Matrix Implanted Laser Desorption Ionization (MILDI) Combined with Ion Mobility-Mass Spectrometry for Bio-Surface Analysis A. Tempez,† M. Ugarov,† T. Egan,† J. A. Schultz,† A. Novikov,‡ S. Della-Negra,‡ Y. Lebeyec,‡ M. Pautrat,‡ M. Caroff,§ V. S. Smentkowski,| H-Y. J. Wang,∇ S. N. Jackson,∇ and A. S. Woods*,∇ Ionwerks, Inc., 2472 Bolsover St., Suite 255, Houston, Texas 77005, Institut de Physique Nucle´aire, CNRS-IN2P3, 91406 Orsay, France, Equipe “Endotoxines”, UMR 8619 du CNRS, IBBMC, Universite´ de Paris-Sud, 91405 Orsay, France, General Electric, Global Research, Niskayuna, New York 12309, and NIDA IRP, 5500 Nathan Shock Drive, Baltimore, Maryland 21224 Received November 22, 2004
The implantation of low velocity massive gold cluster ions allows homogeneous incorporation of a metallic matrix into the near-surface region of rat brain tissues. Subsequent analysis by laser desorption ionization mass spectrometry yields spectra exhibiting molecular ion peaks in the mass range up to 35 kDa similar to those observed by matrix-assisted LDI. Matrix-implanted LDI when combined with ionmobility preseparation promises to be a useful technique for molecular imaging of biotissues with a laser microprobe. Keywords: matrix implanted laser desorption ionization • mass spectrometry • gold • tissue • ion mobility
Introduction Matrix assisted laser desorption ionizations (MALDI)1,2 employing either particulate or organic acid matrixes are extremely useful methods for analyzing molecules from a solid biomolecular sample. However, the use of the dried droplet technique to combine analyte with organic acid matrix often produces a nonuniform spatial distribution of analyte on and within the dried thin film. We have previously described a newly developed alternative method for the homogeneous and quasinondestructive incorporation of Au nanoclusters into bioorganic solid material including rat brain tissue3 using beams of massive Au clusters containing hundreds of atoms produced by a liquid metal ion source (LMIS).4 Previously, a series of SIMS (secondary ion mass spectrometry) experiments employing these heavy and low-velocity projectiles have unexpectedly demonstrated that large fluences of Au4004+ can be implanted into bioorganic samples5 without changing the molecular character of the near-surface region; moreover, these astonishing results have further motivated a recent study which shows that the implanted clusters act as a MALDI matrix for a variety of peptides and proteins.3 In this work, we first present results from a pure test peptide (dynorphin 1-7, MW ) 868.0) which further illustrate the previously reported phenomena of the MILDI3 (matrix implanted laser desorption ionization) production of ions from gold cluster implanted surfaces. We then examine the high and * To whom correspondence should be addressed. E-mail: awoods@ intra.nida.nih.gov. † Ionwerks, Inc. ‡ Institut de Physique Nucle´aire, CNRS-IN2P3. § Equipe “Endotoxines”, Universite´ de Paris-Sud. | General Electric, Global Research. ∇ NIDA IRP.
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low mass spectra from implanted rat brain tissue samples using conventional high vacuum MALDI instrumentations as well as using a prototype MALDI-IM-oTOF MS (MALDI-ion mobilityorthogonal time-of-flight mass spectrometer). The results in the present work are restricted to measurements of positive ions only, partly because we wish to examine high molecular weight proteins and also because our MALDI-IM-oTOF MS prototype is not ready yet for negative ion mode measurements. MILDI from rat brain tissue and our implementation of MILDI-IM-oTOF MS provides some insight into both the capability and potential limitations of MILDI for biotissue and polymer surface imaging applications.
