Matrix-Assisted Laser Desorption Ionization Imaging Mass

Anal. Chem. 2009, 81, 2779–2784

Matrix-Assisted Laser Desorption Ionization Imaging Mass Spectrometry Detection of a Magnetic Resonance Imaging Contrast Agent in Mouse Liver Elena Acquadro,† Claudia Cabella,‡ Simona Ghiani,‡ Luigi Miragoli,‡ Enrico M. Bucci,† and Davide Corpillo*,† Laboratorio Integrato Metodologie Avanzate, Bioindustry Park Canavese S.p.A., and Centro Ricerche Bracco, Bracco Imaging SpA, Bioindustry Park Canavese, Via Ribes 5, 10010 Colleretto Giacosa, Torino, Italy The present paper describes the detection of a magnetic resonance imaging (MRI) contrast agent by matrix-assisted laser desorption ionization imaging mass spectrometry (MALDI-IMS). The contrast agent was analyzed in both frozen and paraformaldehyde-fixed mouse livers explanted after its in vivo administration, and its identity was confirmed by fragmentation experiments. Moreover, a semiquantitative analysis was performed, evaluating its content in livers from mice sacrificed at different postadministration times. To the best of our knowledge, this is the first description of a MALDI-IMS analysis of MRI contrast agents and the first time that results obtained by MALDI-IMS are validated by both an in vivo (MRI) and an ex vivo (inductively coupled plasma atomic emission spectroscopy, ICP-AES) technique. Results shown in the present paper demonstrate the possibility of using MALDIIMS for drug biodistribution analysis. Obviously, this application is particularly interesting in the case of unlabeled compounds, which cannot be detected by any of the other imaging techniques. Matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) has been widely used since its introduction in late 1980s,1,2 especially for protein identification and characterization. About a decade later, due to its high sensitivity, high specificity, and short analysis time, it has also been proposed for ex vivo imaging purposes.3 In MALDI imaging mass spectrometry (MALDIIMS) experiments, tissue sections are attached on a sample plate, covered with a matrix (a compound which is essential for the ionization process), introduced into the mass spectrometer, and scanned by the laser; in this way, for each position a mass spectrum is obtained, providing the spatial distribution of each peak arising from the analyzed tissue. The concept of MALDIIMS was first introduced in 1997 by Caprioli et al.,3 who developed * To whom correspondence should be addressed. E-mail: proteomics@ bioindustrypark.it. † Bioindustry Park Canavese S.p.A. ‡ Bracco Imaging SpA. (1) Karas, M.; Bachmann, D.; Bahr, U.; Hillenkamp, F. Int. J. Mass Spectrom. Ion Processes 1987, 78, 53–68. (2) Tanaka, K.; Waki, H.; Ido, Y.; Akita, S.; Yoshida, Y.; Yoshida, T. Rapid Commun. Mass Spectrom. 1988, 2, 151–153. (3) Caprioli, R. M.; Farmer, T. B.; Gile, J. Anal. Chem. 1997, 69, 4751–4760. 10.1021/ac900038y CCC: $40.75  2009 American Chemical Society Published on Web 03/12/2009

