Dithranol as a MALDI Matrix for Tissue Imaging of Lipids by Fourier

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Dithranol as a MALDI Matrix for Tissue Imaging of Lipids by Fourier Transform Ion Cyclotron Resonance Mass Spectrometry Cuong H. Le,†,‡ Jun Han,† and Christoph H. Borchers*,†,‡ †

University of Victoria-Genome BC Proteomics Centre, University of Victoria, Vancouver Island Technology Park, 3101−4464 Markham Street, Victoria, British Columbia V8Z 7X8, Canada ‡ Department of Biochemistry and Microbiology, University of Victoria, 3800 Finnerty Road, Victoria, British Columbia V8P 5C2, Canada S Supporting Information *

ABSTRACT: To fill the unmet need for improved matrixes for matrix-assisted laser desorption ionization (MALDI) tissue imaging of small molecules, dithranol (DT)a matrix mainly used for the analysis of synthetic polymerswas evaluated for detection of lipids in rat liver and bovine calf lens, using MALDI Fourier transform ion cyclotron resonance mass spectrometry (FTICR MS). The use of DT resulted in better detection of endogenous lipids than did two other commonly used matrixes, α-cyano-4-hydroxycinnamic acid (CHCA) and 2,5-dihydroxybenzoic acid (DHB), with >70 lipid entities (including phosphatidylcholines, phosphatidylethanolamines, sphingomyelins, phosphatidylserines, phosphatidylglycerol, phosphatidic acids, ceramide phosphates, sterol lipids, acyl carnitines, and glycerides) being detected in rat liver and bovine lens tissue sections, using positive-ion detection. Using saturated DT in chloroform/methanol (2:1, v/v), with 1% formic acid in the final mixture, 57 lipid entities were successfully imaged from bovine calf lens, with clear and distinct distribution patterns. In a section across the lens equatorial plane, all compounds showed concentric distributions around the lens nucleus and most showed specific abundance changes, which correlated with lens fiber cell age. As a novel finding, palmitoylcarnitine and oleoylcarnitine were found uniquely localized to the younger lens fiber cell cortex region. This work demonstrates the potential of DT as a new matrix for tissue imaging by MALDI-FTICR MS.

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sublimation,16 and matrix-precoated MALDI MS,17 among others, have been developed for enhancing MSI of small molecules. In addition, the use of ultrahigh-resolution instruments, such as Fourier transform ion cyclotron resonance (FTICR) mass spectrometers, has the ability to resolve analyte signals from matrix signals, and this can partially overcome many problems associated with the matrix background.18 The reduction in the intensities of the metastable matrix clusters19 by FTICR MS can also help to overcome some of the interferences associated with matrix peaks in other instruments.20 The search for new matrixes that enhance the detection of low-molecular weight compounds while generating lowerabundance background signals in the low-mass region remains an important aspect of small-molecule MSI. For example, 9aminoacridine has been successfully used for MALDI-MS of protic analytes,21 as well as for tissue imaging of endogenous compounds such as nucleotides and phospholipids in the negative-ion mode.22,23 2-Mercaptobenzothiazole is another matrix which has been found to give efficient MALDI detection of lipids,24 and this matrix has been successfully used for

ass spectrometry imaging (MSI) allows for simultaneously mapping the spatial localization and distribution patterns (by measuring the ion abundances) of multiple compounds at the surface of a thin tissue section.1 Using matrix-assisted laser desorption/ionization (MALDI), MSI of proteins and peptides has been in development for over a decade, and great improvements have been made in sample preparation, detection sensitivity, spatial resolution, analysis reproducibility, and data processing.2 By correlating MS ion maps with stained histological images, pathologists can link the distributions of specific compounds of interest to pathophysiology.3 In addition to peptides and proteins, MALDI-MS tissue imaging has been also used for the analysis of small molecules, including exogenous drugs4,5 and their metabolites.6−8 Among endogenous compounds, lipids have been extensively investigated using tissue imaging by MALDI-MS,9−14 including the use of MALDI-MS/MS.15 Extensive application of MALDI-MS to the imaging of lowmolecular weight compounds on tissue has been limited by several factors. One of such factors is that most of the commonly used MALDI matrixes generate a multitude of abundant ion signals in the low-mass region (typically m/z < 500) which suppress the ionization and interfere with the detection of the small-molecule analytes. A variety of techniques including solvent-free matrix coating, matrix © 2012 American Chemical Society

Received: July 12, 2012 Accepted: August 29, 2012 Published: August 29, 2012 8391

