Heterogeneity within MALDI Samples As Revealed by Mass

Anal. Chem. 2000, 72, 30-36

Heterogeneity within MALDI Samples As Revealed by Mass Spectrometric Imaging Rebecca W. Garden† and Jonathan V. Sweedler*

Department of Chemistry and the Beckman Institute, University of Illinois, Urbana, Illinois 61801

While matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) has revolutionized the manner by which many large molecules are characterized, the highly variable appearance of MALDI mass spectra remains a concern. We have developed MALDI-based imaging as a diagnostic tool for examining the relationships between preparation strategy, sample morphology, and spectral quality. The imaging protocol involves the automated acquisition of mass spectra at 400-1600 positions within a single sample, followed by off-line processing and image display. Several sample types have been characterized, including a simple peptide mixture prepared in dried droplets of 2,5-dihydroxybenzoic acid and in thin films of r-cyano-4-hydroxycinnamic acid as well as a complex biological sample consisting of intact peptidergic neurons from the marine mollusk Aplysia californica. Imaging experiments provide a wealth of unbiased information concerning sample defects, spectral reproducibility, mass accuracy, differential analyte distributions, and the validity of internal standards. Matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) is one of the most versatile techniques for qualitative characterization of biological and synthetic polymers.1 Sample preparation can be as simple as diluting the analyte in a suitable matrix solution and applying a 0.5-2 µL aliquot to the sample plate.2 Such “dried-droplet” samples often have a diameter of 1-2 mm. During spectrum acquisition, the laser (typical spot size ∼ 200 µm) is moved over the heterogeneous sample surface to locate the position of optimal spectrum quality. The dried-droplet approach has been extended to accommodate the direct profiling of complex mixtures, including crude extracts, cell lysates, bacteria cultures, and individual neurons.1 The appearance of a MALDI mass spectrum is highly dependent on the morphology of the irradiated matrix-sample crystals; significant variations in peak presence, intensity, resolution, and mass accuracy are often obtained by focusing the laser on different regions of the same sample.3-6 To alleviate this concern, considerable efforts have been made toward the production of homoge* To whom correspondence should be addressed. Phone: (217) 244-7359. Fax: (217) 244-8068. E-mail: [email protected]. † Current address: Department of Chemistry, University of Virginia, Charlottesville, VA, 22901. (1) Burlingame, A. L.; Boyd, R. K.; Gaskell, S. J. Anal. Chem. 1998, 70, 647R716R. (2) Karas, M.; Hillenkamp, F. Anal. Chem. 1988, 60, 2299-2301. (3) Cohen, S. L.; Chait, B. T. Anal. Chem. 1996, 68, 31-37.

