Anal. Chem. 2006, 78, 5876-5883
Vacuum Ultraviolet Postionization of Aromatic Groups Covalently Bound to Peptides Praneeth D. Edirisinghe,† Jerry F. Moore,‡ Wallis F. Calaway,‡ Igor V. Veryovkin,‡ Michael J. Pellin,‡ and Luke Hanley*,†
Department of Chemistry, University of Illinois at Chicago, Chicago, Illinois 60607, and Materials Science Division, Argonne National Laboratory, Argonne, Illinois 60439
Experiments demonstrate that peptides with ionization potentials (IPs) above 7.87 eV can be single-photonionized in the gas phase with a molecular fluorine laser following prior chemical derivatization with one of several aromatic tags acting as chromophores. 4-(Dimethylamino)benzoic acid, 1-naphthylacetic acid, and 9-anthracenecarboxylic acid (denoted Benz, Naph and Anth, respectively) behave as chromophores, allowing single-photon ionization for vacuum ultraviolet (VUV) laser light by lowering the IP of the tagged peptide. Anth-tagged peptides that are laser-desorbed from a substrate and subsequently postionized produce mass spectra dominated by the intact radical cation, although protonated ions and fragmented species are also observed. Electronic structure calculations on Anth-tagged peptides indicate that in addition to lowering the ionization potential, the presence of the aromatic tag increases charge localization on and delocalization across the ring structure, which presumably stabilizes the radical cation. Measurements on several tagged peptides confirm this calculation and show that the stabilizing effect of the tag increases with the size of the conjugated system in the order Benz < Naph < Anth. The tagged hexapeptide Anth-GAPKSC exhibits the parent ion, whereas the Benz- and Naph-tagged peptides do not. These results are supported by the experimental comparison of Anth-tagged vs untagged tryptophan, further suggesting that VUV postionization of tagged high-IP species is a promising method for expanding the capabilities of mass spectrometric analyses of molecular species. An important direction for mass analysis is in situ molecular imaging of biological samples and other complex organic materials.1-3 This area presents specific problems for secondary ion mass spectrometry, matrix-assisted laser desorption/ionization (MALDI) mass spectrometry, and related techniques due to constraints of sample preparation, spectral complexity (i.e., chemi* Corresponding author. E-mail:
[email protected]. † University of Illinois at Chicago. ‡ Argonne National Laboratory. (1) Todd, P. J.; Schaaff, T. G.; Chaurand, P.; Caprioli, R. M. J. Mass Spectrom. 2001, 36, 355. (2) Ostrowski, S. G.; Van Bell, C. T.; Winograd, N.; Ewing, A. G. Science 2004, 305, 71. (3) Rubakhin, S. S.; Jurchen, J. C.; Monroe, E. B.; Sweedler, J. V. Drug Discovery Today 2005, 10, 823.
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cal noise), and often low yields of intact analyte ions. A promising alternate analysis technique is postionization of neutral molecules by a second laser pulse fired a microsecond or more after their initial desorption by a first laser pulse. Laser ionization of molecules falls into two broad areas, multiphoton ionization (MPI) and single photon ionization (SPI). SPI has not been used as extensively as MPI because it requires utilizing vacuum ultraviolet (VUV) light sources with photon energies above 10 eV for many molecules, and such sources are relatively uncommon. VUV postionization of neutrals has been observed for gaseous molecular species produced during evaporation, thermal desorption, laser desorption, and keV ion sputtering.4-16 SPI of any gaseous neutral is possible if its ionization potential (IP) is below the photon energy of the light source. SPI can be a general detection method for molecules both because the cross sections for many species are similar and because SPI does not depend on absorptive intermediate states. The relatively high cross sections of SPI also more readily allows saturation of the ionization process, yielding higher sensitivity and facilitating quantification. SPI can control the excess energy left in the photoion and generally produces less fragmentation than other ionization methods. Threshold SPI, using photon energies just above a molecular IP, can further reduce fragmentation by minimizing the internal energy of the photoion that is a radical cation. Tunable VUV sources are required for threshold SP,I14,16,17 since the IP of (4) Van Bramer, S. E.; Johnston, M. V. J. Am. Soc. Mass Spectrom. 