Anal. Chem. 2004, 76, 6734-6742
MALDI Matrices for Biomolecular Analysis Based on Functionalized Carbon Nanomaterials Michael V. Ugarov,† T. Egan,† Dmitry V. Khabashesku,† J. Albert Schultz,† Haiqing Peng,‡ Valery N. Khabashesku,‡ Hiroshi Furutani,§ Kimberley S. Prather,§ H-W. J. Wang,| S. N. Jackson,| and Amina S. Woods*,|
Ionwerks, Inc., 2472 Bolsover, Suite 255, Houston, Texas 77005, Department of Chemistry and Center for Nanoscale Science and Technology, Rice University, Houston, Texas 77005, Department of Chemistry and Biochemistry, University of California, San Diego, San Diego, California 92093, Behavioral Nueroscience, NIDA IRP, NIH, 5500 Nathan Shock Drive, Baltimore, Maryland 21224
When used in small molar ratios of matrix to analyte, derivatized fullerenes and single wall nanotubes are shown to be efficient matrices for matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. The mixing of an acidic functionalized fullerene with a solution of bioanalyte, depositing a dried droplet, and irradiating with a pulsed nitrogen laser yields protonated or cationized molecular ions. Derivatized fullerenes could offer several advantages over conventional MALDI matrices: a high analyte ionization efficiency, a small molar ratios (less than 1) of matrix/analyte, and a broader optical absorption spectrum, which should obviate specific wavelength lasers for MALDI acquisitions. The major disadvantage to the use of fullerenes is the isobaric interference between matrix and analyte ions; however, it is overcome by using MALDI-ion mobility time-of-flight (IM-oTOF) mass spectrometry to preseparate carbon cluster ions from bioanalyte ions prior to TOF mass analysis. However, an alternative to the dried droplet preparation of fullerene MALDI samples is the aerosolization of matrix-analyte solutions (or slurries) followed by impacting the aerosol onto a stainless surface. We also demonstrate that the fullerene matrices can be used to acquire spectra from rat brain tissue. Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry of biomolecular ions was demonstrated by Tanaka, who used metal particles suspended in glycerol,1 and by Karas et al., who used organic compounds as matrices.2 In both cases, the matrix performs the dual function of absorbing the laser light and ionizing the non-light-absorbing biomolecules through specific yet poorly understood chemical reactions. The organic matrices have been more popular due to ease of use and because they can be * Corresponding author. Tel: 410-550-1507. Fax: 410-550-6859. E-mail: awoods@ intra.nida.nih.gov. † Ionwerks, Inc. ‡ Rice University. § University of California, San Diego. | NIDA IRP, NIH. (1) Tanaka, K.; Waki, H.; Ido, Y.; Akita, S.; Yoshida, Y.; Yoshida, T. Rapid Commun. Mass Spectrom 1988, 2, 151. (2) Karas, M.; Bachman, D.; Hillenkamp, F. Int J. Mass Spectrom. Ion Processes 1987, 78, 53.
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tailored to ionize a diverse range of biomolecules such as proteins, peptides, lipids, sugars, and DNA. However, dried droplet organic matrices almost always suffer a nonuniform cocrystallization of matrix and analyte, which causes analyte and matrix segregation into separate regions within the dried droplet. Attempts to use slurries of small particles as alternative MALDI matrices have languished in all but a few laboratories. Shurenberg has reviewed the literature and performed additional experiments,3 which have helped to establish the current understanding of these nanoparticulate matrices. In summary, for protein MALDI up to masses of ∼13 kDa, the particle/glycerol system gives identical spectra though with ∼10 times less sensitivity than organic matrices. Above this mass range, the particle slurry cannot compete with the performance of organic matrices. Any refractory particle seems to work including carbon nanosoot and titanium nitride as long as the particle size is significantly below 1 µm and glycerol is added. The first use of fullerenes for laser desorption of biomolecules involved applying the protein analyte solution directly onto the predeposited fullerene film.5,6 This technique suffered from low sensitivity and the presence of “hot spots” due to poor mixing of a nonpolar matrix and a polar analyte. However, the surface polarity of fullerenes can be significantly increased by derivatization with a variety of functional groups. C60 functionalized with (NHC(CH2OH)3)n, (C4H9SO3Na)n,7 and ((CH2)4SO3)68 have been shown to work as matrices for several amino acids, peptides, and proteins. High nanomole concentrations of these soluble fullerenes were used either to mix with the analyte solutions or to precipitate specific molecular fractions from mixtures by binding with derivatized fullerenes. A detection limit in the low-picomole range was estimated. (3) Schurenberg, M.; Dreisewerd, K.; Hillenkamp, F. Anal. Chem. 1999, 71, 221-229. (4) Taylor, R. Lecture Notes on Fullerene Chemistry: A Handbook for Chemists; Imperial College Press: London, 1988. (5) Michalak, L.; Fisher, K.; Alderdice, D.; Willet, G. Rapid Commun. Org. Mass Spectrom. 1994, 29, 512. (6) Hopweed, F.; Michalak, L.; Alderdice, D.; Fisher, K.; Willet, G. Mass Spectrom. 1994, 8, 881. (7) Huang, J.; Wang, L.; Chiang, L.; Shiea, J. J. Fullerene Sci. Technol. 1999, 7, 541. (8) Shiea, J.; Huang, J.; Teng, C.; Jeng, J.; Wang, L.; Chiang, L. Anal. Chem. 2003, 75, 3587. 10.1021/ac049192x CCC: $27.50
© 2004 American Chemical Society Published on Web 09/29/2004
Figure 1. MALDI-IM OTOF MS diagram.