30 Analytical Chemistry, Vol. 72, No. 1, January 1, 2000

neous, microcrystalline sample surfaces, including the use of matrix additives, solvent mixtures, membranes, and chromatographic cleanup, as well as multilayered, crushed, and electrosprayed preparations.3-18 Perhaps the simplest strategy is the widely utilized “thin-film” or fast-evaporation method, where the matrix is applied to the sample plate in a volatile solvent and the sample solution is applied on top of the microcrystalline matrix film.19,20 Despite these advances with sample handling, spectral reproducibility remains an issue for multicomponent samples and limits the ability to use MALDI for general quantitative purposes. A variety of methods have been used to examine the morphology of prepared MALDI samples. Light and scanning electron microscopies have been the most popularly employed tools for studying MALDI sample surfaces and assessing the effects of different preparation strategies.21,22 Fluorescence microscopy23 and X-ray crystallography24 have been used to evaluate the incorporation of analyte into matrix crystals. Moreover, Hanton and coworkers25 recently studied the segregation of analytes and salts within matrix crystals with time-of-flight secondary ion mass (4) Strupat, K.; Karas, M.; Hillenkamp, F. Int. J. Mass Spectrom. Ion Processes 1991, 111, 89-102. (5) Amado, F. M. L.; Domingues, P.; Santana-Marques, M. G.; Ferrer-Correia, A. J.; Tomer, K. B. Rapid Commun. Mass Spectrom. 1997, 11, 1347-1352. (6) Sadeghi. M.; Vertes, A. Appl. Surf. Sci. 1998, 127, 226-234. (7) Kussmann, M.; Nordhoff, E.; Rahbek-Nielsen, H.; Haebel, S.; Rossel-Larsen, M.; Jakobsen, L.; Gobom, J.; Mirgorodskaya, E.; Kroll-Kristensen, A.; Palm, L.; Roepstorff, P. J. Mass Spectrom. 1997, 32, 593-601. (8) Gusev, A. I.; Wilkinson, W. R.; Proctor, A.; Hercules, D. M. Anal. Chem. 1995, 67, 1034-1041. (9) Chen, H.; Guo, B. Anal. Chem. 1997, 69, 4399-4404. (10) Domin, M. A.; Welham, K. J.; Ashton, D. S. Rapid Commun. Mass Spectrom. 1999, 13, 222-226. (11) Hung, K. C.; Ding, H.; Guo, B. Anal. Chem. 1999, 71, 518-521. (12) Zhang, H.; Caprioli, R. M. J. Mass Spectrom. 1996, 31, 690-692. (13) Zhang, H.; Andre´n, P. E.; Caprioli, R. M. J. Mass Spectrom. 1995, 30, 17681771. (14) Gobom, J.; Nordhoff, E.; Mirgorodskaya, E.; Ekman, R.; Roepstorff, P. 1999, 34, 105-116. (15) Dai, Y.; Whittal, R. M.; Li, L. Anal. Chem. 1999, 71, 1087-1091. (16) O ¨ nnerfjord, P.; Ekstro ¨m, S.; Bergquict, J.; Nilsson, J.; Laurell, T.; MarkoVarga, G. Rapid Commun. Mass Spectrom. 1999, 13, 315-322. (17) Allwood, D. A.; Perea, I. K.; Perkins, J.; Dyer, P. E.; Oldershaw, G. A. Appl. Surf. Sci. 1996, 103, 231-244. (18) Hensel, R. R.; King, R. C.; Owens, K. G. Rapid Commun. Mass Spectrom. 1997, 11, 1785-1793. (19) Vorm, O.; Roepstorff, P.; Mann, M. Anal. Chem. 1994, 66, 3281-3287. (20) Vorm. O.; Mann, M. J. Am. Soc. Mass Spectrom. 1994, 5, 955-958. (21) Westman, A.; Huth-Fehre, T.; Demirev, P.; Sundqvist, B. U. R. J. Mass Spectrom. 1995, 30, 206-211. (22) Kampmeier, J.; Dreisewerd, K.; Schu ¨ renberg, M.; Strupat, K. Int. J. Mass Spectrom. Ion Proc. 1997, 169, 34-41. (23) Dai, Y.; Whittal, R. M.; Li, L. Anal. Chem. 1996, 68, 2494-2500. (24) Strupat, K.; Kampmeier, J.; Horneffer, V. Int. J. Mass Spectrom. Ion Processes 1997, 169, 43-50. 10.1021/ac9908997 CCC: $19.00

