Ionic Matrix for Enhanced MALDI Imaging Mass Spectrometry for

In animal tissue, the primary phospholipids include phosphatidylcholine (PC), ...... Development and Application of Imaging Mass Spectrometry. Mitsuto...
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Anal. Chem. 2010, 82, 8800–8806

Ionic Matrix for Enhanced MALDI Imaging Mass Spectrometry for Identification of Phospholipids in Mouse Liver and Cerebellum Tissue Sections Kamlesh Shrivas, Takahiro Hayasaka, Naoko Goto-Inoue, Yuki Sugiura, Nobuhiro Zaima, and Mitsutoshi Setou* Department of Molecular Anatomy, Molecular Imaging Frontier Research Center, Hamamatsu University School of Medicine, 1-20-1 Handayama, Higashi-ku, Hamamatsu, Shizuoka 431-3192, Japan The ionic matrix (IM) is considered to be versatile for matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) for the identification of a wide range of biomolecules due to its good solubility for a variety of analytes, formation of homogeneous crystals with analytes, and high vacuum stability. When these advantages are exploited, the performance of IM of r-cyano-4-hydroxycinnamic acid butylamine (CHCAB) and 2,5-dihydroxybenzoic acid butylamine (DHBB) was compared with other matrixes for the identification of phospholipids in standard mixtures and mouse liver tissue sections. The results showed that the IM of CHCAB caused higher signal intensity and allowed the detection of a number phospholipids such as phosphatidylethanolamine (PE) and phosphatidylserine (PS) in addition to detection of phosphatidylcholine (PC) on the surface of the liver tissue sample. The IM of CHCAB was also used to identify the species of lipids present in different layers of cerebellum where the greater numbers of biomolecules were detected as compared to DHB matrix. Further, the feasibility of the proposed method was extended for the analysis of tryptic digested cytochrome c for increased signal intensity and number of peptide sequences in MALDI-MS. Thus, the application of IM to MALDI-MS could be a promising tool for imaging biomolecules in tissue sections in high throughput analyses with high sensitivity.

without the time-consuming extraction, purification, or separation steps. Direct MALDI analysis in tissue sections enables the acquisition of cellular expression profiles while maintaining the cellular and molecular integrity. We can now produce a multiplex imaging map of selected biomolecules within tissue sections using imaging software. Thus, direct MALDI analysis of tissue sections is convertible into imaging maps, a method now known as MALDIimaging mass spectrometry (IMS).8-12 The application of matrix to the surface of tissue sections is an important step for obtaining homogeneity, reproducibility, and good resolution of the biomolecule images in IMS analysis. A number of devices are available for the deposition of matrix on the surface of tissue sections in order to obtain a homogeneous crystal formation; these include chemical inkjet printer spotter,13 robotic spotting depositors,14 electrospray depositors,15 and airbrush sprayers.16,17 Murphy and co-workers demonstrated the use of the sublimation method to deposit matrix onto the surface of tissue samples and achieved highly reproducible results in sampleto-sample analysis.18 Caprioli and co-workers described the use of a stainless steel sieve receiving pan for homogeneous deposition of matrix onto a tissue surface and showed that this method achieved a good signal intensity and shot-to-shot reproducibility in MALDI-IMS.19 However, most of the current approaches used for the preparation of samples in order to improve the homogeneity of the sample surface and enhance the signal intensity for the identification of biomolecules prior to MALDI-IMS analysis are tedious, multistep procedures. Here, a simple sample preparation

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) has proven to be one of the most powerful ionization techniques for mass spectrometric analysis of amino acids,1 peptides and proteins,2-4 carbohydrates,5 nucleic acids,5,6 and drugs.7 Today, direct tissue analysis of biological samples is possible using MALDI-MS for the identification of biomolecules