1990, 1, 419. (5) Schlag, E. W.; Levine, R. D. J. Phys. Chem. 1992, 96, 10608. (6) Becker, C. H.; Wu, K. J. J. Am. Soc. Mass Spectrom. 1995, 6, 883. (7) de Vries, M. S.; Hunziker, H. E. J. Photochem. Photobiol., A 1997, 106, 31. (8) Hanley, L.; Kornienko, O.; Ada, E. T.; Fuoco, E.; Trevor, J. L. J. Mass Spectrom. 1999, 34, 705. (9) Muhlberger, F.; Wieser, J.; Ulrich, A.; Zimmerman, R. Anal. Chem. 2002, 74, 3790. (10) Sykes, D. C.; Woods, E. I.; Smith, G. D.; Baer, T.; Miller, R. E. Anal. Chem. 2002, 74, 2048. (11) King, B. V.; Pellin, M. J.; Moore, J. F.; Veryovkin, I. V.; Tripa, C. E. Appl. Surf. Sci. 2003, 203/204, 244. (12) Syage, J. A. J. Am. Soc. Mass Spectrom. 2004, 15, 1521. (13) Oktem, B.; Tolocka, M. P.; Johnston, M. V. Anal. Chem. 2004, 76, 253. (14) Jochims, H.-W.; Schwell, M.; Chotin, J.-L.; Clemino, M.; Dulieu, F.; Baumga¨rtel, H.; Leach, S. Chem. Phys. 2004, 298, 279. (15) Finch, J. W.; Toerne, K. A.; Schram, K. H.; Denton, M. B. Rapid Commun. Mass Spectrom. 2005, 19, 15. (16) Wilson, K. R.; Jimenez-Cruz, M.; Nicolas, C.; Belau, L.; Leone, S. R.; Ahmed, M. J. Phys. Chem. A 2006, 110, 2106. (17) Veryovkin, I. V.; Calaway, W. F.; Moore, J. F.; Pellin, M. J.; Lewellen, J. W.; Li, Y.; Milton, S. V.; King, B. V.; Petravic, M. Nucl. Instrum. Methods Phys. Res., Sect. B 2004, 219-220, 473. 10.1021/ac0605997 CCC: $33.50
© 2006 American Chemical Society Published on Web 07/19/2006
Scheme 1. Aromatic Tags Used for Chemical Derivatization
molecules can vary greatly, but widely tunable high-intensity VUV radiation is only available at synchrotron light sources. The fixed frequency molecular fluorine laser generating 7.87 eV photons (corresponding to a vacuum wavelength of 157 nm) is one of the most convenient laboratory sources of VUV radiation. This laser is perhaps the only laboratory VUV source of sufficient intensity to saturate SPI of molecular species in >1 mm3 volumes; however, the 7.87-eV photon energy of the fluorine laser lies below the IP of many interesting molecular analytes.11 Recent work has demonstrated the utility of the fluorine laser for photodissociation of peptide ions.18 The work reported herein explores the use of the fluorine laser for SPI of desorbed neutral peptides in an effort to minimize photodissociation. An alternate strategy to threshold SPI was previously demonstrated whereby a chemical tag on a molecular species serves as a chromophore that lowers the overall IP of the tagged molecular complex and allows postionization at 7.87 eV of species that are otherwise difficult to detect.19 This IP-lowering effect was shown for Fmoc-derivatized di- and tetrapeptides covalently bound to Si surfaces. Previous work6,20-22 suggested that other aromatic chemical tags in addition to Fmoc should also permit VUV postionization. Carboxylic acid-functionalized naphthalene22 and anthracene,21 as well an aminobenzoic acid and tryptophan, are used in the present work to test the general idea that many aromatics can function as VUV chromophores for chemical derivatization and to extend the application of VUV tags to highIP peptides. Recent work has also examined the use of aromatic labeling of high-IP molecules to facilitate their analysis by mutiphoton ionization.23 The three compounds used as tags here are shown in Scheme 1: 4-(dimethylamino)benzoic acid (denoted Benz), 1-naphthylacetic acid (Naph), and 9-anthracenecarboxylic acid (Anth). Each tag possesses a different number of aromatic rings from one to three, respectively, which are functionalized by a carboxylic acid group. These molecules were selected as tags because it was anticipated that they would reduce the IP of the tagged peptides below the photon energy of the molecular fluorine laser. This paper describes both laser desorption postionization mass spectrometry (LDPI-MS) experiments and electronic structure calculations that show 7.87-eV SPI of analytes can be achieved by their chemical derivatization with the Benz, Naph, and Anth (18) Thompson, M. S.; Cui, W.; Reilly, J. P. Angew. Chem., Int. Ed. 2004, 43, 4791. (19) Edirisinghe, P. D.; Lateef, S. S.; Crot, C. A.; Hanley, L.; Pellin, M. J.; Calaway, W. F.; Moore, J. F. Anal. Chem. 2004, 76, 4267. (20) Anex, D. S.; de Vries, M. S.; Knebelkamp, A.; Bargon, J.; Wendt, H. R.; Hunziker, H. E. Int. J. Mass Spectrom. Ion Processes 1994, 131, 319. (21) Srinivasan, J. N.; Romano, L. J.; Levis, R. J. J. Phys. Chem. 1995, 99, 13272. (22) Houston, C. T.; Reilly, J. P. J. Phys. Chem. A 2000, 104, 10383. (23) Fernandes-Whaley, M.; Muhlberger, F.; Whaley, A.; Adam, T.; Zimmermann, R.; Rohwer, E.; Walte, A. Anal. Chem. 2005, 77, 1.
aromatic tags behaving as VUV chromophores. The electronic structure calculations show that the Anth tag acts not only to lower the IP but also to delocalize charge across the Anth moiety of the radical cation, stabilizing the large, intact parent ions of derivatized peptides. Furthermore, increasing the charge delocalization of the radical cation through increasing the conjugation size of the aromatic tag is shown to allow stabilization of higher molecular weight radical cations, thereby reducing fragmentation. It is also found that proton transfer contributes to the VUV postionization event to varying degrees, depending upon the matrices (or lack thereof) from which the analytes are laserdesorbed. Finally, the potential for combining derivatization with VUV LDPI-MS for applications that are difficult to solve with present analytical techniques is discussed, although more work will clearly be required to develop this as a viable quantitative tool. EXPERIMENTAL DETAILS Samples are analyzed using a LDPI-MS instrument designed and built at Argonne National Laboratory.24,25 The instrument consists of a vacuum system with a base pressure of 10-9 mbar containing a pulsed ion source (Atomika, WF421 S2), a molecular fluorine laser (157 nm, GAM, EX100/F) and a time-of-flight mass spectrometer with an integrated Schwarzchild microscope for optical imaging and laser desorption. Desorption of sample material can be performed with a pulsed nitrogen laser (337 nm, Spectra Physics, VSL-337ND) focused through the coaxial microscope to an ∼5 µm spot on the sample. Alternately, the sample can be sputtered with a microfocused ion beam directed at 60° from the sample normal. The fluorine laser, with vacuum transport and focusing, is arranged to pass a 10-ns pulse of 157-nm light in front of the sample a few microseconds after the desorption laser pulse to photoionize the neutral flux emitted from the sample. The 157-nm beam has a 4 × 2 mm cross section above the target, with ∼0.6 mJ/pulse, resulting in a peak fluence of ∼7 × 105 W cm-2. The 157-nm beam position ranges from 1 to 4 mm above and parallel to the surface. It is possible to study the power dependence of either the desorption or photoionization processes by attenuating the flux of the lasers with neutral density filters, allowing the power range for either process to be varied by 3 orders of magnitude. The nominal kinetic energy of the extracted photoions is typically 1.5 keV, and the total flight path through the reflectron is ∼3.5 m. The maximum achieved resolving power is 2500 (m/∆m), with a mass range up to an m/z of 104 and a mass accuracy of ∼100 ppm. The most remarkable feature of this instrument is its sensitivity, which is derived from its high useful yield of ∼25% (ratio of detected ions to species removed from the sample, calculated for ion sputtered atoms measurements).26 High sensitivity and high useful yield are critical when dealing with samples that contain very small and finite amounts of analyte, such as the case when performing monolayer analyses or obtaining molecule-specific surface images at the micrometer scale. However, to obtain this high useful yield, a large volume (a cube of (24) Veryovkin, I. V.; Calaway, W. F.; Pellin, M. J. Nucl. Instrum. Methods Phys. Res., Sect. A 2004, 519, 353. (25) Veryovkin, I. V.; Chen, C.-Y.; Calaway, W. F.; Pellin, M. J.; Lee, T. Nucl. Instrum. Methods Phys. Res., Sect. A 2004, 519, 345. (26) Veryovkin, I. V.; Calaway, W. F.; Tripa, C. E.; Moore, J. F.; Wucher, A.; Pellin, M. J. Nucl. Instrum. Methods Phys. Res., Sect. B 2005, 241, 356360.