In recent years, advances in fullerene-based chemistry9 have resulted in applications in multiple areas including electronic devices, sensors, and polymers. Since simple ways to derivatize fullerenes and make them water-soluble are now available, multiple biological applications are possible.10 Fullerene surfaces can be derivatized with hydroxyl or acid groups, thus providing a strongly physisorbing surface area, some water solubility, and a source of protons for chemical ionization. Furthermore, although the chemistry of single wall carbon nanotubes is still in its infancy, several methods for sidewall surface functionalization using organic groups ending with COOH, OH, and NH2 moieties have recently been developed.11-13 The fullerene MALDI matrix efficiently absorbs wavelengths other than 337 nm, so the fullerenebased matrices should allow the use of longer wavelength excitation sources including visible range lasers (although we do not specifically investigate this possibility in this work). However, a well-known disadvantage to the use of particulate matrices is the multiplicity of low- and high-mass interferences originating from the particle matrix, which can mask analyte signal. Fortunately, this problem is efficiently solved by recent instrumental advances coupling MALDI with ion mobilityorthogonal-time-of-flight mass spectrometry (MALDI-IM-oTOF MS),14 as shown schematically in Figure 1. Analysis of peptides (9) Taylor, R. Lecture Notes on Fullerene Chemistry: A Handbook for Chemists; Imperial College Press: London 1988. (10) Li, J.; Takeuchi, A.; Ozawa, M.; Li, X.; Saigo, K.; Kitazawa, K. J. Chem. Soc., Chem. Commun. 1993, 1748. (11) Khabashesku, V. N.; Margrave, J. L. Chemistry of Carbon Nanotubes. In The Encyclopedia of Nanoscience and Nanotechnology; Nalwa, S., Ed.; American Scientific Publishers: Stevenson Ranch, CA, 2004; Vol. 1, pp 849861. (12) Peng, H.; Alemany, L.; Margrave, J. L.; Khabashesku, V. N. J. Am. Chem. Soc. 2003, 125, 15174-15182. (13) Stevens, J.; Huang, A. Y.; Peng, H.; Chiang, I. W.; Margrave, J. L.; Khabashesku, V. N. Nano Lett. 2003, 3, 331-336. (14) Gillig, K. J.; Rutolo, B.; Stone, E. G.; Russell, D. H.; Fuhrer, K.; Gonin, M.; Schultz, J. A. Anal. Chem. 2000, 72, 3965.
and other large biomolecules at femtomole loading15 shows MALDI-IM-oTOF to be a very promising alternative to onedimensional MALDI spectrometry, and in fact, trace level fullerenes are often intentionally added as a wide mass range internal calibrant since the fullerene ion fragments are very efficiently separated by IM from the desired bioanalyte ions. MALDI-IMoTOF prototype instrumentation allows separation by IM with a resolution of up to 50 on the basis of ion volume (shape) while retaining the inherent mass accuracy of oTOF MALDI. MALDIIM-oTOF14 first separates laser-desorbed ions according to their drift time, which is determined by each ion’s charge-to-volume ratio (shape). The second stage of the IM-MS system is the timeof-flight mass spectrometer with orthogonal extraction, which provides continuous sampling of the mobility-separated ions with mass resolution of 2500. Data acquisition electronics and software allows data collection and display in the form of two-dimensional plots of intensity versus ion mobility drift time versus m/z ratio. In contrast to conventional MALDI instrumentation, the MALDI-IM-oTOF has the great advantage of enabling complete separation (prior to mass analysis) of the fullerene matrix ions from the analyte ion isobars according to their differing ion mobilities. The MALDI-IM-oTOF instrument allows reexamination of unusual materials that are known to work as matrices but have been heretofore excluded because of such isobaric interferences (as has been the case with the fullerenes). In this work, we examine the matrix properties of either C60((CH2)2COOH)n or C60(C11H23)n (n is nominally 6). In addition, we have derivatized the walls of size-selected fullerenes and nanotubes so that adsorption of biomolecules from solution can be enhanced. Subsequent laser irradiation results in high ionization efficiency of the physisorbed biological analyte molecule from these larger structures. (15) Woods, A. S.; Koomen, J.; Ruotolo, B.; Gillig, K. J.; Russell, D. H.; Fuhrer, K.; Gonin, M.; Egan, T.; Schultz, J. A. J. Am. Soc. Mass Spectrom. 2002, 13, 166-169.