© 2000 American Chemical Society Published on Web 12/03/1999

spectrometry (SIMS) imaging. In an effort to relate sample preparation to the quality of mass spectra, O ¨ nnerfjord and coworkers16 used automated data collection to obtain 36 mass spectra over 600 µm × 600 µm areas within individual MALDI sample spots. Their approach enabled a statistical comparison of spectral variation for different sampling protocols. Studies such as these3-24 have provided important insights about sample heterogeneity, although single analytes or simple mixtures were employed in most cases. Some of the most complicated MALDI samples are those prepared from biological tissues, neurons, and microorganisms. Wang and co-workers26 have investigated the ability of two laboratories to obtain reproducible, strain-specific mass spectra from bacterial samples. They determined that several experimental factors, including solvent composition, salt content, and pH, are critical to spectrum appearance. The effects of matrix, solvent, and morphology have also been studied by Domin and coworkers.10 Furthermore, Arnold and Reilley27 have developed a mathematical algorithm for objectively distinguishing MALDI mass spectra obtained from 25 different Escherichia coli (E. coli) strains. In addition to cell cultures, multiple sampling strategies have also been described that enable MALDI-based profiling of single peptidergic neurons.1 For example, we study peptide signaling in model neurons from the marine mollusk, Aplysia californica (A. california). We have used MALDI to discover new biologically active peptides and to elucidate prohormone processing pathways.28,29 Like the correlation of mass spectra from bacteria,27 normalization of preselected mass spectra appears to allow comparison of different neuronal samples.30-32 However, prior investigations26-32 have not accounted for variability over the sample surface. The goal of the present study is to formulate methods of characterizing a single MALDI sample for heterogeneity to allow the development of improved sample preparation strategies. An important new ability for MALDI is mass spectrometric imaging. First described in 1995, MALDI-based imaging was developed as a detection method for thin-layer chromatographic separations.33 The Caprioli laboratory has described a MALDI imaging approach and associated software for imaging biological samples and membrane-bound capillary electrophoresis eluents.34-36 (25) Hanton, S. D.; Clark, P. A. C.; Owens, K. J. Am. Soc. Mass Spectrom. 1999, 10, 104-111. (26) Wang, Z.; Russon, L.; Li, L.; Rosser, D. C.; Long, S. R. Rapid Commun. Mass Spectrom. 1998, 12, 456-464. (27) Arnold, R. J.; Reilley, J. P. Rapid Commun. Mass Spectrom. 1998, 12, 630636. (28) Garden, R. W.; Moroz, T. P.; Gleeson, J. M.; Floyd, P. D.; Li, L.; Rubakhin, S. S.; Sweedler, J. V. J. Neurochem. 1999, 72, 676-681. (29) Floyd, P. D.; Li, L.; Rubakhin, S. S.; Sweedler, J. V.; Horn, C. C.; Kupfermann, I.; Alexeeva, V. Y.; Ellis, T. A.; Dembrow, N. C.; Weiss, K. R.; Vilim, F. S. J. Neurosci. 1999, 19, 7732-7741. (30) Garden R. W., Shippy S. A., Li L., Moroz T. P., and Sweedler J. V. Proc. Natl. Acad. Sci. U.S.A. 1998, 195, 3972-3977. (31) Li, L.; Moroz, T. P.; Garden, R. W.; Floyd, P. D.; Weiss, K. R.; Sweedler, J. V. Peptides 1998, 19, 1425-1433. (32) Jime´nez, C. R.; Li, K. W.; Dreisewerd, K.; Mansvelder, H. D.; Brussaard, A. B.; Reinhold: B. B.; van der Schors, R. C.; Karas, M.; Hillenkamp, F.; Burbach, J. P. H.; Costello, C. E.; Geraerts, W. P. M. Proc. Natl. Acad. Sci. U.S.A. 1997, 9481-9486. (33) Gusev, A. I.; Vasseur, O. J.; Proctor, A.; Sharkey, A. G.; Hercules, D. M. Anal. Chem. 1995, 67, 4565-4570. (34) Caprioli, R. M.; Farmer, T. B.; Gile, J. Anal. Chem. 1997, 69, 4751-4760. (35) Stoeckli, M.; Farmer, T. B.; Caprioli, R. M. J. Am. Soc. Mass Spectrom. 1999, 10, 67-71.