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up to 4 × 4 × 4 mm) of ions of different kinetic energies and directions must be collected and refocused in time and space through the ion optics that constitute the time-of-flight analyzer. In practice, this cannot be done perfectly and compromises the mass resolution to some extent. Alternately, mass resolution can be improved, but at the sacrifice of useful yield (and thus, instrument sensitivity). For the work presented here, unit mass resolution up to an m/z of 800 is required, so the instrument is optimized above that level. Peptides are synthesized using Fmoc-bound solid-phase synthesis (UIC Research Resources Center),27 then derivatized according to the following procedure. A 100 mM solution of the derivatizing reagent is added to the bead-bound peptide whose N-terminal Fmoc has been previously removed.27 An N,N,-dimethylformamide (DMF) solution that is 100 mM in O-benzotriazoleN,N,N′,N′-tetramethyluroniumhexafluorophosphate (HBTU, GL Biochem, Shanghai, China) and 400 mM in n-methyl morpholine is added to the resin and derivatizing reagent, then allowed to react for 1 h. The resin is washed with DMF and cleaved with 5 mL of trifluoroacetic acid, 50 µL of water, 50 µL of tri-isopropyl silane, and 50 µL of ethane dithiol for 4 h. The derivatized peptide is then precipitated using ethyl ether, lyophilized in 50:50 acetonitrile and water, and purified by high performance liquid chromatography. The derivatization process is confirmed by matrix assisted laser desorption ionization (MALDI) mass spectrometry in a commercial instrument. Samples are prepared for the postionization experiments by dissolution in a 1:1 volume ratio of water/acetonitrile solution (with no trifluoroacetic acid), then 2-5 µL of this solution is deposited as a drop that is allowed to air-dry on an aluminum sample plate. For some experiments, an aliquot of the above solution is first mixed 1:1 by volume with a saturated R-cyano-4-hydroxycinnamic acid solution before being deposited in the same manner. The amount of sample contained in each dried droplet is typically ∼10 pmol. The mass scale of the reported data is calibrated by several means. Gold sputtered by 15 keV Ar+ and postionized with the 157-nm beam provides a convenient set of masses ranging from m/z 197.0 (Au+) to m/z 984.9 (Au5+), allowing for an initial multipoint calibration. Further refinement of the calibration is then made using commercial MALDI peptide mass calibration standards (Sigma Aldrich) and prepared as specified by the manufacturer. Although there is some variation of flight time between experiments due to adjustment of potentials and sample position, the instrument calibration is checked before and after each experiment to verify the mass accuracy of the reported spectra. The maximum observed variation is no more than m/z 0.5 for singly charged ions in the mass range near m/z 800, which is acceptable for the reported study. Single point energy and geometrical optimization calculations are performed using Gaussian 9828 at the B3LYP/6-31G level, and the HOMO orbital is plotted using the Cerius2 graphics package (v. 3.5, Molecular Simulations Inc., San Diego). The geometry of the neutral molecule is optimized, then the vertical ionization potential (fixed geometry) is calculated from the energy difference between the ionized and neutral molecules. (27) Chan, W. C.; White, P. D. Fmoc Solid-Phase Peptide Synthesis: A Practical Approach; Oxford University Press: New York, 2000.
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Figure 1. 337-nm laser desorption, 157-nm (7.87 eV) postionization mass spectra (LDPI-MS) of Benz-, Naph-, and Anth-tagged GAPKSC peptide, prepared with R-cyano-4-hydroxycinnamic acid (CHCA) matrix. Spectra normalized to optimize appearance of significant peaks.