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The main goal of our study is to achieve high sensitivity at low loadings of both analyte and matrix. Also of interest is the use of 1-µm-sized aerosolized particulates comprising intimate mixtures of fullerene matrix and bioanalyte, which allows more uniform analyte ion production. We use the aerosolization as an alternative approach to eliminate the “hot spots”, which are so common to dried droplet preparations of all types of matrices. Finally we examine the potential utility of these new matrices for the MALDI analysis of biological tissue surfaces. MATERIALS AND METHODS Instruments. Data were acquired on an advance prototype MALDI ion mobility o-TOF and on a Voyager DE-pro high-vacuum MALDI TOF (Applied Biosystems Framingham, MA), both using nitrogen lasers. The schematics of the MALDI-ion mobility spectrometer is shown in Figure 1 and described in detail elsewhere.14 Peptides. RRPYIL (817.0 Da), dynorphin fragments 1-7 YGGFLRR (868.0 Da), 1-8 YGGFLRRI (981.2 Da), and 1-9 YGGFLRRIR (1137.4 Da), VRKRTLRRL (1198.5 Da), goosefish angiotensin I NRVYVHPFHL (1281.5 Da), human angiotensin II DRVYIHPF (1946.2 Da), and chicken egg white lysozyme (14305 Da) KVFGRCELAAAMKRHGLDNYRGYSLGNWVCAAKFESNFNTQATNRNTDGSTDYGILQINSRWWCNDGRTPGSRNLCNIPCSALLSSDITASVNCAKKIVSDGNGMNAWVAWRNRCKGTDVQAWIRGCRL were purchased from Sigma (St. Louis MO) Cerebroside sulfate was from Avanti Polar Lipids (Alabaster, AL). Organic Matrices. R-Cyano-4-hydroxycinnamic acid (CHCA) and 2-[(2E)-3-(4-tert-butylphenyl)-2-methylprop-2-enylidene]malononitrile (DCTB) were obtained from Sigma. Functionalized C60. Fullerenes and nanotubes were prepared and characterized according to the procedures introduced and developed at Rice University by Khabashesku et al.12,16 Sample-Solutions. Peptides were dissolved in water and matrix in 50% ethanol, and solution concentrations were in the range of 10 fmol/µL to 1 nmol/µl. Ethanol/water/chloroform solution was used for lipids. Dried Droplet Preparation. C60((CH2)2COOH)n is soluble in water; however, the solubility in ethanol is ∼10 times higher and is estimated to be ∼0.05 mg/mL in the 1:1 ethanol/water solution that was used in these experiments. The pH of a saturated solution of C60((CH2)2COOH)n in ethanol/water (1:1) is 2.0. To generate samples with high analyte concentration, we also used sonicated suspensions with concentrations of up to 1 mg/mL. The suspension’s color was either brown or orange-brown. The water or water/ethanol suspensions of C60((CH2)2COOH)n were relatively stable over days but almost immediately precipitated after only a few seconds of moderate centrifugation. Centrifugation rapidly destabilizes the suspensions, resulting in particulates at the bottom of the tube and an almost clear supernate. (The difference between a solution and a suspension has been dealt with for years by polymer and pharmaceutical chemists, but this distinction seems to often be blurred by purveyors of the fullerene literature, which unfortunately causes much confusion and wasted time.) It was noted that C60(C11H23)n is highly soluble in chloroform and could be used for lipid analysis. Different types of functionalized nanotubes were mostly in the form of black opaque suspensions. (16) Peng, H.; Reverdy, P.; Margrave, J. L.; Khabashesku, V. N. R. Soc. Chem., Chem. Chem. 2003, 362-363.