(8) Chughtai, K.; Heeren, R. M. A. Chem. Rev. 2010, 110, 3237–3277. (9) Caprioli, R. M.; Farmer, T. B.; Gile, J. Anal. Chem. 1997, 69, 4751–4760. (10) Sugiura, Y.; Konishi, Y.; Zaima, N.; Kajihara, S.; Nakanishi, H.; Taguchi, R.; Setou, M. J. Lipid. Res. 2009, 50, 1776–1788. (11) Wang, H. Y. J.; Jackson, S. N.; McEuen, J.; Woods, A. S. Anal. Chem. 2005, 77, 6682–6686. (12) Rubakhin, S. S.; Greenough, W. T.; Sweedler, J. V. Anal. Chem. 2003, 75, 5374–5380. (13) Baluya, D. L.; Garrett, T. J.; Yost, R. A. Anal. Chem. 2007, 79, 6862–6867. (14) Aerni, H. R.; Cornett, D. S.; Caprioli, R. M. Anal. Chem. 2006, 78, 827– 834. (15) Altelaar, A. F.; Klinkert, I.; Jalink, K.; de Lange, R. P.; Adan, R. A.; Heeren, R. M.; Piersma, S. R. Anal. Chem. 2006, 78, 734–742. (16) Hayasaka, T.; Goto-Inoue, N.; Zaima, N.; Kimura, Y.; Setou, M. Lipids 2009, 44, 837–848. (17) Garrett, T. J.; Prieto-Conaway, M. C.; Kovtoun, V.; Bui, H.; Izgarian, N.; Stafford, G.; Yost, R. A. Int. J. Mass Spectrom. 2007, 260, 166–176. (18) Hankin, J. A.; Barkley, R. M.; Murphy, R. C. J. Am. Soc. Mass Spectrom. 2007, 18, 1646–1652. (19) Puolitaival, S. M.; Burnum, K. E.; Cornett, D. S.; Caprioli, R. M. J. Am. Soc. Mass Spectrom. 2008, 19, 882–886.

* Corresponding author. Tel/Fax: +81-53-435-2292. E-mail: setou@ hama-med.ac.jp. (1) Karas, M.; Bachmann, D.; Hillenkamp, F. Anal. Chem. 1985, 57, 2935– 2939. (2) Tanaka, K.; Waki, H.; Ido, Y.; Akita, S.; Yoshida, Y.; Yoshida, T.; Matsuo, T. Rapid Commun. Mass Spectrom. 1988, 2, 151–153. (3) Shrivas, K.; Wu, H. F. Anal. Chem. 2008, 80, 2583–2589. (4) Shrivas, K.; Wu, H. F. Proteomics 2009, 9, 2656–2667. (5) Compton, B. J.; Siuzdak, G. Spectroscopy 2003, 17, 699–713. (6) Butler, J. M.; Jiang-Baucom, P.; Huang, M.; Belgrader, P.; Girard, J. Anal. Chem. 1996, 68, 3283–3287. (7) Su, A. K.; Liu, J. T.; Lin, C. H. Anal. Chim. Acta 2005, 546, 193–198.

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10.1021/ac102422b  2010 American Chemical Society Published on Web 10/12/2010