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imaging gangliosides species in mouse brain.25 In the positiveion mode, 2,5-dihydroxybenzoic acid (DHB) and α-cyano-4hydroxycinnamic acid (CHCA) have been the two most popular matrixes for MALDI MS and for MSI. DHB tends to form large crystals which could cause molecular delocalization and poor spot-to-spot reproducibility in MSI. DHB matrix sublimation has been shown to partially overcome this weakness and has allowed the use of this matrix for sensitive imaging of phospholipids.16,26 Recently, matrix sublimation for several matrixes has been further investigated by Thomas et al.,27 and 1,5-diaminonapthalene was found to be an efficient matrix for profiling the rich lipid signatures in mouse brain and fish tissue, in both positive- and negative-ion modes, with high vacuum stability and sub-20 μm resolution. In addition, ionic matrixes have been used to enhance tissue imaging of phospholipids by MALDI-MS.28 In this work, we have evaluated dithranol (DT: 1,8dihydroxy-9,10-dihydroanthracen-9-one), a common MALDI matrix used for the analysis of synthetic polymers,29 for the MALDI profiling and imaging of endogenous lipids on the surface of mammalian tissue sections, by positive-ion MALDIMS on an ultrahigh-resolution quadrupole−FTICR instrument. To our knowledge, DT has not previously been reported as a MALDI matrix for tissue imaging.

MALDI-MS. All MALDI MS experiments were performed on an Apex-Qe 12T hybrid quadrupole−FTICR mass spectrometer (Bruker Daltonics, Bremen, Germany), which was equipped with an Apollo dual-mode electrospray ionization (ESI)/MALDI ion source. This MALDI source has a 200 Hz solid-state Smartbeam Nd:YAG UV laser (Azura Laser AG, Berlin, Germany). The MS instrument was tuned and calibrated with the “ES tuning mix”, by infusing a 1:200 dilution of this tuning mix in 60:40 isopropyl alcohol/water (containing 0.1% formic acid in the final mixture), at a flow rate of 2 μL/min, introduced from the ESI side of the ion source. Mass spectra were acquired from m/z 200 to 1400 in the positive-ion mode, with broad-band detection. A data acquisition rate of 1024 kBps was used. For on-tissue profiling, simultaneous ESI and MALDI operations were carried out so that each mass spectrum contained the reference mass peaks for post-acquisition internal mass calibration. Because of the larger size of bovine calf lens tissue sections (typically >1 cm in diameter), MSI data sets were acquired at a laser raster step size of 250 μm, and each scan (pixel) was averaged from 100 laser shots. Data Analysis and Processing. For MALDI tissue profiling, mass spectra were processed using Bruker’s DataAnalysis software suite. For tissue imaging, images for all of the lipid entities detected, across the entire tissue section, were generated using Bruker’s FlexImaging software, with a mass filter width of 1 ppm at the peak apex. The MALDI mass spectra were internally calibrated, the ions were deisotoped, and the monoisotopic peaks were picked as described previously, using a customized VBA script.30 The resulting monoisotopic peak lists were then exported and input into the METLIN31 and/or the HMDB32 metabolome databases for mass matching with the measured m/z values. During the database search, the (M + H)+, (M − H2O + H)+, (M + Na)+, and (M + K)+ ions were considered, with an allowable mass error of ±1 ppm. Only compounds which were detected in three replicates (S/N > 10) were included in the analysis. Assignment of the detected lipid entities was based on database searching and tandem mass spectrometry (MS/MS), which will be described below, and spectra from the literature. LC−MS/MS. Because the sensitivity of the 12T FTICR for MALDI-derived ions was not sufficient for MS/MS of many of the lower-abundance lipids, and because of the low-mass cutoff (ca. m/z 130) of the 12T FTICR, liquid chromatography− tandem mass spectrometry (LC−MS/MS) on 10 mg of tissue was performed for verification of analyte identities as described in the LC−MS section of the Supporting Information. For identification of the degradation products of DT that formed at high pH, a degraded matrix solution was infused at 2 μL/min with a syringe, and MS/MS was performed on the FTICR mass spectrometer in the negative-ion mode. Targeted ions were isolated individually using the quadrupole mass filter, with a 2 Da mass separation window around the target masses, and applying collision-induced dissociation (CID) in the hexapole collision cell followed by FTICR detection.