We have also applied MALDI-based techniques to study the spatial distribution of peptides in neurons and neuronal processes.28,37 In all such experiments, images are obtained by acquiring mass spectra stepwise over the desired area of a heterogeneous sample. One usually recreates a spatial map (an image) by selecting the mass of interest, extracting the corresponding intensity value from each spectrum, and displaying the ensemble of intensities at their correct spatial positions to produce the final image. The ability to image samples has prompted this study to determine the effectiveness of MALDI imaging as a diagnostic tool to directly study the distribution of analyte signal over the surfaces of different sample types. Specifically, we have acquired MALDI-based molecular ion images of both simple mixtures and complex samples. Preparations are exhaustively sampled in order to obtain mass spectra every 50 µm, yielding 400-1600 spectra per sample. To accomplish this, we have developed an imaging protocol to study multiple peptides within dried-droplet, thin-film, and cellular samples. We illustrate the types of information one can obtain from such experiments, such as the differential distribution of multiple analytes throughout the sample, mass accuracy over a sample, the validity of an internal standard, and the differential detection of protonated peptides and sodium adducts. We also demonstrate that cellular samples contain distinct regions where different biomolecules are detectable (i.e., peptides and phospholipids). EXPERIMENTAL SECTION Samples. Dried-droplet2,3 and thin-film19,20 sampling methods were employed using 2,5-dihydroxybenzoic acid (DHB; ICN Pharmaceuticals, Costa Mesa, CA) and R-cyano-4-hydroxycinnamic acid (R-CHC) (Aldrich Chemical Co., Milwaukee, WI) matrices, respectively. In each case, the sample was an aqueous mixture of synthetic Aplysia peptide standards, specifically 0.5 µM R-bag cell peptide1-8 (R1-8) (American Peptide Co., Sunnyvale, CA), 0.5 µM acidic peptide (AP) (UIUC Biotechnology Center, Urbana, IL), and 2 µM egg-laying hormone (ELH) (Peninsula Laboratories, Belmont, CA). The dried-droplet samples were prepared by combining 0.5 µL of peptide solution with 0.5 µL of matrix solution (10 mg/mL DHB in water) on the sample plate. For each thinfilm sample, ∼1 µL of R-CHC matrix solution (5 mg/mL in acetone containing 1% water) was spotted onto a clean MALDI sample plate. After rapid solvent evaporation, 0.5 µL of the peptide mixture was applied to the surface of the matrix film. Juvenile A. californica weighing 0.5-5 g were obtained from the Aplysia Research Facility (Miami, FL). The details of preparing marine samples for MALDI analysis have been described elsewhere.28-31 In general, sampling bag cell neurons involved replacing the physiological saline with the DHB matrix solution while dissecting the abdominal ganglion under a stereomicroscope. Tungsten needles were used to isolate and transfer the cell(s) to a standard MALDI sample plate containing 0.5 µL of DHB solution. After drying at ambient temperature, samples were either analyzed immediately or stored at -20 °C. (36) Zhang, H.; Stoeckli, M.; Andren, P. E.; Caprioli, R. M. J. Mass Spectrom. 1999, 34, 377-383. (37) Garden, R. W.; Moroz, T. P.; Li, L.; Gleeson, J.; Sweedler, J. V. Proc. 45th ASMS Conf. Mass Spectrom. Allied Top. 1997, 730.

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Figure 2. Photomicrograph of the thin-film sample after image acquisition: scale bar ) 200 µm. Several film defects, in addition to the rim of the sample, are clearly visible. MALDI mass spectra were acquired over a 29 × 27 array, as evidenced by the “holes” in the R-CHC film.

Figure 1. Representative mass spectra extracted from MALDI images of three different sample types: (A) a mixture of synthetic R1-8, AP, and ELH prepared on a thin film of R-CHC matrix; (B) the same synthetic peptide mixture sampled as a dried droplet with DHB matrix; (C) a group of Aplysia bag cell neurons placed into the middle of an aqueous drop of DHB prior to drying. Detected peaks correspond to previously characterized peptides.30 The average mass resolution, m/∆m, was calculated to be 1800, 1000, and 770 for the R-CHC thin-film, DHB dried-drop, and DHB cellular samples, respectively.

Automated MALDI Image Acquisition. Unless indicated otherwise, positive-ion mass spectra were acquired using a Voyager DE-STR time-of-flight mass spectrometer (PE Biosystems, Framingham, MA) operating with a nitrogen laser (337 nm) in the linear, delayed extraction mode with an accelerating potential of 20 kV, a 94.5% grid voltage, a 0.15% guide wire voltage, and a delay time of 225 ns. The standard laser spot size was reduced by placing a pinhole aperture in front of the existing focusing optics. At laser fluences necessary to ionize and detect peptides, the focused laser spot of our instrument had an effective MALDI sampling diameter of ∼50 µm for DHB and ∼25 µm for R-CHC. To investigate spectral heterogeneity over each sample type, all parameters, including laser power, mass range, calibration, and the number of averaged laser pulses and data points/ spectrum remained constant during data collection. These instrument conditions were established using an external calibration sample that was prepared identically and located adjacent to the test sample. 32 Analytical Chemistry, Vol. 72, No. 1, January 1, 2000