this technique for endogenous peptides and proteins localization in biological samples. Successively, applications of MALDI-IMS were also extended to the analysis of exogenous low molecular weight compounds distribution onto histological sections.4 The most consolidated technique in the ex vivo analysis of drug biodistribution is currently autoradiography. Although characterized by a high sensitivity and a high resolution, this technique has significant limitations:5 first of all, the drug must be labeled with a radioactive isotope, and this process is often expensive, difficult, and time-consuming. Moreover, the tag may alter the pharmacological properties of the compound. Finally, it is not the entire drug but only the tag to be effectively detected, making it difficult to differentiate the intact drug from its possible metabolites. On the contrary, MALDI-IMS does not need labeling, is faster, and most of all, it has a molecular specificity as it detects the precise mass to charge ratio (m/z) of each compound, perfectly discriminating intact drug from metabolites. The first application of MALDI-IMS for drug detection was reported in 1999,4 when paclitaxel was detected in rat liver and human ovarian tumor biopsy (but without any drug localization). Since 2003 so far, some more articles have been published, concerning the MALDI-IMS detection of the distribution of antitumor drugs in mouse tumor and rat brain sections,5 of cocaine, chloroisondamine,6 and clozapine7 in rat brain, of olanzapine and its metabolites in whole-body rat sections,8 and in rat liver and kidney,9 of a proprietary drug candidate in rat tissues,10 of ketoconazole in porcine skin,11 of the cytotoxin AQ4 in mouse (4) Troendle, F. J.; Reddick, C. D.; Yost, R. A. J. Am. Soc. Mass Spectrom. 1999, 10, 1315–1321. (5) Reyzer, M. L.; Hsieh, Y.; Ng, K.; Korfmacher, W. A.; Caprioli, R. M. J. Mass Spectrom. 2003, 38, 1081–1092. (6) Wang, H. Y. J.; Jackson, S. N.; McEuen, J.; Woods, A. S. Anal. Chem. 2005, 77, 6682–6686. (7) Hsieh, Y.; Casale, R.; Fukuda, E.; Chen, J. W.; Knemeyer, I.; Wingate, J.; Morrison, R.; Korfmacher, W. Rapid Commun. Mass Spectrom. 2006, 20, 965–972. (8) Khatib-Shahidi, S.; Andersson, M.; Herman, J. L.; Gillespie, T. A.; Caprioli, R. M. Anal. Chem. 2006, 78, 6448–6456. (9) Cornett, D. S.; Frappier, S. L.; Caprioli, R. M. Anal. Chem. 2008, 80, 5648– 5653. (10) Drexler, D. M.; Garrett, T. J.; Cantone, J. L.; Diters, R. W.; Mitroka, J. G.; Conaway, M. C. P.; Adams, S. P.; Yost, R. A.; Sanders, M. J. Pharmacol. Toxicol. Methods 2007, 55, 279–288. (11) Bunch, J.; Clench, M. R.; Richards, D. S. Rapid Commun. Mass Spectrom. 2004, 18, 3051–3060.

Analytical Chemistry, Vol. 81, No. 7, April 1, 2009

2779

Figure 1. Structure of the gadolinium complex B22956/1.

tumor tissues,12 of erlotinib and its metabolites in rat tissue sections,13 of a β-peptide in whole-body mouse sections,14 of imatinib in mouse brain glioma,9 and of vinblastine in whole-body rat sections.15 In some of these studies, MALDI-IMS results were validated ex vivo by autoradiography or high-performance liquid chromatography coupled with tandem mass spectrometry (HPLC/ MS/MS). In the present study, a magnetic resonance imaging (MRI) contrast agent was detected by MALDI-IMS in mouse liver after its in vivo administration, and results were validated by both in vivo MRI and ex vivo inductively coupled plasma atomic emission spectroscopy (ICP-AES). EXPERIMENTAL SECTION Materials. MRI contrast agent gadocoletic acid trisodium salt, B22956/1, was kindly provided by Bracco Imaging Spa (Milan, Italy). All other chemicals were purchased from Sigma Chemical Co. (St. Louis, MO). Animal Models. Adult wild-type C57BL/6 male mice (24-28 g) were supplied by Charles River Italia Laboratories (Calco, LC, Italy). Animals were kept in limited access, air-conditioned facilities. Their care was in accordance with the national guidelines for animal experimentation. MRI. Mice were anesthetized with 0.5% isofluorane gas (95% O2). During imaging experiment, anesthesia was maintained by adjustment of gas level in function of their measured breath rate. The experiments were performed on a 7T MRI system (Pharmascan 300, Bruker, Germany) dedicated to small animals. B22956/1 was intravenously administered at a dose of 0.1 mmol/kg, corresponding to an administration volume of 4 µL/kg. Coronal T1-weighted spin echo images (TR/TE/NEX: 168 ms/8.4 ms/6) were acquired before contrast agent administration and then at regular intervals of about 3 min, averaging results deriving from six regions of interest (ROI) drawn in different areas of each liver. Percentage of enhancement was calculated as relative signal-to-noise (S/N) increase. (12) Atkinson, S. J.; Loadman, P. M.; Sutton, C.; Patterson, L. H.; Clench, M. R. Rapid Commun. Mass Spectrom. 2007, 21, 1271–1276. (13) Signor, L.; Varesio, E.; Staack, R. F.; Starke, V.; Richter, W. F.; Hopfgartner, G. J. Mass Spectrom. 2007, 42, 900–909. (14) Stoeckli, M.; Staab, D.; Schweitzer, A.; Gardiner, J.; Seebach, D. J. Am. Soc. Mass Spectrom. 2007, 18, 1921–1924. (15) Trim, P. J.; Henson, C. M.; Avery, J. L.; McEwen, A.; Snel, M. F.; Claude, E.; Marshall, P. S.; West, A.; Princivalle, A. P.; Clench, M. R. Anal. Chem. 2008, 80, 8628–8634. (16) Schwartz, S. A.; Reyzer, M. L.; Caprioli, R. M. J. Mass Spectrom. 2003, 38, 699–708. (17) Chaurand, P.; Cornett, D. S.; Caprioli, R. M. Curr. Opin. Biotechnol. 2006, 17, 431–436.