procedure is proposed for MALDI imaging using ionic matrix (IM) for the detection of biomolecule signals in mouse tissue sections. This is a new class of MALDI matrix which has been proposed by several groups for the analysis of several molecules in MALDIMS. The IM exhibited enhanced sensitivity and good shot-to-shot and sample-to-sample reproducibility due to the more homogeneous mixing of matrix with analytes.20-23 Lemaire et al. reported the use of IM in MALDI-IMS for direct tissue analysis to identify peptides in rat brain tissue sections with better shot-to-shot reproducibility, improved ionization efficiency, and better images of molecules.24 Chana et al. also proposed the application of IM to know the localization of different types of gangliosides in mouse brain.25 In the present study, IM of R-cyano-4-hydroxycinnamic acid butylamine (CHCAB) and 2,5-dihydroxybenzoic acid butylamine (DHBB) were synthesized and exploited for the analysis of phospholipids in MALDI imaging to obtain an enhanced signal intensity and better ion image quality and number of phospholipids on the surface of tissue sections. Phospholipids are essential cellular constituents possessing multiple, distinct critical roles in cellular function. The majority of cellular phospholipids form a membrane bilayer whose integrity and physical characteristics are vital for life processes. In animal tissue, the primary phospholipids include phosphatidylcholine (PC), lyso-phosphatidylcholine (LPC), phosphatidylserine (PS), phosphatidylethanolamine (PE), phosphatidylinositol (PI), and sphingomyelin (SM).26 Various matrixes have been reported for identification and characterization of lipids in MALDI-MS, including 2,5-dihydroxybenzoic acid (DHB), 2,6-dihydroxyacetophenone (DHAP), p-nitoroaniline (PNA), and 9-aminoacridine (AA). However, PNA and DHAP were reported to be unstable under high vacuum conditions and started to sublimate after introduction into the MALDI-MS.27,28 In the present work, we compared the use of CHCAB and DHBB IM with other matrixes for the identification of phospholipids from standard phospholipid mixtures and liver tissue sections in MALDI-IMS analysis. In addition, DHB and CHCAB IM were also applied to analysis of the mouse cerebellum to identify the species of lipids in different layers of tissue sections. Finally, we demonstrated the application of CHCAB IM for the analysis of tryptic digested cytochrome c in MALDI-MS. EXPERIMENTAL SECTION Chemicals and Solution Preparations. Trifluoroacetic acid (TFA) was obtained from Sigma (St. Louis, MO). DHB, DHAP, and R-cyano-4-hydroxycinnamic acid (CHCA) were purchased from Bruker Daltonics (Bremen, Germany). AA and PNA were purchased from Acros Organics (Morris Plains, NJ) and Fluka (20) Armstrong, D. W.; Li-Kang, Z.; He, L.; Gross, M. L. Anal. Chem. 2001, 73, 3679–3686. (21) Laremore, T. N.; Zhang, F.; Linhardt, R. J. Anal. Chem. 2007, 79, 1604– 1610. (22) Tholey, A.; Heinzle, E. Anal. Bioanal. Chem. 2006, 386, 24–37. (23) Crank, J. A.; Armstrong, D. W. J. Am. Soc. Mass Spectrom. 2009, 20, 1790– 1800. (24) Lemaire, R.; Tabet, J. C.; Ducoroy, P.; Hendra, J. B.; Salzet, M.; Fournier, I. Anal. Chem. 2006, 78, 809–819. (25) Chana, K.; Lanthiera, P.; Liua, X.; Sandhua, J. K.; Stanimirovica, D.; Li, J. Anal. Chim. Acta 2009, 639, 57–61. (26) Nagle, J. F.; Tristram-Nagle, S. Biochim. Biophys. Acta 2000, 1469, 159– 195. (27) Wang, H. Y. J.; Jackson, S. N.; Woods, A. S. J. Am. Soc. Mass Spectrom. 2007, 18, 567–577. (28) Estrada, R.; Yappert, M. C. J. Mass Spectrom. 2004, 39, 412–422.