Experimental Section Production of Cluster Beams. Massive gold cluster ion implantation was performed at the Institut de Physique Nucle´aire in Orsay (IPNO), France with an ion beam produced by a Au-Si LMIS used since the early nineties at the IPNO to investigate secondary emission from various solid samples.6,7 Recently, it has been shown that beams of mass-selected Au clusters containing hundreds of atoms can be produced by this Au LMIS.4 After acceleration of the clusters by a 10 kV electric potential a Wien filter is used for velocity selection of the clusters so that Aun+ beams are well separated up to n ) 9. Above n ) 9, portions of a continuous distribution of massive cluster beams can, nevertheless, be selected with broader mass distributions but fixed values of n/q (q is the cluster charge). The Wien filter was adjusted to n/q ) 100 for the experiments reported in this paper. The mean charge of these clusters has been measured elsewhere4 to be 〈q〉 ) +4. Thus the mean number of atoms in the clusters used in this work is 〈n〉 ) 400 and the mean total energy 〈E〉 ) 40 keV (i.e., 100 eV/Au atom). 10.1021/pr0497879 CCC: $30.25
2005 American Chemical Society
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MILDI for Bio-Surface Analysis
Figure 1. Mobility instrument with MALDI at elevated pressure (left) and orthogonal extraction time-of-flight mass spectrometer (right).
Sample Preparation. Dynorphin 1-7 (YGGFLRR, MW ) 868 Da) was purchased from Bachem and droplets containing 2 µL of a 1 nmole/µL solution of the pure peptide in water were deposited on a stainless steel plate and dried. The resulting dried droplet samples have a diameter of 2-3 mm. These samples were either implanted with Au400 clusters or were kept as an unimplanted control for comparison LDI (Laser Desorption/Ionization) MS analysis. Since we want to mimic the environment of the brain tissue the salt in the peptide solution was not removed prior to deposition. Sprague Dawley rat brain tissue slices were cut into a 14 µm thickness by a cryostat, placed on stainless steel targets, and dried in a vacuum chamber for 5 min. One of the brain slices was implanted with massive gold clusters and analyzed within 48 h. A saturated solution of 2,6-dihydroxyacetophenone (DHA) in 50% ethanol was used as matrix for another of the brain slices. Spectra were obtained both with a DE-Pro MALDI-TOF instrument (from Applied-Biosystems) and a prototype MALDIIM-oTOF MS(see below). Au4004+ Cluster Implantation Procedure. Both dynorphin 1-7 films and the rat brain tissue slices were irradiated with 40 keV Au4004+ clusters at a normal incidence. Since the energy per gold atom was only 100 eV/atom and although the physical processes of energy loss of larger clusters in matter are not wellknown one can roughly estimate that the depth of penetration into an organic sample is limited to some 10 nm. Samples for implantation were introduced into a small vacuum chamber (1-2 × 10-6 mbar) connected to the end of the gold cluster beam line where a beam of uniform fluence homogeneously implanted the projectiles into the sample over a 3 mm diameter area. Fluences varied from 2.5 × 1011 to 2.5 × 1013 Au400/cm2. Using a beam intensity of about 100 pA required implantation times ranging from 2 to 200 min. LDI MS Analysis. Analysis of the implanted samples was done using a DE-Pro MALDI-TOF instrument (from AppliedBiosystems) equipped with N2 lasers (photon wavelength 337 nm, spot size 200 µm). MILDI-IM-oTOF Combined Analysis. The prototype MALDI-IM-oTOF MS instrument (described in detail in ref 8 and references therein)8 is shown in Figure 1. A N2 laser (photon wavelength 337 nm, spot size 300 µm) was also utilized. It integrates a laser ablation target inside an ion mobility cell.