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Table 1. Signal Intensity (mV) of the MH+(MK+) Ions from YGGFLRR (Top Entry) and Angiotensin (Bottom Entry) Peptides Deposited as Premixed Slurries of Analyte and C60((CH2)2COOH)n Matrix deposited peptide, pmol deposited matrix, pmol 50 5 0.5 0.05 0.005
50 5 0.5 0.05 0.005
100 96(489) 116(350) 41(161) 35(158) 8(11) 10(9) 13(12) 12(8) 123(216) 131(233) 101(850) 233(950) 38(123) 35(163) 45(48) 52(108) 88(58) 68(72) 125(42) 138(93)
10
1
0.1
0.01
96(80) 67(80) 57(160) 249(267) 71(50) 110(43) 22(35) 60(47) 48(40) 42(41)
33(40) 13(28)
Laser Power “2300” 126(483) 161(216) 113(330) 106(199) 19(41) -18(40) 68(50) 41(27) 17(12) 12(7) Laser Power “2500” 128(366) 45(155) 194(382) 36(87) 126(243) 63(53) 118(366) 35(73) 316(334) 216(400) 500(157) 400(467) 366(483) 199(67) 334(570) 83(108)
37(27) 33(18)
The MALDI targets were prepared using conventional dried droplet technique. After mixing, 1-µL aliquots were deposited onto the sample plate and air-dried. Serial dilutions were prepared spanning a concentration range of 10 fmol/µL-100 pmol/µL of both matrix and analyte (Table 1). Formation of Aerosolized Matrix/Analyte Particles for Deposition. Matrix coating of aerosol particulates has been pioneered over the past few years as a way to detect large airborne biomolecules on the surface of aerosol particulates.17-20 Therefore, we tested an alternative fullerene matrix preparation and deposition scheme as follows: (1) aerosolization of the matrix/peptide solution, (2) drying the liquid droplets while suspended in a carrier gas, and (3) concentrating and impacting the particles on the MALDI target. Aqueous solutions of dynorphin and C60((CH2)2COOH)n (∼4-10 mol/L) were prepared using HPLC grade water and later mixed together in the desired ratios. The solution of acid-derivatized C60 was sonicated prior to and after mixing with the peptide. Mixed solutions (∼10 µL) were atomized by using a homemade collision atomizer to form droplets of the solution (diameter ∼10 µm). The composition in each atomized droplet was thus the same as in the suspension. To prevent contamination ultrahigh purity (UHP) nitrogen was used for the atomizing carrier gas (flow rate of 1.2 L/min). The aerosol droplets were then introduced to a flow tube (i.d. 48 mm, length 1500 mm), where another flow of UHP nitrogen (5 L/min) was added to promote water evaporation in the droplets. Since both dynorphin and C60 derivative are nonvolatile, both compounds remained in the droplet (17) Murray, K. K.; Russell, D. H. Anal. Chem. 1993, 65, 2534-2537. (18) He, L.; Murray, K. K. J. Mass Spectrom. 1999, 34, 909-914. (19) Stowers, M. A.; van Wuijckhuijse, A. L.; Marijnissen, J. C.; Scarlett, B.; van Baar, B. L.; Kientz, C. E. Rapid Commun. Mass Spectrom. 2000, 14, 829833. (20) Jackson, S. N.; Murray, K. K. Anal. Chem. 2002, 74, 4841-4844.
Figure 2. MALDI spectrum of four peptides at femtomole loadings of peptide and matrix: I, RRPYIL (818.0 amu); II, dynorphin 1-7 YGGFLRR (868.0 amu); III, dynorphin 1-9 YGGFLRRIR (1138.4 amu); and IV, goosefish angiotensin I NRVYVHPFHL (1281.5 amu). Concentration of each peptide is 25 fmol/µL (water solution); concentration of the C60((CH2)2COOH)n matrix is 100 fmol/µL (water/ethanol solution). Peak V is potassiated (IV +K+) ion signal for the angiotensin I, can be seen at 1320.4 amu.
during drying. Solid particles with the desired mixture of matrix and analyte were formed after complete drying. The dried particles have a narrow particle size distribution (fwhm of 1.0 µm centered around 1.5 µm). Laser light scattering was used to track the dried particulates either prior to depositing on stainless steel or introduction into a single-particle laser ablation aerosol mass spectrometer.21 The aerosolized solid matrix/peptide particles were deposited onto a stainless steel MALDI sample plate by using a gas stream containing these particles. The particulate-loaded gas stream was injected into a thin steel tubing (o.d. 1/8 in., i.d. 1/ in.), which accelerated the gas and the particles and directed 16 them onto a stainless steel MALDI plate where the particles impacted and were deposited. Alternatively, the particles were sized by an aerodynamic lens and introduced one by one into the single-particle mass spectrometer. Rat Brain Tissue Slices. The 14-µm-thick Sprague-Dawley rat brain slices were cut, placed on stainless steel targets, and then dried for 5 min in a vacuum chamber. A solution containing 100 pmol of C60((CH2)2COOH)n matrix was deposited over 1-cm2 area. RESULTS AND DISCUSSION C60((CH2)2COOH)n as a MALDI Matrix. The compound is believed to have up to six attached alkylcarboxyl chains (as shown by solid-state NMR). Laser desorption/ionization (LDI) mass spectra from pure samples of the functionalized fullerene molecule showed that the side-chain bonding does not survive direct laser desorption, and only a strong signal of a bare C60 molecular ion and dimer fragments are observed. MALDI analysis of functionalized fullerenes is an effective approach22,23 compared to LDI24 if (21) Gard, E.; Mayer, J. E.; Morrical, B. D.; Dienes, T.; Fergenson, D. P.; Prather, K. A. Anal. Chem. 1997, 69, 4083. (22) Ballenweg, S.; Gleiter, R.; Kraetschmer, W. Synth. Met. 1996, 77, 209212. (23) Brown, T.; Clipston, N.; Simjee, N.; Luftmann, H.; Hungerbuhler, H.; Drewello, T. Int. J. Mass Spectrom. 2001, 210/211, 249-263. (24) Beck, R.; Weis, P.; Hirsch, A.; Lamparth, I. Phys. Chem. 1994, 98, 96839687.