RESULTS AND DISCUSSION Laser Desorption Postionization Mass Spectrometry. Mass spectrometric measurements are carried out on tagged and untagged peptides and amino acids laid down on aluminum surfaces both neat and in a matrix. Figure 1 shows the mass spectra obtained when the GAPKSC hexapeptide is tagged with Benz, Naph, and Anth, incorporated into an R-cyano-4-hydroxycinnamic acid (CHCA) matrix, and analyzed by 7.87-eV LDPI-MS. Figure 1 shows that the Anth-GAPKSC-tagged hexapeptide yields a large signal at the intact parent ion (m/z ) 766), whereas the highest-mass ions observed for Benz-GAPKSC and NaphGAPKSC appear below the parent ion mass (at m/z 708 and 730, respectively). When examined at higher gain, both the Benz- and Naph-tagged peptide spectra are seen to contain high molecular weight fragments that are not observed when no tag is attached to the peptide. It was previously shown that Anth-KRSTC-tagged peptides also display a strong parent ion signal from LDPI-MS analysis using 7.87 eV postionization.29 Various control experiments are performed to confirm that the observed ion signal results from VUV SPI and not MALDI or some other process. Measurements under conditions similar to those used for Figure 1 but on untagged GAPKSC produce few or no significant peaks above m/z ∼ 300 (data not shown). Furthermore, no peaks above m/z ∼ 300 for the various tagged peptides are observed unless both the fluorine laser and the desorption laser (28) Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.; Robb, M. A.; Cheeseman, J. R.; Zakrzewski, V. G.; Montgomery, J. A., Jr.; Stratmann, R. E.; Burant, J. C.; Dapprich, S.; Millam, J. M.; Daniels, A. D.; Kudin, K. N.; Strain, M. C.; Farkas, O.; Tomasi, J.; Barone, V.; Cossi, M.; Cammi, R.; Mennucci, B.; Pomelli, C.; Adamo, C.; Clifford, S.; Ochterski, J.; Petersson, G. A.; Ayala, P. Y.; Cui, Q.; Morokuma, K.; Malick, D. K.; Rabuck, A. D.; Raghavachari, K.; Foresman, J. B.; Cioslowski, J.; Ortiz, J. V.; Stefanov, B. B.; Liu, G.; Liashenko, A.; Piskorz, P.; Komaromi, I.; Gomperts, R.; Martin, R. L.; Fox, D. J.; Keith, T.; Al-Laham, M. A.; Peng, C. Y.; Nanayakkara, A.; Gonzalez, C.; Challacombe, M.; Gill, P. M. W.; Johnson, B. G.; Chen, W.; Wong, M. W.; Andres, J. L.; Head-Gordon, M.; Replogle, E. S.; Pople, J. A. Gaussian 98, revision A.9; Gaussian, Inc.: Pittsburgh, PA, 1998. (29) Hanley, L.; Edirisinghe, P. D.; Calaway, W. F.; Veryovkin, I. V.; Pellin, M. J.; Moore, J. F. Appl. Surf. Sci. 2006, in press.