This sample target has typically been a dried droplet of matrix/ analyte, but this work includes also the use of implanted Au clusters as a matrix. After pulsed laser irradiation, the ablation plume is collisionally cooled within microseconds by interaction with the pure mobility carrier gas (e.g., helium or nitrogen (or air) at 2 Torr). The desorbed bio-ions drift through the helium gas to the end of the mobility cell under the force of a high voltage field. Ion mobility separates ions according to their drift velocities which are determined primarily by their geometric collision cross-section with the helium gas “chromatographic column”. The ions’ drift time through the mobility cell is thus related to their physical shape which is determined by their gas phase conformation. The separation of isobaric (same mass) molecular ions is achieved on the basis of their different shapes. The mobility separated bio-ions are focused through the differential pumping aperture, leaving the 2 Torr pressure region of the mobility cell, exiting through the differential pumping region, and entering as a nearly parallel beam into the orthogonal time-of-flight mass spectrometer. The extractor region is allowed to fill for 20 µs after which time a high voltage pulse is applied to deflect the ions orthogonally into the reflector optics and then onto the detector where their mass is determined by their arrival flight time relative to the time of the extraction pulse. The mobility drift times are typically several milliseconds while the flight times within the mass spectrometer are typically twenty microseconds or less. Therefore, several hundred mass spectra can be obtained after each laser pulse at 20 µs increments and then stored as a twodimensional array of mass spectra and mobility drift time. Then, to improve signal-to-noise, these spectra can be summed over multiple laser shots so that the ion mass as a function of mobility can be measured. Custom data acquisition electronics and software allows collection of the ion intensity as a function of mass-shape information in two dimensions as shown in Figure 5.
Results and Discussion Dynorphin 1-7 Test Peptide Thin Film for Demonstrating MILDI. Figure 2 shows two MILDI linear time-of-flight positive ion mass spectra obtained at two different spots from the surface of a pure thin film of dynorphin 1-7 (YGGFLRR, MW ) 868.0 Da) implanted with a fluence of 1.7 × 1013 Au400/cm2. Journal of Proteome Research • Vol. 4, No. 2, 2005 541
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Figure 2. Laser desorption mass spectra in positive mode were obtained with the same number of shots at the same laser power (2800) from an unimplanted pure dynorphin 1-7 thin film (a) and from a pure dynorphin 1-7 thin film that was implanted with 1.7 × 1013 Au4004+ clusters at 40 keV (b) and (c). Spectrum (c) is vertically offset for clarity from (a) and (b). Spectra (b) and (c) from implanted dynorphin 1-7 were acquired from the same sample at two different positions one of which was at the extreme edge of the sample. The MH+ peak is prominent in spectrum (b) whereas in (c), the potassiated and sodiated adducts dominate.
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Figure 4. Linear MALDI-MS and MILDI-MS results from rat brain tissue. The MILDI spectra labeled Au 250 ns and Au 210 ns were acquired from two different spots on the same Auimplanted tissue sample using extraction delay times of 250 ns and 210 ns, respectively. The MALDI spectrum was acquired from a second tissue slice to which dried droplet DHA matrix was added.
Figure 5. MILDI-IM-oTOF spectrum from Au4004+ implanted Sprague Dawley rat brain tissue slice.
Figure 3. Variations of MILDI intensity as a function of accumulated laser shots. MILDI spectra were acquired on a single spot of a dynorphin 1-7 thin film that was implanted with 40 keV 1.7 × 1013 Au4004+/cm2+ at the same laser power (2700).