Automated data acquisition was achieved through modification of the autosampler mode within the Voyager Biospectrometry Workstation software. To control the position of the MALDI sample plate with respect to the laser, the standard methods and search patterns were customized to raster the sample under the laser spot. For the representative images presented herein, mass spectra were obtained at 50 µm increments, typically over a 20 × 20-40 × 40 array (400-1600 spectra per spot), and the same external calibration was applied to each mass spectrum within an image. Offline Image Processing and Display. After unattended data acquisition was complete (typically 1-4 h to collect 4001600 spectra), the PE Biosystems Convert program was used to convert each mass spectrum file to ASCII data pairs, where the elements of each data pair correspond to the mass (m/z) and the arbitrary intensity number. To facilitate spectral analysis, we used a computer program written in-house with Lab Windows CVI (National Instruments, Austin, TX). The program was designed to extract intensity and/or mass values within a specified mass range and to place these values into an ordered x, y spatial array which directly corresponds to the coordinates of the original sample. Several x, y arrays were constructed for each molecular ion of interest, including an intensity array (the maximum intensity over a specified mass range), a mass array (the mass which corresponds to the maximum intensity in the range), and a background array (the average or standard deviation of the intensity within several daltons of a peak). The arrays were manipulated with a standard spreadsheet program, and the images were generated using SigmaPlot 5.0 (SPSS, Chicago, IL). Statistical comparisons, based on the nonparametric Mann-Whitney rank sum test, were calculated using SigmaStat 2.0 (SPSS).

Figure 3. MALDI ion images showing intensities for selected peptides using (A) the thin-film, R-CHC sample depicted in Figure 2 and (B) the dried-droplet, DHB sample. Approximately 250 fmol of R1-8, 250 fmol of AP, and 1 pmol of ELH were prepared with each matrix. The blackto-red color map corresponds to the arbitrary intensity values specified for each peptide, while the grid lines correspond to 50 µm increments within each image. Typical mass spectra are shown in Figure 1A,B for each respective sample type.

RESULTS AND DISCUSSION As illustrated by previous reports, MALDI-based imaging promises to be an excellent approach toward characterizing the distribution of peptides in separations33,36 and in heterogeneous biological samples.34,35,37 Here we adapt MALDI imaging as a diagnostic tool. We examined a mixture of synthetic peptides as well as a highly heterogeneous biological sample. Figure 1 shows representative mass spectra acquired from a single position within each of the three sample types; these spectra are similar to others obtained using the respective preparation methods. Molecular ion images are straightforward to generate and process through modification of the existing autosampler software and utilization of a custom program designed to extract and assemble mass and intensity information. We automatically acquire, calibrate, save, and assign coordinates to the mass spectra at each position over the sample. Obviously, by adjusting instrument parameters at any given position, it is possible to obtain mass spectra of different qualities, but consistent use of the acquisition method across the entire image enables the examination of spatial variation. To determine if MALDI imaging is useful for assessing differences within individual samples, a three-component peptide mixture was prepared using both DHB dried-droplet and R-CHC thin-film approaches. Figure 2 is a photomicrograph of the thinfilm sample obtained after imaging. As a result of placing a pinhole in front of the nitrogen laser, the laser used for R-CHC samples

provides ∼25 µm diameter spots, considerably less than the 200 µm beam diameter of the laser as supplied on the commercial instrument. Because the mass spectra were acquired at 50 µm intervals, “bullet holes” are easily observed over the sample. In other words, the sampling process appears to exhaust the thin film at each spot. Also note that several areas lacking the thin film (defects) are readily observed in Figure 2. For MALDI imaging, each acquired mass spectrum spans the m/z mass range 500-5000. The molecular ion images shown in Figures 3 and 4 represent the background-subtracted intensities of selected peptide signals for the standards and the neurons, respectively. Except for the noted defects, the R-CHC thin films are microcrystalline and uniform, and the resulting images (Figure 3A) indicate that peptide signals are detected at all positions where the sample drop contacted the matrix film. The largest defect in the thin film is also observed in each peptide image as an area with no signal intensity. For the DHB dried-droplet sample, microscopic examination reveals larger nonuniformaties of crystal morphology and localization (not shown, but see examples in refs 4 and 14). The long, needlelike crystals exist mostly at the periphery, whereas the shorter, thinner crystals are distributed nonuniformly throughout the sample interior. The heterogeneous surface is reflected in the peptide images (Figure 3B); the areas with no crystals obviously yield no peptide signals. The threshold laser fluence is much higher for DHB dried-droplet samples; Analytical Chemistry, Vol. 72, No. 1, January 1, 2000