an appropriate matrix is chosen. Although the standard MALDI matrices typically used for peptides give no improvement for detection of derivatized fullerenes, the use of 9-nitroanthracene and DCTB on the other hand seems to work well for producing positively charged parent fullerene MALDI ions.23 Indeed, the MALDI of the C60((CH2)2COOH)n functionalized fullerene mixed with DCTB yielded some signal from derivatized C60 with one and two acid attachments. The mass range was limited to 900 amu (instead of 1174 amu, which would be the nominal mass for six attachments), and the spectra also showed a strong degree of fragmentation of the derivatized fullerene molecule. By contrast, typical MALDI matrices such as CHCA and sinapinic acid did not work at all. Although the attached functional groups do not survive direct laser desorption, we nevertheless demonstrate that they play a crucial role in matrix performance. A series of control tests were performed with a variety of analytes and concentrations using either a suspension of pure C60 in water or ethanol or in mixtures with simple aromatic acids such as (C6H5)2CHCOOH and C6H5CH2COOH. No analyte signal was measurable using these combinations (spectra not shown). By contrast, the premixing of C60((CH2)2COOH)n and peptide at very low concentrations (down to 2-fmol depositions of matrix with analyte) showed the ability of the acid-derivatized fullerenes to enhance peptide desorption and ionization. The most important point in this paper is demonstrated by the data in Figure 2, which shows a high-vacuum MALDI spectrum of a 1-µL deposit of a solution containing four peptides at a concentration of 25 fmol/µL each and 1 µL of fullerene matrix solution (100 fmol/µL). All four MH+ ion peaks are observed with good signal-to-noise (S/N) ratio after only a few laser shots. The surface deposit was still not depleted after acquisition of the Figure 2 data. In contrast to conventional organic matrices where high matrix-to-analyte ratios (typically 1000:1) are needed in order to incorporate the analyte into the matrix crystal, the situation is reversed for C60-based matrices. Reliable spectra can be obtained Analytical Chemistry, Vol. 76, No. 22, November 15, 2004
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with matrix-to-analyte ratios of between 10:1 and 0.001:1. In fact, we have determined that the best quality spectra, i.e. highest S/N ratio, low fragmentation, and predominance of the MH+ peaks (rather than the MNa+ and MK+), were obtained at matrix-toanalyte ratio between 0.01:1 and 0.001:1 and at very dilute concentrations of peptide. The predominance of the MH+ signal at dilute peptide concentrations is partly a result of the salt contamination of peptides being serially diluted. At higher matrix concentrations and loadings, a slightly larger peptide signal could be obtained, but the spectra are noisier, and sodiated ions predominate. The increase in peptide signal was not significantly larger as the ratio was increased to 1:1, the matrix signal appears strong compared to that of the analyte at high concentration, but at low matrix-to-analyte ratio the matrix signal is often almost absent. Control experiments, with no fullerene matrix added, shows no peptide signal. By contrast, we have obtained peptide signal at a S/N ratio of 100 from samples containing ratios of 20 fmol of fullerene/200 fmol of peptide and 2 fmol of fullerene/1 pmol of peptide. Very little matrix signal appeared from either of these spots. These results suggest that fullerenes could be efficacious at high-attamole loadings with femtomole or less peptide; however, these low-level experiments have not been attempted due to the difficulty of finding and laser irradiating such a small amount of randomly distributed analyte-matrix sample deposit. It is essential that the fullerene carbon clusters be in solution or suspension when mixed with analyte. Superior peptide signals were obtained from spots prepared by drying the premixed analyte and matrix mixture solution droplet when compared to depositing a droplet of peptide solution on top of the dried matrix. The data in Table 1 show the dependence of the test peptide signal on the fullerene concentration. The solutions were prepared by serialy diluting and premixing of solutions of both matrix and solutions of equal concentrations of YGGFLRRIR (dynorphin 1-8) and angiotensin peptides. A 1-µL droplet of each dilution of the premixed peptides and matrix solutions was deposited into 1-mm diameter wells comprising a Teflon wall and stainless steel bottom surface. The peak intensities of both the MH+ and MK+ peptide ion signals were acquired from a series of 10 shots at different locations on the surface. Theoretical “monolayer coverage” for both peptide and matrix are ∼1 pmol. Therefore, only for higher loadings