Also shown for comparison is a background LDI spectrum (labeled (a)) from an unimplanted control dynorphin thin film 542
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that was obtained using identical instrumental settings and identical number of laser shots as used to acquire the spectra from Au implanted samples (labeled (b) and (c)). Spectra (a) and (b) have a common zero intensity while spectrum (c) has been shifted vertically for clarity by adding 4000 arbitrary intensity units. The MILDI spectrum (b) obtained from the extreme edge of the Au implanted dynorphin dried droplet shows (M + H)+ as the dominant peak. MILDI spectrum (c) is from a broad central spatial region of the implanted dynorphin surface and shows predominantly alkali cationized dynorphin 1-7. It was found previously that the peak intensities of MILDI ion spectra from different spots in this broad central region were within 15% of each others.3 The data clearly show that gold cluster implantation serves the function of a UV laser matrix as previously reported.3 Additionally, we see that Au and alkali elements can all create adduct ions with the peptide. Whether the gold forms adduct
MILDI for Bio-Surface Analysis
to the parent (as in Figure 2b) or to NH3, as in Figure 2c seems correlated to the degree of alkali adduction to the parent molecule. We have also performed extensive X-ray photoelectron spectroscopy (XPS) analysis of both the gold cluster implanted and the unimplanted dynorphin 1-7 surfaces. The sampling depth of the XPS is between 1.5 and 2.0 nm. In this region, we find almost identical C, N, and O stoichiometries and chemical shifts for both the implanted and unimplanted films. The only difference between the two XPS spectra from the unimplanted and implanted films is the trace presence of Au (around one atomic percentage) in the near-surface region of the implanted film. We calculate that if all the implanted gold atoms were localized in the first 2 nm, then we would expect a 40% atomic Au signal in the XPS. Taken together, these results show that little decomposition of the peptide structure and/or fragments occurs within the top few nm and that the majority of the gold clusters reside below a depth of around 1.5 nm. In Figure 3 are shown MILDI spectra as a function of the number of laser shots on one spot from a dynorphin 1-7 thin film that was implanted with 40 keV gold clusters at a fluence of 1.7 × 1013 Au4004+/cm2. Consecutive spectra containing five laser shots each were acquired at the same position on the sample. Plots of the intensities of molecular adduct ions (MNa+ and MNaK+), a fragment ion (b6 + H2O + Na+) and gold related ions (AuNa+ and AuK+) as a function of number of accumulated shots are also shown. All peaks associated with molecular adducts and fragments of dynorphin 1-7 disappear within 1025 laser shots. By contrast the remaining peaks associated with the implanted gold (AuNa+ and AuK+) slightly increase in intensity with the number of laser shots. All peptide analyte molecules and fragments are preferentially desorbed and totally depleted after 25 laser shots, while gold related signals persist throughout the laser irradiation. This trend is strongly confirmed by negative ion mode data,9 in which an intense Aupeak (a more stable species than AuK+ and Au Na+) is also clearly observed with an intensity increasing throughout laser irradiation up to 300 laser shots. At this point we do not know how much peptide is removed (consumed) to obtain the spectra in Figure 2; nor do we know how much gold still remains within the film after multiple laser shots. The number of gold clusters initally distributed throughout the first few nanometers of the peptide film is obviously sufficient to create very strong MILDI emission spectra. However, the spatial distribution of the gold clusters within the film, before, during, and after laser ablation, remains a very important question. MILDI Detection of High Mass Proteins from Rat Brain Tissues. Figure 4 shows that MILDI is not restricted to analysis of small molecules. Two MILDI spectra are shown from a 14 µm thick Sprague Dawley rat brain tissue sample implanted with a fluence of 5 × 1012 Au400/cm2. Also shown in Figure 4 is the more intense MALDI spectrum from an adjacent tissue slice from the same rat brain which is covered with an approximately 5 micron thick dried droplet of DHA matrix. The MALDI spectrum using DHA is the sum of fifty shots. By contrast, the MILDI spectra are acquired from a second gold implanted tissue slice (from the same brain) at two different spots from 10 laser shots each. However, most of the protein ion signal is gone after the first two shots. Some common protein ions such as histone H4 are seen at different intensities in all three spectra while the haemoglobin is seen only in the MALDI spectrum.