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EXPERIMENTAL SECTION Samples and Reagents. Rat liver and bovine calf lens specimens were obtained from Pel-Freez Biologicals (Rogers, AR). According to the accompanying sample information sheet, these specimens, once harvested, were flash-frozen with the aid of liquid nitrogen. These samples were shipped on dry ice and were stored at −80 °C until used for the MS experiments. Dithranol, CHCA, DHB, reserpine, and terfenadine were analytical reagent grade. Formic acid, ammonium formate, ammonium hydroxide, trifluoroacetic acid (TFA), water, methanol, acetonitrile, ethyl acetate, and isopropyl alcohol were LC−MS grade. Chloroform, acetone, and ethanol were HPLC grade. These solvents and reagents were purchased from Sigma-Aldrich (St. Louis, MO). The “ES tuning mix” standard solution, used for MS tuning and calibration, was obtained from Agilent Technologies (Santa Clara, CA). Matrix Coating. Various pH-adjusted solvents, including acetonitrile, 70:30 acetonitrile/water, or chloroform/methanol (2:1, v/v) were compared for preparing the matrix solutions. In all cases, the TFA, formic acid, or ammonium hydroxide was added after mixing, to give final solutions containing either 0.01% TFA, 1−10% formic acid, or 1−10% ammonium hydroxide. For matrix coating, the pH-adjusted acetonitrile or 70:30 acetonitrile/water solvents were applied to the surfaces of tissue sections using an ImagePrep electronic matrix sprayer (Bruker Daltonics, Bremen, Germany). Twenty cycles of matrix coating (2 s spray, 30 s incubation, and 60 s drying time) were used on the ImagePrep for the acetonitrile and 70:30 acetonitrile/water solvents. Due to the incompatibility of chloroform with the ImagePrep sprayer manufacturing material, a pneumatically assisted Eclipse HP CS airbrush sprayer (Anest Iwata Inc., West Chester, OH) was used for manual application of the chloroform-containing matrix solutions onto the bovine lens sections. To minimize any possible spread of the analytes due to the organic solvents, only the minimum amount of matrix solution was applied during each cycle of matrix spray so as to barely coat the tissue section. Approximately 10 cycles of matrix spray were applied in this way.

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RESULTS AND DISCUSSION Performance of DT as a MALDI Matrix for On-Tissue Detection. To determine whether DT when used “on tissue” generated significant matrix-related signals by MALDI-FTICR MS, a saturated solution of DT in 70:30 acetonitrile/water (with 0.01% TFA in the final mixture) was spotted onto a rat liver section and onto a tissue-free region of the same indium 8392

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Figure 1. Comparison of dithranol (a), DHB (b), and CHCA (c) as the MALDI matrixes for on-tissue detection of endogenous lipids in a liver tissue section. ∗ denotes the ESI-generated mass calibrant ions of the “ESI tuning mix”.

compared to 18 for CHCA and 17 for DHB from each of the tissue sections. The lipid identities were assigned using database searching, the MS/MS spectrum, and the literature, and most were shown to be polar lipids. The list of the identified lipid entities is shown in Supporting Information Table 1. All of the lipid entities detected with CHCA and DHB were also detected with DT, with the exception of a diacylglyceride (DG(22:4/ 14:0/0:0)) which was only detected with DHB. The use of DT resulted in the detection of many more lipid entities in the liver tissue than had been reported in a recent publication where only 13 phospholipids were detected from a mouse liver by MALDI-FTICR using CHCA as the matrix.34 The largest group of polar lipids detected corresponded to phosphatidylcholines (PCs), with 30 of the 70 lipid entities being PCs. Other lipid classes detected included sphingomyelins (SMs), phosphoethanolamines (PEs), phosphatidylglycerol (PGs), phosphatidic acids (PAs), and oxysterols. This preliminary comparison showed that, with the FTICR, DT was able to detect more lipid entities than the two most commonly used MALDI matrixes, DHB and CHCA. This indicates that DT could be a potentially useful matrix for MALDI tissue imaging using this instrument. The ability for a matrix to cocrystallize well with an analyte is a prerequisite for high-sensitivity MALDI-MS analysis, and the solid-phase solubility of an analyte in the matrix is important in the MALDI process, so the best analyte signal intensities come from MALDI matrixes with solubilities similar to those of the desired analytes.35,36 DT is a weak organic acid, as well as a very hydrophobic organic compound, which is expected to favor ionization of positively charged and less-polar compounds in the gas phase, based on classical Brønsted−Lowry acid−base neutralization theory37 and the theory of solubility.38 It is therefore not surprising that polar lipids dominated the detected compounds in the positive-ion mode when DT was used as the matrix.

