substrate. Upon deposition of gramicidin S onto the thin silver film substrate, SPR activity lessens as indicated by a higher reflected light Analytical Chemistry, Vol. 70, No. 11, June 1, 1998
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Figure 2. SPR angular profile of (a) a thin silver film substrate and (b) gramicidin S deposited on a thin silver film substrate, obtained using a red light source.
Figure 3. Typical SPR-LDI-TOFMS spectrum of a thin silver film substrate.
intensity for the dip, which is accompanied by a small shift in the SPR angle. SPR-LDI-TOFMS spectra of a thin silver film substrate typically consist of peaks corresponding to desorbed silver monomer and cluster cations, Agn+ (1 e n e 3), as shown in Figure 3. The other two peaks correspond to sodium and calcium cations which most likely originate from the microscope slide support. Usually, the silver monomer cation at 4.06 µs dominates the spectrum, and the 5.76-µs silver dimer cation abundance is lower than the 7.06-µs silver trimer cation abundance. The reverse occurs for the mass spectrum of gramicidin S deposited on a thin silver film substrate, where the silver dimer cation abundance is greater than the silver trimer cation abundance, as shown in Figure 4. More importantly, the abundant 1141 amu gramicidin S molecular cation at 13.26 µs indicates successful SPR-LDI of gramicidin S which accompanies the SPR-LDI of the underlying silver. Since a second laser is not used for postionization of desorbed neutral gramicidin S molecules and silver clusters, the cation intensities in these mass spectra are relatively weak. Three processes that occur in SPR-LDI are deformation, desorption, and ionization. Deformation yields slight abnormalities in the thin silver film substrate, desorption produces an ablation hole, and ionization generates cation peaks in TOFMS spectrum. First, deformation within the silver film structure is 2362 Analytical Chemistry, Vol. 70, No. 11, June 1, 1998
Figure 4. Typical SPR-LDI-TOFMS spectrum of gramicidin S deposited on a thin silver film substrate.
visually inspected while the laser energy is slowly increased. The minimum laser fluences required for silver deformation at various angles of incidence are shown in Figure 5. The minimum laser fluence is found to be the lowest (33 ( 3 mJ/cm2) at the SPR angle of ∼48.5°. This result is in good agreement with independent photoacoustic spectroscopy (PAS) measurements which produce a maximum signal amplitude at the same angle. Apparently, SPR assists in the deformation of thin metal film substrates via electronic excitation.13 SPR activity in the thin silver film substrate is confirmed with the application of a +20 kV high voltage. The applied voltage does not seem to hinder the collective electron density oscillations along the film surface. Upon deposition of gramicidin S, the minimum laser fluence necessary for silver deformation is generally greater (48-73 mJ/cm2), but no lowest value can be found at any specific angle of incidence in Figure 5. This is expected because absorption of p-polarized 532nm laser light by the thin silver film substrate under SPR conditions is decreased, in accordance with Figure 2. By comparison, back illumination without SPR requires a greater minimum laser fluence of 67 ( 1 mJ/cm2 to yield silver deformation for thin silver film substrates and 76 ( 2 mJ/cm2 for gramicidin S deposited on the same thin silver film substrates. Next, the visually deformed spots on the silver film substrate are inspected for desorption. Desorption yields an ablation hole in the thin silver film substrate, which is found at the SPR and adjacent angles. A measurement of desorption efficiency is the change in He-Ne laser light absorption by the silver film substrate within each deformed spot. Figure 6 plots the silver desorption (in absorbance units) versus the angle of incidence for the p-polarized 532-nm laser beam. The plot indicates that maximal silver desorption also occurs at the SPR angle. A similar analysis of desorption efficiency for the thin silver film substrate with deposited gramicidin S indicates a smaller degree of silver desorption in Figure 6, again in accordance with its lower SPR activity. These results support the SPR-enhanced desorption of thin aluminum film substrates via electronic excitation first reported by Lee et al.13 The present desorption angular profiles coincide with the SPR angular profile measured by PAS, which can be attributed to a greater amount of energy being absorbed by the thin silver film substrate at the SPR angle. The absorbed
Figure 5. Minimum laser fluence for deformation of a thin silver film substrates with and without gramicidin S deposited at various angles of incidence (left). PAS signal of a thin silver film substrate at various angles of incidence (right).
Figure 6. Silver desorption (AU) for a thin silver film substrate with and without gramicidin S deposited at various angles of incidence (left). PAS signal of a thin silver film substrate at various angles of incidence (right).
energy is presumably transferred to bond breaking in the thin silver film substrate.
The validity of SPR in the enhanced laser desorption of thin silver film substrates is confirmed by a comparison between Analytical Chemistry, Vol. 70, No. 11, June 1, 1998
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Figure 7. Variations of the ablation hole size, measured through the mass of the ablated hole, for a thin silver film substrate for a s-polarized and a p-polarized 532-nm laser beam. Figure 9. Variations of the fwhm of the silver monomer, dimer, and trimer cluster cation peaks using p-polarized 532-nm laser light at various angles of incidence.