(Steinheim, Germany), respectively. n-Butyle amine (BA) was obtained from Nacalai Tesque Inc. (Kyoto, Japan). The standard phospholipids mixture of SM from bovine heart, PC from bovine liver, PS from porcine brain, and PI were obtained from Avanti Polar Lipids Inc. (Alabaster, AL). The methods to prepare the standard phospholipid solutions and matrix solutions are described in the Supporting Information. Safety steps were maintained during the handling of organic solvents and chemicals used in the experiments. Synthesis of Ionic Matrixes. IM of DHB and CHCA were synthesized according to the available protocols.20,29 Equivalent molar ratios of matrixes (i.e., DHB and CHCA) with BA were added separately to 10 mL of methanol in a polyethylene vial and shaken for 1 h at room temperature. Each mixture was placed in a vacuum distillation apparatus until the evaporation of methanol. The resulting products of IM, i.e., the mixtures of DHB and BA and CHCA and BA, were named DHBB and CHCAB, respectively. IM were further placed into desiccators to eliminate the remaining solvent from the IM and stored in a refrigerator until the MALDIIMS analysis. Preparation of Mouse Cerebellum and Liver Tissue Sections. Experimental procedures for mouse samples were performed in accordance with the Animal Experiment Regulations of Hamamatsu University School of Medicine, which were approved by the Science Council of Japan. A male mouse purchased from Japan SLC (Shizuoka, Japan) was sacrificed. The cerebellum and liver tissue were immediately dissected from the body and stored in liquid nitrogen at -80 °C until the preparation of tissue sections. The cerebellum and liver tissue samples were sliced into 15 µm-thick sections by a cryostat (CM 1950; Leica Microsystems, Wetzler, Germany) and placed onto an indium tin oxide (ITO) glass substrate (Bruker Daltonics). The prepared matrix solutions were applied to the surface of the tissue sections using a 0.2 mm nozzle caliber airbrush (Procon Boy FWA Platinum, Tokyo). In the present study, serial sections of tissue were used to minimize the sample variability in IMS analysis using different types of matrixes. The effects of solvents in the matrix solution on the identification of phospholipids are described in the Supporting Information (see Figure S1A-H). Procedure for the Analysis of Standard Phospholipids Mixtures. Standard phospholipid mixtures (1 µL containing 40 pmol) were spotted onto the wells of a target plate by a pipetter. The droplets of the sample were coated with matrix solution using an airbrush and kept for vacuum drying at room temperature followed by MALDI-IMS analysis to investigate the homogeneity, signal enhancement, and reproducibility. Operating Conditions for MALDI-IMS. All MALDI-IMS studies were performed by an ultraflex II TOF/TOF (Bruker Daltonics). A 355 nm wavelength was used for desorption and ionization of samples with a repetition rate of 200 Hz. Four hundred shots were collected from each measurement point in the positive or negative ion mode. The setting of laser energy, detector gain, and random walk function were optimized in order to achieve better sensitivity of the target molecules during the IMS analysis. The calibration of the instrument was performed with bradykinin and angiotensin II at m/z 757.39 [M + H]+ and 1046.54 [M + H]+ in positive ion mode and 755.34 [M - H](29) Mank, M.; Stahl, B.; Boehm, G. Anal. Chem. 2004, 76, 2938–2950.

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Figure 1. Crystal formation of (A) DHB, (B) CHCA, (C) DHBB, and (D) CHCAB matrixes with phospholipids onto a MALDI target plate. The pictures were taken with an Ultraflex II TOF/TOF. The scale bar (white color line) is 100 µm. E-H shows the ion image of phospholipids reconstructed obtained using (E) DHB, (F) CHCA, (G) DHBB, and (H) CHCAB matrix at m/z 703, 731, 760 and 786. I-L shows the signal enhancement. 3-7-fold enhancement of signal intensity when DHBB IM (J) was used as a matrix compared to DHB matrix (I) and 50-100fold improvement of signal intensity using CHCAB IM (L) as compared to CHCA matrix (K). M-O shows the six replicate analyses of samples with RSD, % using (M) DHB: (20.5-40.8%; (N) CHCA: (29.5-45.8%; (O) DHBB: (14.5-21.8%; and (P) CHCAB: (7.5-10.0%.

and 1044.54 [M - H]- in negative ion mode prior to MALDIIMS analysis of each sample. flexImaging software (Version 3.0) was used to reconstruct the ion images of phospholipids. The mass peaks (at m/z) obtained were normalized to the total ion current, and then, the peak intensity was taken in account to study the phospholipids in MALDI-IMS. Thirty points of the region of interest (ROI) were exported from each ion image of phospholipids to compare the enhancement of signal intensity and reproducibility using different matrixes in MALDIIMS (the script program was included in the flexAnalysis, Bruker Daltonics). RESULTS AND DISCUSSION Sample Homogeneity for the Identification of Phospholipids by MALDI-IMS. The crystal formation of the DHB, CHCA, DHBB, and CHCAB matrixes with standard phospholipids mixture is shown in Figure 1A-D. Figure 1A shows rough and uneven distribution of crystal when the mixtures of DHB and phospholipids are deposited onto a MALDI target plate. Similarly, the heterogeneous distribution of crystals of CHCA with phospholipids can be 8802