was it possible to reliably obtain analyte signal from any location within the nominal 1-mm-diameter area of the deposited spot. Thus, submonolayer deposits of both matrix and analyte were interrogated by only accepting signal from individual laser shots if either peptide or fullerene signal was present. The peptide detection limit appears to be in the low-femtomole range and seems to be limited by the ability to find the material on the surface. We were unable to measure the laser power in the DE -Pro. The laser power at “2300” corresponds to the midrange of the power typically used in this instrument for desorbing/ionizing from traditional chemical matrices. As one can see from the data in Table 1, the ion signal only weakly depends on the peptide concentration in the low-picomole range. This is likely due to the mechanism of the analyte-matrix attachment in solution when only a limited number of peptide chains can be attached to one fullerene. A large excess of peptide does not lead to any increase in peptide signal intensity. Likewise, 6738
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a weak dependence of the signal upon the fullerene concentration can be explained either by the shortage of peptide (at high matrixto-analyte ratio) or by the low fluency of the laser ablation, which involves only the topmost monolayers of the sample (at high loads). The performance of the fullerene matrices is certainly comparable to that of a typical organic matrix such as CHCA; moreover, the fullerene matrices are still far from being optimized with respect to the choice of functional groups, concentration ranges, and sample preparation techniques. When the laser beam is scanned across a fullerene matrixanalyte spot prepared by the dried droplet technique, there is a variation in the signal intensity of ∼1 order of magnitude. This variation is consistent with an uneven thickness and coverage of the sample spot (observable with the unaided eye from the 100pmol deposits of matrix-analyte). Such preferential deposition of the solid matrix from the solution is caused by the unevenness and roughness of the sample plate surface as well as the low solubility of the matrix in water. Another part of the problem lies in the inadequate mixing and segregation of matrix and analyte molecules during drying due to very different solution solubilities of peptide and matrix. This causes aggregation of fullerene in certain regions and of peptide in others. To avoid the limitations of the dried droplet approach, we employed an alternative technique that produces a fine uniformly sized aerosol of the matrix/analyte suspension, which is subsequently dried and impacted onto the MALDI sample plate surface. MALDI experiments were performed with samples prepared by aerosolization of the matrix-peptide solution, drying the liquid droplets, and impacting the particles on the target (see details in the Experimental Section). As long as the spectra were acquired from the regions that had been deposited for longer periods of time and visually had the most accumulation of particles, the ratio of matrix/analyte MALDI peaks was very close to constant over the entire sample area irrespective of where the laser was focused. These results show that by ensuring both an intimate mixing of the sample components and a uniform deposition of the aerosols onto the target, one can then obtain a constant signal response as a function of laser spot position. The contrasting “sweet spots” in conventional MALDI dried droplet experiments are thus eliminated by this procedure. Furthermore, we have also seen weak parent ion production in single-particle spectra during 266-nm laser ablation of these pure dynorphin 1-8 aerosols. The dynorphin 1-8 peptide is of a structure similar to other peptide molecules that are well known to photofragment at this wavelength.25 Simultaneous positive and negative mass spectra containing a very weak parent ion signal as well as prominent peptide fragments were recorded from each particle even though each pure peptide aerosol particle comprised ∼1 fmol of peptide. By contrast, when the aerosol particle was prepared from a suspension containing a 1:5 ratio of peptide to C60((CH2)2COOH)n, the resulting aerosol particle contained only ∼100 amol of peptide. We could nevertheless still see a very weak peptide parent ion signal; however, the fragment ions were obscured by strong signals from the fullerene matrix ion decomposition. These results make the point that single-particle ablation (25) Tecklenburg, R. E., Jr.; Russell, D. H. Mass Spectrom. Rev. 1990, 9, 405451.
Figure 3. 2D mobility-mass spectrum of angiotensin II with C60((CH2)2COOH)n matrix in water/ethanol solution used as a matrix. Different colors denote different intensity of the signal, for example, red being stronger than green.