2780


Figure 2. Distribution (in red) obtained integrating the 840.5 ( 2 m/z range (in order to include all B22956/1 ligand isotopic peaks) onto a B22956/1-treated (left) vs control (right) mouse liver section, as determined by MALDI-IMS analysis (analyzed areas are delimited by dotted lines). The treated mouse was sacrificed 1 h after contrast agent administration.

Tissue Preparation. At 8, 30, 60, 90, or 150 min after contrast agent administration, mice were sacrificed. Briefly, animals were anesthetized by intramuscular injection of 0.4 mL/kg tiletamine/ zolazepam (Zoletil) plus 0.25 mL/kg xylazine (Rompun), then mice were perfused with phosphate-buffered saline (PBS), and livers either were or were not paraformaldehyde (PAF)-fixed by in vivo perfusion to be finally frozen immediately after explantation. For each time point, at least two animals were analyzed. Tissues were stored at -80 °C until use. Frozen liver sections of 12 µm were obtained using a cryostat microtome (CM 1900UV, Leica Microsystems, Germany) and immediately thaw-mounted to precooled (-20 °C) conductive indium-tin oxide (ITO)-coated slide glasses (Bruker Daltonics, Germany). Care was taken to mount tissues with minimum tissue freezing medium to prevent section contamination with the optimal cutting temperature medium (OCT).16,17 After sectioning, sections were dehydrated into a vacuum desiccator for at least 1 h at room temperature before matrix deposition. MALDI-IMS. Optical images were taken using a gel scanner at a resolution of 1500 dpi, prior to MALDI matrix application. Sections were spray-coated with 25 mg/mL 2,5-dihydroxybenzoic acid (DHB) in 50% methanol containing 0.1% trifluoroacetic acid, using a pneumatic TLC sprayer. All imaging analyses were performed on an Ultraflex II TOF-TOF (time-of-flight) mass spectrometer (Bruker Daltonics, Bremen, Germany) equipped with a 200 Hz all-solid-state (SmartBeam) laser and controlled by the FlexControl 2.4 software package. The instrument was operated in positive polarity in reflectron mode under optimized delayed extraction conditions, and measurements were done in a mass range of 500-4000 Da. Settings were as follows: ion acceleration voltage (IS1), 25.00 kV; reflector voltage, 26.30 kV;

Figure 4. Distribution (panel A, in red) determined by MALDI-IMS analysis integrating the 840.5 ( 2 m/z range onto a paraformaldehyde-fixed B22956/1-treated mouse liver section and corresponding average spectrum (panel B, arrow indicates B22956/1 ligand peak). The mouse was sacrificed 1 h after contrast agent administration.