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observed on the surface of MALDI target plate (Figure 1B). Though a smooth surface is obtained using DHBB IM (Figure 1C), some white colored crystals, distributed unevenly, are noticed. On the other hand, the most homogeneous and transparent thin layer was obtained when CHCAB IM was applied for the analysis of phospholipids in MALDI-IMS (Figure 1D). The sample homogeneity was confirmed by ion images formed for SM at m/z 703 [SM(d18:1/16:0) + H)]+ and 731 [SM(d18:1/18:0) + H)]+ and PC at m/z 760 [PC(16:0/ 18:1) + H]+ and 786 [PC(18:1/18:1) + H]+ using DHB, CHCA, DHBB, and CHCAB matrixes in the MALDI-IMS analyses (Figure 1E-H). A wide variation of the signal intensity for the ion images of SM and PC was observed when DHB (Figure 1E) and CHCA (Figure 1F) were used as matrixes, probably due to heterogeneous crystal formation of matrix with analyte molecules, as can be seen in Figure 1A,B. In the case of DHBB IM, the signal responses were not similar at all the regions of ion image for the respective SM and PC (Figure 1G). On the other hand, a nearly equal signal intensity of ion images of phospholipids was observed when CHCAB IM was used as a matrix in the MALDI-IMS analyses (Figure 1H).

Figure 2. MALDI-IMS spectra of phospholipids of mouse liver tissue sections obtained with (A) DHB, (B) DHBB, (C) CHCA, and (D) CHCAB IMs matrixes, where high signal intensities of lipids were obtained when CHCAB IM used as matrix as compared to DHB. The identified molecular species of phospholipids obtained by different matrixes are summarized in Table S1, Supporting Information.

The acidic phospholipids such as PI and PS were selected for MALDI-IMS analysis using CHCA, DHB, DHBB, and CHCAB as matrixes in negative ion mode. The ion images formed using these matrixes at m/z 861 [PI(18:0/18:2) - H]- and 885 [PI(18:0/20: 4) - H]- for PI and m/z 788 [PS(18:0/18:1) - H]- and 834 [PS (18:0/22:6) - H]- for PS are shown in Figure S2A-D (see Supporting Information). Better ion images and 6-18-fold improvements in the signal intensity were obtained when CHCAB IM was used as a matrix for desorption and ionization of PI and PS in MALDI-IMS as compared to other matrixes. The detailed information can be found in the Supporting Information. Signal Enhancement for the Identification of Phospholipids by MALDI-IMS. Signal enhancement was calculated from the values obtained by summation of a 30 ROI point from ion images of SM and PC species against the corresponding matrixes. The representative mass spectra are shown in Figure 1I-L. Improvements of 3-7-fold in the signal intensity were obtained when DHBB IM (Figure 1J) was used as a matrix as compared to DHB (Figure 1I). However, when CHCAB IM (Figure 1L) was used as a matrix for the determination of SM and PC, 50-100-fold improvements in the signal intensity were obtained as compared to the use of CHCA as a matrix (Figure 1K). The enhancement of the signal intensity of phospholipids when IM was used was attributed to the high solubility of analytes and the formation of a homogeneous thin

layer of matrix, which might have caused the effective desorption and ionization of molecules from the surface of the sample.30 Reproducibility for the Identification of Phospholipids by MALDI-IMS. The homogeneity in the crystal formation with phospholipids and different matrixes can again be confirmed by calculating the relative standard deviation percentage (% RSD) by six replicate analyses of the same concentrations of phospholipids in MALDI-IMS (Figure 1M-P). The peaks at m/z 703 and 731 for SM and m/z 760 and 786 for PC were the most intense signals and, thus, were selected for RSD determination (sample-to-sample reproducibility). A higher value of RSD was obtained using CHCA (29.5-45.8%, in Figure 1N) than DHB (20.5-40.8%, in Figure 1M). This type of discrepancy was also observed by Mank et al., who suggested that it was due to a single scattered isolated matrix-sample cocrystallization for CHCA matrix.29 The RSD values obtained using DHBB IM were between 14.5% and 21.8% for the analysis of SM and PC, as shown in Figure 1O. The finding that higher values of RSD were obtained with DHBB IM might have been due to the formation of some white crystals, as shown in Figure 1C. However, good values of RSD (7.5-10.0%) were obtained with CHCAB IM for MALDI-IMS analysis of SM and PC, as shown in Figure 1P. Similarly, we determined that the shot-to-shot reproducibilities for DHB, CHCA, (30) Li, Y. L.; Gross, M. L. J. Am. Soc. Mass Spectrom. 2005, 16, 679–682.