research articles Although the MILDI spectra are substantially weaker than the MALDI, it is worth comparing the two matrix preparation techniques to establish a perspective on the two approaches. In MILDI, a number of massive gold clusters are introduced specifically into approximately the top 10 nm of the 14 µm thick rat brain tissue slice. In MALDI, on the other hand, multiple 1 µL-matrix solution droplets are allowed to dry over a 30 min period resulting in an approximately 5 µm thick overcoating matrix film on top of the tissue slice. This overcoat thin film comprises a mixture of organic matrix molecules and solvent extracted biomolecules. The molar amounts of active matrix material in MILDI and MALDI differ by approximately seven to eight orders of magnitude. What is unclear from our study and remains to be determined in future work is the disposition of the gold atoms after laser ablation of the gold implanted tissue surface. If the removal of analyte molecules is restricted to a region comparable to the implantation depth of the gold clusters, then it may be possible to repeatedly implant and desorb so that the molecular layers atop the tissue slice are slowly peeled away with a depth resolution of a few 10’s of nm. Signal averaging of the molecular signals as a function of depth could then both improve detection sensitivity of the analyte proteins while simultaneously providing unique information about the depth of origin of the biomolecules within the tissue slice. MILDI-IM-oTOF Detection and Isolation of Low Mass Lipids and Peptides from Rat Brain Tissue. The twodimensional plot of ion mobility vs m/z seen in Figure 5 was obtained from Sprague Dawley rat brain tissue implanted with a fluence of 5 × 1012 Au400/cm2. The MILDI tissue sample was inserted into a relatively new combined spectrometer8 which provides ion mobility preseparation prior to orthogonal timeof-flight mass spectrometry. Previous work with known test mixtures of isobaric brain lipids, peptides, and oligonucleotides has shown mobility separations of about 15% between each molecular type.8,10,11 Thus, two major classes of brain tissue molecules which are resolved by mobility in Figure 5, can be quickly and rigorously assigned to lipids and peptides based simply on their slope in the ion mobility-m/z chromatogram. Figure 5 shows a trend line which extends weakly up to 6 kDa and can be assigned to peptides and small proteins.8,10,11 On the top axis is an m/z projection of the two-dimensional spectrum. This derived mass spectrum contains all bioanalyte ions as well as any chemical and instrumental noise. As seen in the insert showing masses below 1 kDa, an additional trend line assigned to lipids is well resolved from isobaric peptides that elute faster from the mobility cell. Instrumental noise originates from a discharge within the source and has been removed by smoothing and thresholding pixels in the 2D IMm/z plot insert in Figure 5. Our inability to detect a substantial signal above 5 kDa may be due to these discharges. On the other hand, our data do not exclude the possibility that the MILDI desorbed proteins shown in Figure 4 may have a metastability which prevents their surviving the transit through the mobility cell. In linear MALDI (no mobility cell), metastable ions are also formed, but the detection probability may be higher. In the region below 2 kDa, the peptide isobaric “interference” with the lipid spectra can be stripped away by plotting only those ions associated with the lipid trend line.8 The results of this procedure are shown in Figure 6 which is a derived mass spectrum comprising only those ions in the region along the lipid trend line in Figure 5. Unfortunately, the mobility resolving Journal of Proteome Research • Vol. 4, No. 2, 2005 543
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Figure 6. Derived lipid MILDI mass spectrum comprising only the ions along the lipid trend line of the 2D ion mobility vs m/z plot (Figure 5) from the Au-implanted rat brain tissue sample. Table 1. Possible Assignments for Lipids Peaks of MILDI Mass Spectrum (Figure 6) Extracted from the 2D Ion Mobility Mass Spectrum Plot of the Au-Implanted Rat Brain Tissue Sample name
formula
MW
Phosphatidic acid Phosphatidic acid Phosphatidyl ethanolamine Phosphatidylcholine Cerebroside Cerebroside Cerebroside Cerebroside Cerebroside Phosphatidylserine Sphingomyelin(16:0) Sphingomyelin(18:0) Sphingomyelin(20:0) Sphingomyelin(22:0) Sphingomyelin(24:0) Sphingomyelin(24:1)
C37H70O8P C37H70O8P C39H80NO8P
673.9 673.9 717.6
C40H80NO8P C40H75NO9 C44H85NO9 C44H77NO9 C46H89NO9 C48H93NO9 C46H75NO10P C39H79N2O6P C41H83N2O6P C43H87N2O6P C45H91N2O6P C47H95N2O6P C47H93N2O6P
733.6 713.7 771.7 763.7 799.7 827.7 832.5 703.1 731.1 759.1 787.1 815.1 813.1
MH+
MNa+
MK+