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Figure 4. MALDI images for selected molecular ions from the cells sampled with DHB matrix. Refer to Figure 3 for the color map and scaling details. The “lipids” image corresponds to the putative phospholipid at m/z 747. “RTotal” is the average intensity distribution for R1-9, R1-8, and R1-7, while ELH is the intensity at m/z 4385. The peptide AP is imaged in terms of its protonated [M + H]+ and sodium-adduct [M + Na]+ signals. A representative mass spectrum extracted near the periphery of the DHB crystals is shown in Figure 1C. Table 1. Mass and Intensity Variations over a MALDI Sample sample type thin film, R-CHC

dried droplet, DHB

bag cell neurons, DHB

peptide

[M + H]+

n

m/z jx ( σ

jx ( % RSD

n

m/z jx ( σ

jx ( % RSD

n

m/z jx ( σ

intensityc jx ( % RSD

R1-8

1009.6a 1010.2b 2961.3b 4385.2b

440 357 386

1009.5 ( 0.1 2961.2 ( 0.7 4385.2 ( 0.9

62000 ( 11 4200 ( 44 4600 ( 91

375 335 402

1010.0 ( 0.4 2961.4 ( 1.5 4385.1 ( 1.9

1700 ( 49 930 ( 115 5100 ( 148

245 262 262

1010.3 ( 0.5 2961.9 ( 0.6 4385.2 ( 1.2

2900 ( 43 29000 ( 37 23000 ( 95

AP ELH a

intensityc

intensityc

Monoisotopic. b Average. c Arbitrary value.

hence, the effective laser spot size appears larger (∼50 µm). However, the thicker DHB crystals are not exhausted as observed with the R-CHC film. An important question concerns the reproducibility of peptide signals for each sample preparation method. For example, as shown in Table 1, AP intensities have 44 and 115% relative standard deviations (RSDs) for the R-CHC and DHB samples, respectively. There have been several reports of the use of MALDI for quantitation (comparing the peak intensity to a calibration curve).1 To allow such comparisons, most quantitative studies use intensities that have been normalized to an internal standard. Such normalization is believed to account for spectral/sampling differences. To test the effectiveness of this approach, we calculated the AP/ELH ratio for every spot on each sample, which, in 34 Analytical Chemistry, Vol. 72, No. 1, January 1, 2000

essence, is designating the ELH peak as the internal standard. Figure 5A shows a histogram of the AP/ELH ratios observed for the two sample types (Figure 3) for all spectra which contain signals for both AP and ELH. In other words, we did not use the location of “holes” in the matrix. A wide range of ratios were calculated over each sample. For the thin film, AP/ELH values varied more than 100%, with 80% of the AP/ELH ratios between 80 and 160%. The DHB dried-drop approach had a smaller absolute range, with 80% of the values between 10 and 50%. These results indicate that, even with internal standards and apparently uniform samples, peak intensities are not absolute and are difficult to compare, even within a single spot. This appears to be especially true for peptides such as AP and ELH that have different ionization properties.30

Figure 5. Histograms of normalized peptide intensities from the MALDI images: (A) ELH-normalized AP for the two synthetic peptide samples shown in Figure 3 and (B) R1-9-normalized intensities for R1-8 and R1-7 from the cellular sample shown in Figure 4.

In addition to using MALDI imaging for determining the distribution of intensities across the sample,33-37 the spectra obtained during image acquisition can be used to examine variations in mass accuracy. The images were calibrated externally, and the masses corresponding to the peptide peaks were extracted at each laser position. Table 1 lists the mass accuracy for each sample at all positions where R1-8, AP, and ELH were detected (resulting in 245-440 spectra used in the calculations). Of course, each mass spectrum could be internally calibrated during image acquisition, but the goal here is to compare the effects of external calibration for each sample type. Our findings of better mass accuracy (Table 1) and improved resolution (Figure 1) for the thin-film samples are consistent with previous results.19,20 Both samples discussed thus far consisted of a model mixture of synthetic peptides; to examine a more complex and heterogeneous sample, a small group of neurons was placed into a drop of DHB on the sample plate. These neuroendocrine cells are much more complex than the peptide standards and literally contain thousands of compounds. The bag cell neurons from A. californica are well-characterized by MALDI, and the 13 peptides labeled in Figure 1C are expected, including the R1-8, AP, and ELH.30 Selected molecular ion images of this sample are shown in Figure 4. One consistent observation of the cell samples is that the small, hydrophilic peptides have enhanced intensities in mass spectra