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Figure 3. (A) HE-stained mouse cerebellum showing three layers with 1.5 mm scale bar (white color). The ion images of lipids in mouse cerebellum tissue section obtained using (B) CHCAB and (C) DHB as a matrix at m/z 734.5 [(PC(16:0/16:0) + H)]+, 770.5 [PC(16:0/16:1) + K]+, 772.5 [PC(16:0/16:0) + K]+, 798.5 [PC(16:0/18:1) + K]+, 834.5 [PC(18:0/22:6) + H]+, and 870.5 [PC(18:1/22:6) + K]+ were localized in the molecular layer of cerebellum; at m/z 760.5 [PC(16:0/18:1) + H]+, 832.5 [PC(18:0/20:4) + Na]+, 844.5 [PC(16:0/22:6) + K]+, and 846.5 [PC(18: 1/20:4) + K]+ were specific to the granular layer; and at m/z 756.50 [PC(16:0/16:0) + Na]+, 810.5 [PC(18:0/18:1) + Na]+, 824.5 [PC(18:0/18:2) + K]+, 826.5 [PC(18:0/18:1) + K]+ were found to be concentrated in the white matter of cerebellum. The ion images at m/z 769.5 [SM(d18:1/ 18:0) + K]+ and 835.6 [SM(d18:1/24:1) + Na]+ illustrated that the molecules were distributed in the region of molecular layer of tissue. The ion images at m/z 822.6 [GalCer(d18:1/22:0) + K]+ and 850.6 GalCer(d18:1/24:0) + K]+ were localized in the white matter of mouse cerebellum. “ND” means the molecules were not detected.

DHBB, and CHCAB were in the ranges of 17.5-37.5%, 25.4-38.8%, 12.5-18.6%, and 4.8-7.0%, respectively. These results demonstrated that the exploitation of CHCAB IM is very useful for obtaining a homogeneous crystal with phospholipids, yielding good reproducibilities in MALDI-IMS analyses. Limit of Detection in MALDI-IMS. The optimized experimental conditions of MALDI-IMS were used to calculate the limit of detection (LOD) for standard PC (16:0/18:1) and PI (18:1/18: 1) in positive and negative ion modes, respectively, using DHB and CHCAB matrixes. The reason for the selection of DHB and CHCAB matrixes is due to know the performance of conventional matrix (DHB) against the IM for the analysis of phospholipids in MALDI-IMS. The LOD was based on a signal-to-noise ratio (S/ N) of 3 obtained from flexAnalysis software when 30 ROI points were selected from the ion image of phospholipids. The LODs obtained for PC using DHB and CHCAB as a matrix were 200 and 40 fmol in positive ion mode. Similarly, the LODs obtained for PI using DHB and CHCAB as a matrix were 500 and 80 fmol in negative ion mode (data are not shown). 8804

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Imaging of Phospholipids in the Mouse Liver Tissue Sections by MALDI-IMS. Mouse liver was selected to evaluate the potentiality of IM for imaging of phospholipids in tissue sample by MALDI-IMS. The mouse liver is known to be an almost homogeneous structure at all regions and ideal for analyzing ion images obtained from tissue sections. In this study, we tested three other matrixes, i.e., AA, PNA, and DHAP, for the analysis of phospholipids in mouse liver tissue sections in addition to the use of DHB, CHCA, DHBB, and CHCAB matrixes. Figure 2A-D shows the MALDI-IMS spectra of phospholipids obtained from mouse liver tissue sections using DHB, DHBB, CHCA, and CHCAB matrixes. The mass spectra obtained using AA, PNA, and DHAP are shown in the Supporting Information (Figure S3A-C). The ion images of the respective phospholipids reconstructed from MALDI-IMS spectra of mouse liver tissue sections (ion images are not shown) using different types of matrixes and the mass peaks (m/z) observed are given in Tables S1 and S2 (Supporting Information). The listed molecules were verified by MS/MS analyses (Figure S4A-D, Supporting Information), and a detailed