of such aerosols should be possible particularly if such studies are repeated at a laser wavelength that is absorbed exclusively in the matrix and eliminates the strong absorption at 266 nm within the aromatic side chains of the peptide analyte. MALDI-IM-oTOF. Two-Dimensional Acquisition of the Ion Mobility-Mass Spectra. Figure 3 shows an example of 2D ion mobility-m/z data from a sample prepared by the dried droplet method from a mixture of angiotensin II and C60((CH2)2COOH)n matrix. All angiotensin II parent ions and their fragments are aligned along an average “trend line” typical for all peptides and proteins. In addition to the parent ion, minor ions are also present at higher mass and were identified as MK+ or other adducts. Meanwhile, the C60 ion signal, and signals of C2 losses and higher mass derivatives (not visible in this figure), have much higher mobility (at least 20% shorter drift time) and are well separated from the peptide trend line. Mobility separation of the fullerene matrix applies not only to peptides but to all other common biological ions since they all have much less dense structure than fullerenes. The most compact of all high-mass complex biological molecules are oligonucleotides. We have nevertheless shown that they still have lower mobility then fullerenes and their complexes. Therefore, the use of ion mobility preseparation eliminates virtually any interference between the analytes of interest in biological milieu and the potentially interfering fullerene carbon matrix signal. Fullerene Matrix Analyses of Lipids with C60(C11H23)n Matrix. Derivatized Nanotube Matrices for Peptide Analysis. Lipid detection (and by extension other non-water-soluble analytes) using C60(C11H23)n solubilized in chloroform is possible. We have used a saturated solution of the matrix in order to obtain a
mobility-mass spectrum of cerebroside sulfate (data not shown). In this case, the 2D plot again allows the easy separation of the lipid signal from that of the matrix, since the lipids have even lower mobility than peptides.26 Although this fullerene matrix can contain up to six C11H23 chains (all are retained in the mass spectrum), the mobility of the complex is still at least 25% higher than that of lipids of the same mass. Functionalized Carbon Nanotubes. The nanotubes were also used as MALDI matrices for small peptides. Single wall nanotubes (SWNTs) with attached (CH2)2COOH groups12 were suspended in water (the solubility of these functionalized SWNTs is very low). The samples were prepared using the dried droplet technique, by combining equal amounts of the suspension and the peptide solution. It was found that spectra of peptides that can be obtained with these types of functionalized nanotube matrices are very similar to those obtained using the C60((CH2)2COOH)n. An important advantage of the SWNTs is the absence of low-mass matrix signal in part due to the large size and relative stability of single nanotubes. Figure 4 shows a 2D mass-mobility plot of the sample containing 100-pmol loading of dynorphin 1-7. There is only one trend line on the plot, which is formed by the peptide and its adducts/fragments. Nanotubes could also prossibly have some advantages for protein analysis because of their larger sizes. Furthermore, we have performed similar tests with nanotubes carrying dissimilar functional groups at different sites. The presence of multiple acidic (CH2)2COOH groups attached to the wall appears to be critical for the matrix operation. For example, (26) Woods, A. S.; Ugarov, M.; Egan, T.; Koomen, J.; Gillig, K. J.; Fuhrer, K.; Gonin, M.; Schultz, J. A. Anal. Chem. 2004, 76, 2187.
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Figure 4. 2D spectrum of dynorphin 1-7 with single-walled carbon nanotubes functionalized with (CH2)2COOH groups used as a matrix.
Figure 5. 2D MALDI IM-TOF MS spectrum obtained from Sprague-Dawley rat brain tissue using C60((CH2)2COOH)n matrix: (A) original spectrum; (B) same 2D spectrum after the matrix signals were filtered out.
nanotubes with the functional groups attached to the nanotube ends do not perform as MALDI matrices. Nor did nanotubes work when the COOH end groups and multiple sidewall fluorine substituents were used. Therefore, the presence of abundant proton source on the nanotube sidewall seems indispensable for both the nanotubes and fullerenes to function as matrices. Rat Brain Tissue Analysis Using Dried Droplet Fullerene Matrix. Figures 5 shows the MALDI-IM-oTOF spectra obtained from dried droplet addition of C60((CH2)2COOH)n solution deposited onto a 14-µm-thick rat brain tissue section. In both panels of 6740 Analytical Chemistry, Vol. 76, No. 22, November 15, 2004
Figure 5, the one-dimensional mass spectrum is derived by summing all ions at each m/z irrespective of their ion mobility and plotting the result on the top of the MALDI-IM-oTOF 2D plot. As seen in Figure 5A, the peptide and lipid ions below m/z 2000 are ionized from the tissue surface by pulsed UV laser irradiation and are separated by the MALDI-IM-oTOF into two distinct trend lines, which have been previously assigned to the two types of biochemical molecules.26 Figure 5B further shows the spectrum after the fullerene ions are subtracted from the data. The lipids and peptides can be easily separated along their individual trend
Figure 6. Linear mode MALDI spectrum obtained from a mixture of 100 pmol of chicken lysozyme and 170 pmol of C60((CH2)2COOH)n. The MH+ ion for lysozyme is seen at mass 14 306. Note the significant amount of in-source fragmentation.