obtained around the rim of the DHB crystals. For example, R-bag cell peptide (APRLRFYSL) exists in the bag cell neurons as fulllength R1-9 and as C-terminally truncated R1-8 and R1-7. Interestingly, these three peptides have very similar ionization properties and are simultaneously detected at consistent relative intensities, as indicated in the histogram of R1-9-normalized intensities in Figure 5B. In contrast to the somewhat even distribution of AP and ELH signals, the absolute intensities for R-bag cell peptide detected in the outermost 100 µm of the sample are significantly higher than those detected in the interior of the sample (P e 0.001). The consistent intensity ratios from these three peptides suggests that if one chooses the correct internal standard, it may be possible to make semiquantitative comparisons of MALDI peak intensities, even for heterogeneous samples. To obtain MALDI mass spectra from marine organisms with >500 mM intercelluler cation concentrations, we use a matrix rinsing protocol30 that requires aqueous DHB. The reduction in salt is critical to successfully acquiring mass spectra from Aplysia neurons as well as bacterial samples;26 thus, we are interested in the distribution of salts over the sample surface. To measure this, we generated images corresponding to the [M + H]+ and the [M + Na]+ signals for AP (Figure 4). Consistent with previous observations,4,18,25 the distributions of protonated peptide and sodium adduct are distinct. By expressing the sodium adduct peak as a percentage of the protonated peak (i.e., [AP + Na]+/[AP+H]+ × 100), the difference between the outermost 100 µm of the sample and the remaining interior is statistically significant (P e 0.001) with an outside average of 7.6% (range 1-18) and an inside average of 19.5% (range 3-67). These data show that, even with salty samples, it may be possible to find regions of the sample with reduced adducts. This finding may also partially account for the distribution of normalized intensity ratios (Figure 5). For example, the MALDI peak intensity for ELH tends to be more salt-dependent in comparison to AP;30 hence, a variable distribution of cations would dramatically affect the AP/ELH ratio. This sample heterogeneity also explains one reason for the need to “search” a sample spot to find a spectrum with the desired characteristics. In addition to detecting peptides from neuronal samples, a group of peaks between m/z 500 and m/z 900 is sometimes observed and is generally attributed to phospholipids.31 Phospholipids are present in the cell samples, and it is well-known that they ionize well in DHB.38-40 We imaged the putative phospholipids by plotting the background-subtracted intensity of several peaks selected from this mass region. As one example, the “lipid” image in Figure 4 is the intensity distribution at m/z 747. Interestingly, the phospholipid peaks are observed predominantly within the middle of the sample where the virtually intact cell bodies were placed and can still be observed using light microscopy. Unlike the solubilized cations and peptides which can segregate within specific areas of the matrix crystals, the lipids appear to remain near the original placement of the cell somas. Figure 6A shows an extracted mass spectrum from the central area of the sample. Note that these peaks are not present in Figure 1C, as that spectrum was extracted from the periphery. Figure (38) Harvey, D. J. J. Mass. Spectrom. 1995, 30, 1333-1346. (39) Marto, J. A.; White, F. M.; Seldomridge, S.; Marshall, A. G. Anal. Chem. 1995, 67, 3979-3984. (40) Schiller, J.; Arnhold, J.; Benard, S.; Mu ¨ ller, M.; Reichl, S.; Arnold, K. Anal. Biochem. 1999, 267, 46-56.

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Figure 6. MALDI mass spectra of phospholipids in Aplysia bag cell neuron samples. In A, the linear-mode mass spectrum was extracted from the central region of the cell image. In B, the data acquisition parameters were optimized for detection of low-mass components in the reflectron mode. The most intense spectral peak at m/z 746.581 is within 2 ppm of the mass calculated for oleoylstearoylphosphatidylethanolamine, a phospholipid expected to exist in the Aplysia nervous system.41

6B shows the same m/z region of a reflectron MALDI mass spectrum obtained from another cell sample. Many of the peaks correspond, within