696.8 713.0 740.6 734.6 714.7 772.7 786.7 800.7 822.7 850.7 833.5 855.5 704.1 726.1 732.1 754.1 760.1 782.1 788.1 810.1 816.1 838.1 814.1 836.1
752.7
866.7 742.1 770.1 798.1 826.1 854.1 852.1
power of 25 in this instrument is insufficient to further differentiate these ions between fatty acid chain fragments and possible alkali or gold adduction to the brain lipids or fatty acids. Our previous lipid data8 were acquired from test molecules using a mobility cell with a resolution of 40. In those studies,8 it was shown that within the overall “lipid” trend line we could further resolve isobaric components from the lipidss such as free fatty acid fragments (1-2% slower) and organic matrix/alkali adducted lipids (1-2% faster). Moreover, there seemed to be subtle differences in the mobilities of lipid isobars originating from lipids with different types of headgroups8 (however much additional work will be required to determine this effect). Possible types of lipids which would be expected in the rat brain and are consistent with the masses shown in the derived lipid spectrum (Figure 6) are listed in Table 1. Ultimately, the utility of the MALDI-IM-oTOF MS for analysis of brain lipids will depend on achieving mass resolutions of several thousand, mass accuracies of a few ppm, and mobility resolving powers on the order of between 75 and 100. It may also be necessary to further corroborate structural assignments by fragmentation mass spectrometry after elution from the mobility cell. Some 544
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(or perhaps all) of these instrumental improvements should be possible in a future prototype instrument. Direct mass spectrometric analysis of native biological products and/or tissues is one of the exciting prospects in analytical biochemistry. Recent investigations on tissue imaging using MALDI are beginning to yield important molecular information in many areas of biological and medical research.12 MALDI imaging of peptides and proteins expressed in tumor and healthy tissue may reveal correlations between certain marker proteins/peptides and the disease state. However, the uniform incorporation of organic MALDI matrix remains probably the greatest difficulty for a successful MALDI image analysis. Wet matrix treatment of the tissue sample surface suffers from inhomogeneous matrix crystallization and solvent extraction effects during drying. The spatial distribution of the targeted proteins can also be easily perturbed. The new MILDI technique offers significant advantages as an alternative method for homogeneous, nondestructive, and uniform matrix incorporation into near-surface regions of biotissues. The data in Figure 4 is particularly tantalizing. Recent studies with derivatized fullerene matrixes have shown that the ratio of peptide/nanoparticulate matrix is in the range of 100 peptides/1 fullerene (acid derivatized C60).13 This is reversed from the ratio of 10 000 matrix molecules/1 peptide and suggests an extremely high efficiency of peptide desorption and ionization from the nanoparticulate. Peptide ions have been efficiently measured from as little as a few femtomoles of deposited peptide and matrix (i.e., “submonolayer” amounts of matrix + peptide).13 Even more pertinent to the data in Figure 4 is recent work which shows that the molar efficiency of colloidal gold nanoparticles may be an even more efficient matrix than the fullerene nanoparticulates.14 Furthermore, desorption wavelengths of 355 nm were used in both of these studies;13,14 however, it is likely that these nanoparticulate matrixes will perform even better using wavelengths up to and including IR. If we assume that the efficiencies of the MILDI gold clusters are similar to the colloidal gold system, then we are truly seeing monolayer type protein signals from the tissue surface which have a per shot intensity equal to or greater than those coming from the thick organic matrix layer overcoat in Figure 4. Nevertheless, possible caveats to the application of MILDI are this unknown efficiency of the gold nanocluster matrix as well as the propensity toward increased cation and gold adduction to positive ions. This may be partially mitigated when the analytical problem can be solved by restricting analysis to negative ion spectra. Another practical consideration is that the gold cluster ion currents are insufficient for implantation of large areas (few mm diameter) in times less than about an hour. A more important use of the gold cluster LMIS technology may be the focused implantation into regions of interest on the surface. It may be possible to focus the massive gold clusters to an implantation spot diameter of less than a few microns. Thus, the point of origin of the MILDI ions would then possibly be controlled by the focal spot of the implanter rather than the focal spot size of the desorption laser.