lines so that detailed assignments of the lipid and peptide components can be made. We did not see strong signals above m/z 2000. The issue of protein detection from tissue surfaces using the smaller fullerene particulate matrices remains an open question that will be the subject of much future work. We will also need to examine the applicability of SWNT as matrices. MALDI Detection of High-Mass Molecules Using C60((CH2)2COOH)n as Matrix. Figure 6 is a linear mode MALDI spectrum obtained from a mixture of 100 pmol of chicken lysozyme and 170 pmol of C60((CH2)2COOH)n. The MH+ ions at masses 14 and 17 kDa are observed. There is also significant insource fragmentation as seen from the peaks in Figure 6 and Table 2. We were surprised as to the extent of in-source decay, which could possibly be due to laser fluency higher than the one used for peptides. However, considering the extensive range of the fragments obtained, most MH+ seen in the spectrum were within one mass unit of the expected MH+. Note that no systematic attempt has been made to optimize the molecular emission from this matrix by adjusting either the ratio of matrix to analyte or the laser power since this is outside the scope of the present study. However, our results are consistent with previous work which suggests that particulate MALDI is less suited for protein analysis than small aromatic matrices.3
Table 2. Egg White Chicken Lysozyme Fragments Generated by In-Source Decay MH+ spectrum
MH+ calc
fragment residues
MH+ spectrum
MH+ calc
fragment residues
6508.8 6817.0 7013.8 7295.5 7506.4 7628.3 7856.6 8159.2 8375.8 8609.2 8855.1 9049.6 9439.4
6508.4 6816.6 7013.7 7296.0 7507.3 7629.4 7857.6 8160.1 8375.6 8609.4 8854.8 9050.0 9440.5
57-115 47-110 21-83 31-97 31-99 4-71 12-80 47-121 54-129 17-94 25-106 17-98 32-117
9882.2 10656.5 10827.5 11251.4 11499.3 12345.4 12535.3 12729.6 13021.2 13230.0 13607.0 14306.0
9881.9 10654.9 10827.1 11253.5 11502.8 12347.8 12535.0 12732.2 1320.5 13231.8 13608.3 14306.1
16-106 33-128 27-124 20-121 14-117 11-122 6-119 7-122 6-123 11-129 1-128 1-129
Two open questions are left for future work by the data of Figure 5 and Figure 6. One is, can the surface engineering of the fullerene particulate improve the production of high-mass ions and minimize the in-source decay in linear mode MALDI? An equally important and related issue is the production of large MALDI ions that would survive in the ion mobility-oTOFMS application. So Analytical Chemistry, Vol. 76, No. 22, November 15, 2004
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far the data obtained from the tissue are restricted to only a few kilodaltons, as shown in Figure 5. Either proteins are not prevalent on the outermost tissue surface or they are not being desorbed and ionized by the C60((CH2)2COOH)n in a form that survives the 1-ms transit through the ion mobility cell. Our attempts to measure the lysozyme parent ion in the MALDI-IM-oTOF spectrometer using C60((CH2)2COOH)n matrices have so far yielded too many peptide fragment ions. CONCLUSIONS Functionalized fullerenes and nanotubes were shown to work as MALDI matrices for peptides and lipids at low-femtomole concentrations of both matrix and analyte. Our experimental data suggest that very low relative amounts of matrix might be necessary to achieve good signals from biological samples; no recrystallization of the matrix is required. In the case of acidic functional groups, no additional components will be needed to decrease the pH of the solution to promote efficient ionization as is often the case with conventional matrices. At present we have not obtained protein signals from intact tissue. This remains a potential disadvantage to the fullerene matrices; however, we have not exhaustively tested carbon matrices for this application. Additional work using test proteins and further optimization of matrices for the analysis of biological tissues remain an extremely important topic for future work. While our present study has convincingly shown the utility of the particulate matrix combined with MALDI-IM-oTOF for smallmolecule analysis in biotissues, a potential limitation would be if we cannot find experimental conditions whereby the particulate matrices can function for the analysis of large molecules as well. We see a number of other potential advantages of the fullerenebased matrices over conventional chemical MALDI matrices. We suggest the possibility of derivatizing larger size fullerenes as well as single wall carbon nanotubes. Larger sizes of these materials may lead to different mechanisms for the adsorption/desorption processes for large peptides or proteins. The effect on the desorption/ionization efficiency on oligonucleotides is also unknown.
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Fullerene-based materials in combination with MALDI-IMoTOF might be beneficial in cases where interference from organic matrix should be avoided or where thorough mixing and cocrystallization of organic matrix and analyte is impossible. Potential applications of fullerene matrices include biological tissue imaging by MALDI microprobe analysis as well as applications requiring MALDI surfaces that are prepared in advance and serve as surface sensors for the detection of airborne species in aerosol form or in adsorption of analyte directly from solution. In both applications, both the low detection limits and signal uniformity over the entire sample area are crucial. An existing problem for biological tissue imaging with MALDI lies in the inability to uniformly deposit conventional chemical matrix solution onto the tissue slice. This limitation could be eliminated if it is possible to achieve a uniform deposition or implantation of a superthin layer of functionalized fullerene matrix. The successful incorporation of such fullerenes would thus ionize biomolecules present on tissue surfaces irrespective of their solubility in matrix solution and of chemical type (as seen herein with lipids and peptides). This approach to tissue imaging with MALDI might also limit the cell lysis that occurs when an acidic organic matrix solution is directly added to the tissue. Thus, the fullerene might allow discrimination between extracellular and intracellular components. Such attempts to deposit or inject fullerene particulate matrices will therefore be the subjects of future work. The existing caveat is the inability (so far) to measure large proteins with particulate matrices when combined with MALDI-IM-oTOF. ACKNOWLEDGMENT J.A.S. thanks Marjorie Schultz for the personal funds used to support Ionwerks’ participation in this study. A.S.W. thanks ONDCP for instruments funding, without which this and other projects could not have been done. Received for review June 2, 2004. Accepted August 4, 2004. AC049192X