description can be found in the Supporting Information. We found that the use of CHCAB IM in MALDI-IMS can increase the signal intensity and the number of phospholipids, such as PE and PS, in the mass spectrum in addition to the detection of PC. However, the use of AA, DHAP, DHB, and PNA matrixes resulted in the detection of PC molecules in the MALDI-IMS analysis. The reason for the higher signal intensity and detection of a greater number of phospholipid signals in the mouse liver tissue when CHCAB was used as a matrix was probably attributable to the good solubility of analytes, the formation of a homogeneous thin layer of matrix, and the high vacuum stability of the IM, which might have caused effective desorption and ionization of a greater number of molecules from the surface of the tissue sections. Thus, the IM of CHCAB was selected for further tissue analysis. Imaging of Phospholipids in the Mouse Cerebellum Tissue Sections by MALDI-IMS. The cerebellum is known to be an interesting structure for the visualization of various types of lipids in positive ion mode. The cerebellum is a trilobed structure of the brain situated posterior to the pons and medulla oblongata and is responsible for the regulation and coordination of complex voluntary muscular movement as well as the maintenance of posture and balance.31 The cerebellum is composed of three main layers. The outer gray matter (i.e., the cerebral cortex) makes up the molecular layer (ML) and the granular layer (GL), while the third layer, known as the white matter (WM), is found within the gray matter. Figure 3A shows and hematoxylin and eosin (HE)-stained mouse cerebellum tissue section with the three different layers labeled. In the present study, we applied CHCAB and DHB matrixes to the surface of the cerebellum tissue section and analyzed at a scan pitch of 50 µm using MALDI-IMS. The mass peaks (m/z) obtained using CHCAB IM and DHB in MALDI-IMS analysis were reconstructed to form an ion image of lipids in the different layers of cerebellum tissue as relative signal intensities, as shown in Figure 3B,C. The ion images of lipids, such as PC, SM, PS, and galactosylceramide (GalCer), found in the mouse cerebellum tissue sections were verified using MS/ MS analyses, and the results were consistent with the results of previous studies10,16,19 (Table 1). All the ion images of phospholipids visualized the trilobed structure of the cerebellum with distinguishable distribution of biomolecules in the tissue sections. The molecular species of PC with protonated, sodiated, and potassiated ions were major components present in the cerebellum obtained using CHCAB and DHB as the matrix in the MALDI-IMS analysis. The ion images at m/z 734.5, 770.5, 772.5, 798.5, 834.5, and 870.5 showed that the PC species found were localized in the molecular layers of the cerebellum; at m/z 760.5, 832.5, 844.5, and 846.5 were specific to the granular layer; and at m/z 756.5, 810.5, 824.5, and 826.5 were found to be concentrated in the white matter of the mouse cerebellum tissue, as shown in Figure 3B. The ion images at m/z 769.5 and 835.6 illustrated that the molecules were distributed in the region of molecular layer of tissue section. Next, we evaluated the results of GalCer found in the cerebellum at m/z 822.6 and 850.6 which were localized in the white matter of the mouse cerebellum. The results obtained were similar to those reported by Jackson et al., who found that the GalCer was detected (31) Timmann, D.; Daum, I. Cerebellum 2007, 6, 159–162.

Table 1. Species of Lipids Identified in Mouse Cerebellum Tissue Section Using CHCAB and DHB as a Matrix in Positive Ion Modea experimental experimental mass (m/z) mass (m/z) theoretical by CHCAB by DHB mass (m/z) 734.52 756.50 760.55 769.54 770.54 772.50 782.53 788.57 798.51 800.52 804.53 806.56 810.56 820.49 822.59 824.53 826.52 832.54 834.56 835.65 844.49 846.51 848.53 850.62 852.48 856.55 864.60 870.50 872.54 a

734.53 756.52 760.56 769.51 770.52 772.51 798.49 806.52 810.56 820.50 822.55 824.50 826.51

844.48 848.51

734.57 756.55 760.58 769.56 770.51 772.53 782.57 788.61 798.54 800.56 804.55 806.57 810.60 820.53 822.62 824.56 826.57 832.58 834.60 835.67 844.53 846.54 848.56 850.65 852.52 856.58 864.63 870.54 872.56