Table 1. Peak Assignments for Tagged GAPKSC 7.87-eV LDPI-MS Displayed in Figure 1, Using Standard Notation for Peptide Fragmentation,30 but also Including Attached Tags molecule (mass)
mass peak (m/z)
identification
Anth-GAPKSC (765 Da)
765 617 645 530 665 660 588 560 501 473 581 494
M+• a4 + Anth b3 + Anth a3 + Anth (M - N(CH3)2)+ impurity b4 + Benz a4 + Benz b3 + Benz a3 + Benz a4 + Naph a3 + Naph
Benz-GAPKSC (708 Da)
Naph-GAPKSC (708)
are present, confirming the separation of the desorption and photoionization processes. Intensities of peaks at lower masses (m/z < 300) in the absence of the postionization laser are very low and are difficult to identify, despite the presence of the CHCA matrix (i.e., there are few MALDI ions formed under single laser desorption conditions here, and none when no matrix is added, as shown below). This is expected for LDPI-MS, since low desorption laser intensities are used to maximize neutral desorption (typically, 600 K internal energy, yet the major peak observed is the m/z 408 parent ion. A 600 K temperature for tryptophan corresponds to an internal energy in excess of 1 eV, according to published ab initio calculations.16 The internal Analytical Chemistry, Vol. 78, No. 16, August 15, 2006
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energy will, therefore, be somewhat larger for Anth-tryptophan laser desorbed under the same conditions, given its greater number of degrees of freedom. The data in Figure 4 show that the presence of the Anth tag on tryptophan clearly suppresses fragmentation, despite the high internal energy of the Anthtryptophan neutral. The strong parent ion for Anth-tryptophan can, therefore, be taken as evidence of charge stabilization on the Anth moiety following SPI, which is further supported by the appearance of the m/z 205 and 220 ions, which both contain the Anth moiety (see Figure 4). To observe large intact parent ions, soft photoionization, such as SPI, is necessary but not sufficient. In many cases, the photoionization process creates an unstable ion that fragments.6 By attaching a tag such that the highest molecular orbital of the new molecule is localized on the tag, fragmentation can be reduced or eliminated in at least some cases. An aromatic tag behaving as a chromophore for VUV photoionization is a general but often unrecognized phenomenon that is underappreciated and has yet to be widely exploited for MS analyses. The work here has shown that Benz, Naph, and Anth behave in this fashion. Work to be published will demonstrate that Benz tagging of bisphenol-functionalized silane self-assembled monolayers by 7.87-eV LDPI-MS reduces fragmentation when compared to 10.5-eV LDPI-MS of the untagged counterpart.38 Tryptophan also behaved as a localized chromophore in peptides such as KRSTCW, GGW, and GGWGG when using 7.87-eV postionization.29 By contrast, the similar peptide sequences KRSTC and GGG without tryptophan did not undergo 7.87-eV photoionization. It could be argued that the appearance of the m/z 130 methylene indole fragment for Anth-tryptophan in Figure 4 results from SPI of the methylene indole ring, in competition with SPI of the Anth group. The common fluorescence tag fluorescein also served as a postionization tag in 7.87-eV LDPI-MS,29 and it is expected that the many other fluorescence tags whose ionization potentials are below 7.87 eV will behave similarly. A selection of aromatic compounds with an amine side group (similar to the Benz tag) or fused ring systems (similar to the Naph tag) have been selectively photoionized intact using 7.75-eV energy photons from mixtures.15 Phenol groups on perfluoropolyethers up to 7 kDa behaved as chromophores and permitted multiphoton ionization with minimal fragmentation.20 Anthracene, naphthalene, and other aromatic tags have been shown to behave as chromophores for MPI of nucleotides, peptides, and other analytes.21-23 However, naphthalene tagging only led to the formation of intact ions for internally cooled tripeptides, whereas larger peptides fragmented.22 Use of the Anth or other larger tags and VUV SPI should reduce fragmentation in many cases. CONCLUSIONS These results have significant implications for mass spectrometric analysis of molecular species. The high sensitivity of LDPIMS, which has been demonstrated for elemental analysis, has yet to be realized for molecular analysis. If the potential for molecular analysis could be realized, the high sensitivity of LDPI-MS could be used for molecular compositional imaging of surfaces. The difficulties that have limited LDPI-MS in the past are fragmentation, sensitivity, and selectivity. The use of tags has the potential