Figure 3. Average spectra from MALDI-IMS analysis of frozen B22956/1-treated mouse liver (A) and control mouse liver (B). The arrow indicates the B22956/1 ligand peak (where present). The treated mouse was sacrificed 1 h after contrast agent administration.

first extraction plate voltage (IS2), 21.75 kV; detector voltage, 1623 V; laser intensity, around 55%. All spectra were the sum of 200 individual laser shots collected in 50-shot increments at each spot position at a laser frequency of 200 Hz. Spectra calibration was performed externally using a peptide mixture (Bruker Daltonics, Bremen, Germany) deposited on the glass slide surface and covering the range from 757.4 to 3147.5 m/z. Imaging spatial resolution was set to 100 µm. Two-dimensional (2-D) ion intensity maps and average spectra were created by FlexImaging 2.0 software (Bruker Daltonics, Bremen, Germany), employing the software to normalize the data set. B22956/1 ligand peak S/N was evaluated manually in average spectra. Each liver analysis was performed at least in triplicate. Untreated mice were used as control. MALDI-TOF-TOF. Fragmentation analysis was performed on both a B22956/1-treated mouse liver section covered with DHB (see above) and a 10 µM solution of B22956/, mixed 1:1 with DHB and directly spotted on a MALDI target plate. MS/MS spectra were acquired using the LIFT module on the Bruker Ultraflex II TOF-TOF. Instrument settings were as follows: IS1, 8.00 kV; IS2, 7.15 kV; LIFT 1, 19.00 kV. Neither delayed extraction nor collision gas were used. Settings for laser intensity were adjusted manually for optimal fragmentation. A timed ion gate was used for precursor ion selection (parent ion at 840.5 ± 2 m/z),

and generated fragments were further accelerated in the LIFT cell and detected following passage through the reflectron. ICP-AES. Portions of the livers were subjected to ICP-AES analyses in order to evaluate gadolinium content (expressed as Gd ng per tissue mg). ICP-AES assay was carried out on an Optima 2100 DV Perkin-Elmer spectrometer after sample digestion in 65% nitric acid by a microwave system (MDS-2000 CEM Corporation). The following experimental conditions were employed for the emission spectrometer: rf power, 1300 W; argon plasma flow rate, 15 L min-1; argon auxiliary gas flow rate, 0.2 L min-1; argon carrier flow rate, 0.8 L min-1; sample flow rate, 1.5 mL min-1; emission line, 342.247 nm. Each analysis was performed at least in triplicate, sampling different portions of each liver. RESULTS AND DISCUSSION B22956/1 is a highly stable small molecular weight gadolinium complex (see Figure 1) that combines the well-known gadopentetate moiety (Gd-DTPA) as contrast enhancer with the deoxycholic acid moiety as an albumin-binding promoter. When intravenously administered, it is able to bind albumin, preventing its rapid renal excretion. It instead accumulates in liver, where it is excreted in bile without any metabolization.18 A quantity of B22956/1 (0.1 mmol/kg) comparable to contrast agents dosage routinely used for humans was administrated to wild-type C57BL/6 mice, which were then monitored by MRI. In the first experiments, animals were sacrificed 1 h after contrast agent administration. PBS-perfused livers were explanted and immediately frozen, to finally be analyzed by MALDI-IMS. As evident in Figure 2, the comparison between B22956/1-treated and control mouse liver evidenced in the former the appearance of a peak, uniformly distributed over (18) De Hae¨n, C.; Anelli, P. L.; Lorusso, V.; Morisetti, A.; Maggioni, F.; Zheng, J.; Uggeri, F.; Cavagna, F. M. Invest. Radiol. 2006, 41, 279–291.


2781

Figure 5. MALDI-TOF-TOF fragmentation analysis of the peak in the 840.5 ( 2 m/z range from B22956/1-treated mouse liver section (A) and from a 10 µM B22956/1 solution (B).