Figure 8. Variations of (a) silver monomer signal intensities and (b) gramicidin S signal intensities at various angles of incidence.
s-polarized and p-polarized 532-nm laser light. At constant laser fluence, no ablation hole due to silver desorption can be readily produced at any angle of incidence in the range of 38-51° using s-polarized light. In contrast, an ablation hole is distinctly formed at the SPR angle of ∼47.5° using p-polarized light, as shown in Figure 7. Last, LDI of thin silver film substrates with and without gramicidin S deposition is investigated by TOFMS. With a constant laser fluence of 55 ( 4 mJ/cm2 (higher than the minimum required), mass spectra could be obtained at various angles of incidence, both inside and outside the SPR range. Using the silver monomer and gramicidin S molecular cation peaks, the largest intensities occur at their corresponding SPR angles, as shown in Figure 8. These results fully confirm SPR enhancement of the LDI process, for the first time in the case of gramicidin S (1141 amu). Note that the gramicidin S molecules do not undergo desorption in the strict sense the word. Rather, the silver film decomposes under laser irradiation (and ablation) so that the substrate surface for adsorption of the gramicidin S molecules disappears. This capability to perform SPR-LDI on a larger molecular weight analyte represents a new milestone beyond the previous achievement with rhodamine B (479 amu). By comparison, conventional front-illumination LDI-TOFMS requires a minimum laser fluence of 50 ( 2 mJ/cm2 for thin silver film substrates 2364 Analytical Chemistry, Vol. 70, No. 11, June 1, 1998
and 100 ( 5 mJ/cm2 for gramicidin S deposited on thin silver film substrates. SPR-LDI thus requires a lower minimum laser fluence, which is an important benefit because ions produced by a decreased laser fluence usually have a lower energy distribution.11 Furthermore, the SPR-LDI yield increases linearly with laser fluence from 35 to 80 mJ/cm2. A better understanding of the SPR mechanism is gained in three aspects. First, silver is a better thin film substrate than aluminum in producing a stronger gramicidin S molecular cation signal (∼125 times). Second, laser ablation in the present desorption process vaporizes the thin silver film substrate on a mesoscopic scale, which forms a larger plume of ejected material than the conventional microscopic-scale LDI. Last, gas-phase ion/ molecule reactions in the large ablation plume can have a significant degree of chemical ionization, producing abundant gramicidin S molecular cations without the instrumental complexity of multiphoton postionization. The full width at half-maximum (fwhm) of cation peaks observed in the mass spectrum reflects on not only the resolution of the instrument but also the kinetic energy distribution. Silver cluster cation peaks exhibit drastically different fwhm at various angles of incidence, as shown in Figure 9. At the SPR angle, the fwhm of the silver dimer and silver trimer cation peaks deviate the most from their regular fwhm. Specifically, the silver trimer cation peak becomes 1.5 times broader, while the silver dimer cation peak becomes 1.5 times narrower. The fwhm (70 ( 7 ns) of the silver monomer cation peak does not seem to be affected by the SPR angle, although this peak becomes slightly narrower at larger angles of incidence. Similarly, the gramicidin S cation peak varies only slightly in fwhm (150 ( 20 ns) with different angles of incidence. These fwhm results indicate that the absorption of p-polarized 532-nm laser light energy by the thin silver film substrate under SPR conditions does not significantly affect the mass resolution in analytical applications. CONCLUSIONS The absorption of p-polarized 532-nm laser light by thin silver film substrates at the SPR angle assists in the desorption and
ionization of silver clusters and gramicidin S molecules. This is the first successful demonstration of SPR-LDI-TOFMS analysis on a biomolecule with a molecular weight greater than 1000 amu. The analysis requires at least 2 times lower minimum laser fluence than the conventional front-illumination LDI-TOFMS approach. No significant fragmentation of molecular ions or deterioration of mass spectral resolution occurs. One potential benefit from these performance merits would be direct mixture analysis of medium-molecular-weight peptides by SPR-LDI-TOFMS. Recent advent of matrix-assisted laser desorption/ionization (MALDI) has afforded a host of mass spectrometric methods for the analysis of gramicidin S and high-molecular-weight biomolecules. Under SPR excitation conditions, the addition of a matrix is offering an even lower laser fluence requirement for the abundant formation
of pseudomolecular gramicidin S cations in our latest experiments. Research is continuing in our laboratory to further develop this new technique (SPR-MALDI-TOFMS) for enhanced analysis of high-mass biomolecules. ACKNOWLEDGMENT This work was funded by the Natural Sciences and Engineering Research Council of Canada. The authors thank Michel Grenier and Wayne Mullett for their assistance in the SPR-LDI-TOFMS construction. Received for review October 22, 1997. Accepted March 19, 1998. AC971166U
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