to address all of these problems. Tags that act as VUV chromophores allow a wide range of difficult to detect molecules to be single photon ionized with a high-intensity, fixed frequency VUV laser, thereby improving photoionization efficiency and limiting photofragmentation. In addition, if the tag molecule also serves to stabilize the parent ion, then large molecules can be detected intact with minimal fragmentation. Both of these improvements work in tandem to enhance sensitivity and eventually will aid in quantification by this method. Another application for 7.87-eV LDPI-MS with chemical derivatization would be identification of complex molecular species from mixtures without prior chromatographic separation. Chemical derivatization of an individual analyte or a group of analytes with an aromatic tag in a complex mixture will allow their selective detection by 7.87-eV LDPI-MS. Prior work with 7.75-eV postionization of aromatic pharmaceutical compounds permitted their selective detection from mixtures prepared by only a simple extraction rather than full chromatographic separation.15 The 7.87eV photons can be used to detect many organic species via bound tags, such as Anth or Benz, which serve to localize the initial VUV postionization event. Furthermore, this effect is not expected to be confined to the Benz, Naph, Anth, tryptophan, or Fmoc19 tags. VUV postionization is probably possible for many of the fluorescence probes utilized in optical spectroscopy and microscopy,39,40 allowing selective detection via VUV postionization combined with a wide range of established chemical derivatization techniques. Molecular species that are efficient fluorescence probes should also serve as tags for VUV postionization, since they display highest occupied molecular orbitals with extended π-conjugation, relatively low ionization potentials, and the ability to stabilize the net positive charge of the radical cations. A perfect example is that the common fluorescence tag fluorescein serves as a postionization tag in 7.87-eV LDPI-MS.29 VUV postionization is also more readily quantifiable, proceeds by fewer fluctuations in cross section, and induces less fragmentation than is generally observed for multiphoton ionization. Widely tunable VUV radiation,11 such as is available at some synchrotron light sources,14,16 may be a superior method for VUV postionization studies; however, such experiments require a trip to a synchrotron, which many experimentalists would prefer to and can avoid through the use of the commercially available 7.87-eV fluorine laser and the chemical derivatization techniques discussed here. This work demonstrates that single photon ionization of peptides with whose IPs exceed the photon energy is possible with the aid of an aromatic tag. It is shown here computationally that the aromatic group contains the charge in the radical cation, and if it is large enough, it can stabilize that cation, presumably by delocalizing the charge enough to minimize charged initiated fragmentation. This hypothesis is further supported by the tagging of tryptophan with Anth, which displayed an intense parent ion peak, as compared with the untagged amino acid, which fragmented extensively. Measured spectra of GAPKSC with and without matrix show that postionization is truly a dual-mechanism process, with both direct 7.87-eV single photon ionization producing M+• and secondary indirect photon-induced protonation accounting for a significant fraction of the (M + 1)+ ion signal.
(38) Zhou, M.; Wu, C.; Edirisinghe, P. D.; Drummond, J. L.; Hanley, L. J. Biomed. Mater. Res. 2006, 77A, 1.
(39) Craig, D. B.; Dovichi, N. J. Anal. Chem. 1998, 70, 2493. (40) Haugland, R. P. Handbook of Molecular Probes and Research Products, 9th ed.; Molecular Probes: Eugene, OR, 2004.
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The method described is not proposed as a general analytical tool for peptide analysis. Clearly, for femtomole and larger quantities of peptides, there are excellent routine methods, such as electrospray ionization and MALDI-MS. For example, it is wellknown that a set of matrix conditions can be found for the GAPKSC (or virtually any other intermediately sized) peptide that will allow its efficient detection by traditional MALDI with a single laser. It therefore makes little practical sense to use derivatization followed by VUV postionization to replace MALDI in such cases. Rather, the method presented here allows detection of analytes under circumstances that prove difficult by MALDI or other methods, such as secondary ion mass spectrometry. The larger implications of this work include ultratrace detection of specific peptides, self-assembled monolayers, and any other species for which methods are available that allow selective derivatization. Other biomolecules, including carbohydrates, lipids, and nucleic acids, may also be similarly tagged and postionized. Pharmaceutical compounds may be detected directly or following derivatization, as required. The sensitivity and low background made possible by the high useful yield of this instrument suggests its use for selected applications in imaging mass analysis. An estimate shows that at typical biological concentrations (∼1 ppm), an analyte such as
the hexapeptide analyte in this paper presents only 1000 molecules for analysis in a cubic micrometer of sample. Nonetheless, to fully develop this approach as an analytical microprobe, it is clear that further work is required to demonstrate quantitation and application to biological systems by either in situ derivatization (analogous to labeling with fluorescent tags for optical microscopy) or selective detection of low-IP analytes. Such work is progressing and will be reported separately. ACKNOWLEDGMENT This work is supported by the U.S. Department of Energy, BES-Materials Science, under Contract W-31-109-ENG-38. P.D.E. is supported by subcontract 5-KD73-P-00153-00 from Argonne National Laboratory. The authors acknowledge the assistance of Cindy Harwood in performing the Gaussian calculations. 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 March 31, 2006. Accepted June 15, 2006. AC0605997
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