the histological section (as expected from the high degree of molecular homogeneity exhibited by liver9). This peak resulted to have a monoisotopic m/z value of 839.5 ± 0.2 (Figure 3A), corresponding to that expected for monoprotonated B22956/1 ligand. This result was not surprising, as it was already verified by a MALDI-TOF analysis of pure B22956/1 (data not shown) that the extremely acidic conditions due to the presence of DHB matrix solution cause the almost complete dissociation of the gadolinium complex during sample preparation for MALDI analysis. This peak was absent in control mouse (Figure 3B). As histological samples are often stored after PAF fixation, the experiment was repeated on an analogous B22956/1-treated mouse liver, which was perfused with PAF immediately before explantation. The ligand peak still resulted detectable in the histological section (Figure 4A), even if with a lower intensity (see the average spectrum in Figure 4B), thus demonstrating the possibility of analyzing drug biodistribution in PAF-fixed tissues by MALDI-IMS, as already reported for protein analysis.19-22 However, it must be stressed that although protein analysis of PAF-fixed tissues requires some unlocking procedures (e.g., trypsin digestion), no additional procedure (19) Lemaire, R.; Desmons, A.; Tabet, J. C.; Day, R.; Salzet, M.; Fournier, I. J. Proteome Res. 2007, 6, 1295–1305. (20) Stauber, J.; Lemaire, R.; Franck, J.; Bonnel, D.; Croix, D.; Day, R.; Wisztorski, M.; Fournier, I.; Salzet, M. J. Proteome Res. 2008, 7, 969–978. (21) Groseclose, M. R.; Massion, P. R.; Chaurand, P.; Caprioli, R. M. Proteomics 2008, 8, 3715–3724. (22) Ronci, M.; Bonanno, E.; Colantoni, A.; Pieroni, L.; Di Ilio, C.; Spagnoli, L. G.; Federici, G.; Urbani, A. Proteomics 2008, 8, 3702–3714.

2782


was applied to detect B22956/1 ligand peak in the presence of PAF. In order to confirm the identity of this peak, a manual MALDI-TOF-TOF analysis was performed directly on the liver section: the peak was isolated and fragmented, resulting in the fragmentation pattern showed in Figure 5A. The same experiment was then repeated on a 10 µM solution of B22956/1, which showed the same pattern (Figure 5B), demonstrating the correct assignment of the peak. Once the identification of B22956/1 ligand was confirmed, it was chosen to go on with MALDI-IMS experiments in MS mode, as there were no matrix interference ions present at the m/z region of interest. Although MS/MS experiments, besides improving the quantitative analysis by filtering isobaric ions resulting from biological matrixes, also add a higher degree of confidence in peak identification, on the other hand the MS-only mode allows one to simultaneously acquire signals from endogenous metabolites, which in the future could be analyzed to gain information on the possible organism response to the treatment.12,23,24 With this strategy, a semiquantitative analysis was attempted, evaluating B22956/1 ligand peak S/N in normalized average spectra from MALDI-IMS experiments related to the analysis of mice sacrificed at five different times (namely, 8, 30, 60, 90, and 150 (23) Stoeckli, M.; Staab, D.; Schweitzer, A. Int. J. Mass Spectrom. 2007, 260, 195–202. (24) Reyzer, M. L.; Caprioli, R. M. Curr. Opin. Chem. Biol. 2007, 11, 29–35. (25) Desiderio, D. M.; Wirth, U.; Lovelace, J. L.; Fridland, G.; Umstot, E. S.; Nguyen, T. M. D.; Schiller, P. W.; Szeto, H. S.; Clapp, J. F. J. Mass Spectrom. 2000, 35, 725–733.

explanted livers. As shown in Figure 6, there is a good correlation among these three techniques, as they all revealed the same trend: MRI contrast agent content in mouse liver grows rapidly in the first minutes, reaching its maximum 30-90 min after administration and then slowly decreasing to finally be completely excreted after about 8 h (data not shown). This validation of MALDI-IMS data by both an in vivo (MRI) and an ex vivo (ICP-AES) technique is of particular interest, as these techniques monitored different species: MALDI-IMS detected B22956/1 ligand peak deriving from complex dissociation during matrix deposition, whereas MRI and ICP-AES detected gadolinium. The good agreement among data from these three techniques, in our opinion, demonstrates both the absence of any in vivo contrast agent degradation process and the high reliability of MALDI-IMS results. Finally, it must be stressed that the average spectra related to the time point with the lowest B22956/1 content (namely, the one at 150 min after administration, see Figure 6) still show a ligand peak with a good S/N value (about 7, data not shown). Even if an evaluation of its limit of detection was not attempted, it can be estimated that at least a B22956/1 content in liver corresponding to one-fifth of that found for the 150 min postadministration analysis should still be detectable. This means that, in the case of this Gd complex, MALDI-IMS sensitivity is at least as good as that of MRI, as for the latter it can be estimated that only signals with an enhancement higher than 20% (about one-fifth of that measured for the 150 min postadministration analysis) can be discriminated from noise (data not shown).