Conclusions In conclusion, we suggest that the combination of a massive cluster ion source and laser desorption ionization mass spectrometrysparticularly when coupled with ion mobility preseparationsis an efficient and reproducible method for analyzing the near-surface region of biological thin films. The
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combination of MILDI and ion mobility-oTOF MS already offers an advantage for separating the low mass region into lipids and peptides (as well as nucleotides and aromatic drug molecules8,10). However, the utility of the MILDI-IM-oTOF MS combination for analysis of higher mass protein bioanalytes which are laser desorbed from complex biomatrixes such as tissue remains an open question.
Acknowledgment. JAS would like to thank Marjorie Schultz for the use of personal funds to completely support Ionwerks’ participation in this work. References (1) Karas, M.; Hillenkamp, F. Anal. Chem. 1988, 60, 2299-2301. (2) Tanaka, K.; Waki, H.; Ido, Y.; Akita, S.; Yoshida, Y.; Yoshida, T. Rapid Commun. Mass Spectrom. 1988, 2, 151-153. (3) Novikov, A.; Caroff, M.; Della-Negra, S.; Le Beyec, Y.; Pautrat, M.; Schultz, J. A.; Tempez, A.; Wang, H. W. J.; Jackson,S. N.; Woods, A. S. Anal. Chem. 2004, 76, 7288-7293. (4) Bouneau, S.; Brunelle, A.; Della-Negra, S.; Depauw, J.; Jacquet, D.; Le Beyec, Y.; Mouffron, J. P.; Novikov, A.; Pautrat, M. Nucl. Instrum. Methods. Phys. Res. B 2004, B225, 579-589.
(5) Tempez, A.; Schultz, J. A.; Della-Negra, S.; Depauw, J.; Jacquet, D.; Novikov, A.; Le Beyec, Y.; Pautrat, M.; Caroff, M.; Ugarov, M.; Bensaoula, H.; Gonin, M.; Fuhrer, K.; Woods, A. S. Rapid Commun. Mass Spectrom. 2004, 18, 371-376. (6) Della-Negra, S.; Joret, H.; Le Beyec, Y.; Blain, M.; Schweikert, E. A. Phys. Rev. Lett. 1989, 63, 1625-1628. (7) Benguerba, M.; Brunelle, A.; Della-Negra, S.; Depauw, J.; Joret, H.; Le Beyec, Y.; Blain, M.; Schweikert, E. A.; Ben Assayag, G.; Sudraud, P. Nucl. Instrum. Methods. B 1991, 62, 8-22. (8) Woods, A. S.; Fuhrer, K.; Egan, T.; Ugarov, M.; Koomen, J.; Gonin, M.; Gillig, K. J.; Schultz, J. A. Anal. Chem. 2004, 76, 2187-2195. (9) Novikov, A.; Caroff, M.; Della-Negra, S.; Le Beyec, Y.; Pautrat, unpublished data. (10) Gillig, K. J.; Ruotolo, B.; Stone, E. G.; Russell, D. H.; Fuhrer, K.; Gonin, M.; Schultz, J. A. Anal. Chem. 2000, 72, 3965-3971. (11) 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 Spectrosc. 2002, 13, 166-169. (12) Todd, P. J.; Schhaff, T. G.; Chaurand, P.; Caprioli, R. M. J. Mass Spectrom. 2001, 36, 355-369. (13) Ugarov, M. V.; Egan, T.; Khabashesku, D. V.; Schultz, J. A.; Peng, H.; Khabashesku, V. N.; Furutani, H.; Prather, K. S.; Wang, H.-W. J.; Jackson, S. N.; Woods, A. S. Anal. Chem. 2004, 76, 6734-6742 (14) McLean, J.; Stumpo, K.; Russell, D. H. submitted to J. Am. Chem. Soc. (Oct 2004).
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