molecular species of lipids PC(16:0/16:0) + H PC(16:0/16:0) + Na PC(16:0/18:1) + H SM(d18:1/18:0) + K PC(16:0/16:1) + K PC(16:0/16:0) + K PC(16:0/18:1) + Na PC(18:0/18:1) + H PC(16:0/18:1) + K PC(16:0/18:0) + K PC(16:0/20:4) + Na PC(16:0/22:6) + H PC(18:0/18:1) + Na PC(16:0/20:4) + K GalCer(d18:1/22:0) + K PC(18:0/18:2) + K PC(18:0/18:1) + K PC(18:0/20:4) + Na PC(18:0/22:6) + H SM(d18:1/24:1) + Na PC(16:0/22:6) + K PC(18:1/20:4) + K PC(18:0/20:4) + K GalCer(d18:1/24:0) + K PS(18:0/20:3) + K PC(18:0/22:6) + Na GalCer(d18:1/h24:1) + K PC(18:1/22:6) + K PC(18:0/22:6) + K

The listed m/z confirmed by MS/MS analyses.

by MALDI-ion mobility-TOFMS.32 Therefore, we conclude that the use of CHCAB is helpful for the identification of a greater number of lipids (such as GalCer and PS in addition to PC and SM), and a higher intensity of ion images of molecules in the cerebellum is obtained as compared to the use of DHB as a matrix in MALDI-IMS. The reason is the same as stated for the identification of phospholipids in liver tissue sections with CHCAB IM. Next, we compared our results with those reported by Puolitaival and co-workers for MALDI imaging of phospholipids in mouse brain tissue sections using a dry-coating technique for the deposition of matrix on the tissue samples.19 We found that the ion images of lipids at m/z 769.5 (SM), 772.5 (PC), 826.5 (PC), and 844.5 (PC) in the mouse cerebellum were comparable between the two methods, but the number of lipids detected by CHCAB IM was greater than that detected by the dry coating method. Thus, the ion images formed by MALDI-IMS were used to differentiate the different species of phospholipids found in the three layers of mouse cerebellum using CHCAB IM and DHB matrixes. Taking these results into consideration, we concluded that the IM of CHCAB was better for imaging of phospholipids present in mouse cerebellum tissue sections in order to localize the specificity particular to biomolecules in different layers of the tissue sample. Analysis of Tryptic Digested Protein. We also investigated the application of CHCAB IM to the analysis of digested cyto(32) Jackson, S. N.; Ugarov, M.; Egan, T.; Post, J. D.; Langlais, D.; Schultz, A.; Woods, A. S. J. Mass Spectrom. 2007, 42, 1093–1098.

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Figure 4. MALDI-MS spectra of tryptic digested cytchrome c using (A) CHCA and (B) CHCAB as a matrix. The mixture solution (containing 100 nM cytochrome c with 1:30 of trypsin to protein ratio) was mixed followed by reduction and alkylation process for protein treatment. The solution was incubated overnight at 37 °C and then analyzed using CHCA and CHCAB as a matrix in MALDI-MS. The observed peaks (m/z) and peptide sequence of digested cytochrome c is listed in Table S3, Supporting Information.

chrome c by MALDI-MS analysis. Figure 4A,B shows the mass spectrum of tryptic digested cytochrome c using a CHCAB and CHCA matrix. The use of IM for the analysis of digested cytochrome c increased the signal intensity (10-fold) and number of peptide sequences in the mass spectrum as compared to CHCA matrix (see Table S3 in Supporting Information). CONCLUSIONS The use of CHCAB IM in MALDI-IMS has been successfully demonstrated for the imaging of various species of phospholipids in mouse liver and cerebellum tissue sections. Improvements (10-12-fold) in the signal intensity with a greater number of signals of molecules in tissue sample can be observed when CHCAB IM used as a matrix as compared to other matrixes. The study of the distribution of these biomolecules in different layers of mouse cerebellum tissue samples can provide critical insights into biological processes in both healthy and diseased tissue

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samples. In the near future, we anticipate that this proof-of concept work could be certainly useful for MALDI-IMS analysis of protein and peptides in biological tissue sections. ACKNOWLEDGMENT We would like to thank the Japanese Society for the Promotion of Science, Japan, for a postdoctoral fellowship (to K.S). This work was also supported by a Grant-in-Aid for SENTAN from the Japan Science and Technology Agency (to M.S.). SUPPORTING INFORMATION AVAILABLE Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org. Received for review April 13, 2010. Accepted September 23, 2010. AC102422B