Figure 6. Time-course analysis of B22956/1-treated mice: B22956/1 ligand peak relative S/N, as determined in normalized average spectra after MALDI-IMS analysis of explanted livers (upper panel); in vivo liver MRI enhancement (middle panel); gadolinium content in explanted livers, as determined by ICP-AES (lower panel).

min) after contrast agent administration. Even if quantification by MALDI-MS is always a challenging task, quantitative evaluation of exogenous small molecular weight compounds,25 and in particular Gd-containing MRI contrast agents,26 has already been reported. Moreover, it has been shown by comparison with autoradiography or HPLC/MS/MS data that MALDI response reflects the relative amount of drug in tissues.5,13 In the present study, MALDI-IMS data were compared to in vivo MRI data acquired analyzing the same mice and to ICP-AES data related to Gd quantification in their (26) Corpillo, D.; Cabella, C.; Geninatti Crich, S.; Barge, A.; Aime, S. Anal. Chem. 2004, 76, 6012–6016.

CONCLUSIONS The present paper describes the MALDI-IMS detection of an MRI contrast agent, B22956/1, in mouse liver. The choice of an MRI contrast agent as a model for drugs/diagnostic agents analysis onto histological sections was due to the fact that it allowed the subsequent validation of MALDI-IMS results by more consolidated techniques such as MRI and ICP-AES. Moreover, this particular Gd-containing complex was selected because of its high stability in vivo, its known accumulation in liver, and the fact that it is excreted without any metabolic degradation. B22956/1 was detected in both frozen and PAFfixed mouse livers explanted after its in vivo administration, and its identity was confirmed by fragmentation experiments. Moreover, a semiquantitative analysis was performed, evaluating its content in livers from mice sacrificed at different postadministration times. To the best of our knowledge, this is the first description of a MALDI-IMS analysis of MRI contrast agents and the first time that results obtained by MALDI-IMS are validated by both an in vivo (MRI) and an ex vivo (ICPAES) technique. Recently, indeed, MRI has been suggested as the technique of choice for cross-validation, interpretation, and visualization of data deriving from MALDI-IMS analysis of endogenous proteins in whole mouse heads.27 The possibility of coregistering in 2-D and ultimately in 3-D space these two imaging approaches via the contrast agent could allow a direct (27) Sinha, T. K.; Khatib-Shahidi, S.; Yankeelov, T. E.; Mapara, K.; Ehtesham, M.; Cornett, D. S.; Dawant, B. M.; Caprioli, R. M.; Gore, J. C. Nat. Methods 2008, 5, 57–59. (28) Hsieh, Y.; Chen, J.; Korfmacher, W. A. J. Pharmacol. Toxicol. Methods 2007, 55, 193–200.


2783

overlay of MRI contrast images with drug, metabolite, (phospho)lipid, and protein localization information acquired by MALDIIMS. Results shown in the present paper demonstrate, once again, the possibility of using MALDI-IMS for drug biodistribution analysis, a key issue in pharmaceutical discovery and development.13 Obviously, this application is particularly interesting in the case of unlabeled compounds, which cannot be detected by any of the other imaging techniques. If further developed and integrated within the pharmaceutical industry, MALDI-IMS could dramatically reduce costs of drug development by removing the need to synthesize radiolabeled drug candidates and the health and safety issues arising from handling radioactive material.15 Finally, MALDI-IMS could also

2784


represent a diagnosis, screening, or discovery tool, when looking for changes induced by drugs in the comparison between treated and control tissues.28 ACKNOWLEDGMENT This work was supported by the “I-TECHPLAT Project” (Docup 2000-2006, Misura 3.4 from Regione Piemonte local government) and by “Progetto Lagrange Fondazione CRT” Grants. Received for review January 7, 2009. Accepted February 18, 2009. AC900038Y

Matrix-Assisted Laser Desorption Ionization Imaging Mass

Recommend Documents