tin oxide (ITO)-coated glass slide. At minimum laser power, it was shown that DT generated abundant DT-related background signals from the tissue-free matrix spots (Supporting Information Figure 1a), as had been previously observed.33 These background signals were identified as coming from DT oligomers and their corresponding sodium and potassium adducts. However, when DT was applied to the tissue sections, many of these background ions were much lower in abundance, or were not observable in the mass spectra under the same experimental conditions, as shown in Supporting Information Figure 1b. As a result of this observation, DT was then compared to two most commonly used MALDI matrixes, DHB and CHCA, for in situ detection of low MW compounds to determine what detection sensitivity could be achieved. To do this, terfenadine and reserpine were chosen as test compounds, and 1 μL aliquots of the standard solutions containing these two compounds, at different concentrations (1 μM to 1 pM), were spotted onto the surface of a liver tissue section. Again, DT produced more abundant signals for terfenadine and reserpine within the m/z 200−1400 mass range than did CHCA and DHB. Spotting 1 μL aliquots of a standard solution onto the surface of the liver tissue section generated a spot of ca. 1 mm in diameter. With optimal laser energies (i.e., those at which the strongest signals were observed for each of the compounds), the limits of detection (S/N = 3) were determined to be 0.01 and 1 pmol for terfenadine and reserpine, respectively, from a single sample spot with signals accumulated from an average of 100 laser shots with a 50 μm laser spot size. In comparison, the limits of detection (LODs) for terfenadine and reserpine were 10 and 100 pmol with DHB and 0.1 and 1 pmol with CHCA, respectively. Next, DT was compared with DHB and CHCA for the ontissue profiling of lipids by MALDI-FTICR MS. As shown in Figure 1, the use of DT on rat liver tissue sections led to the detection of 70 lipid entities from a single spectrum acquired as 8393

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Figure 2. Differentiation between lipids that are close in mass by MALDI-FTICR MS. (a) A zoomed-in mass spectrum acquired from the outer cortex region of a bovine calf lens section, with the phospholipid entities annotated. (b) The zoom-in (panel a) with a mass window of 0.2 Da for m/ z 744.4−744.6. Two peaks can be seen. The ions at m/z 744.49393 and m/z 744.530614 were assigned as [PC(30:0) + K]+ and [PE(34:0e) + K]+, respectively. (c) MALDI images showing different distribution patterns of [PC(30:0) + K] and [PE(34:0e) + K] on the equatorial tissue section. (d−m) MALDI images showing distribution patterns of the representative lipids which are summarized in Supporting Information Table 1. (d) The optical image of a bovine calf lens tissue section before MALDI-MSI; panels e−m are the ion maps of these lipids: (e) [PS(36:1) + K]+, (f) [PC(34:1e) + K]+, (g) [PE(35:1) + K]+, (h) [PC(32:1) + K]+, (i) [PA(34:1) + K]+, (j) [CerP(d18:1/24:1) + K]+, (k) [SM(18:0/24:1) + K]+, (l) [lyso-PAF(16:0) + K]+, and (m) [7-ketocholesterol + K]+. In each case, the area shown is 18.9 mm × 18.4 mm.

Effect of the DT Matrix Solvent on MALDI-MS Detection. The solvents used for preparing a matrix solution play an important role in direct tissue analysis by MALDIMS.39 To determine whether different pH matrix solvents would have an effect on the MALDI detection of lipids in a bovine calf lens section, several low-pH and high-pH solutions were tested by preparing DT in 70:30 acetonitrile/water with different concentrations of TFA, formic acid, or ammonium hydroxide in the final mixture. With 1% ammonia hydroxide in the matrix solution, 49 endogenous lipid signals were detected, 44 of which were the same as had been observed with TFA and

formic acid. However, the high-pH matrix solution underwent a visible color change, and a precipitate was observed several minutes after a fresh solution was prepared. DT is known to form a compound known as danthron as well as a condensed DT dimer under photodegradation.40 Negative-ion ESI/MS and MS/MS using CID on the FTICR instrument confirmed the presence of danthron (m/z 239.03515) and the DT dimer (m/z 449.10317) in the ammonium hydroxide containing DT matrix solution (data not shown). The formation of these compounds caused problems with reproducibility, and the precipitate could also cause major problems if mechanical 8394

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tissue section and the corresponding MALDI-MS ion maps of some representative lipid entities. Interestingly, although protonated and sodiated adducts were detected, the lipid entities were primarily detected in the form of potassium adducts. Strong potassium and sodium adducts have been previously reported in MALDI experiments on lens tissue.49 The use of the ultrahigh-resolution FTICR mass spectrometer was important not only for resolving the analyte signals from matrix signals but also for providing sufficient mass accuracy to resolve lipid signals with very similar m/z. For example, the difference between the theoretical m/z of the 2 × 13 C isotope peak for SM(18:1/16:0) and the monoisotopic peaks from SM(18:1/24:1) is ∼8 ppm, and this requires a resolving power of 125 000 (fwhm) for baseline separation. Using a lower-resolution instrument, these peaks would not have been successfully resolved and could not have been separately imaged, because the ion maps would contain intensities from both lipid entities. Similarly, SM(18:0/16:0) and SM(18:0/24:1) were also successfully imaged in our experiment because of the high resolution of FTICR MS. In an earlier study, these two entities were also localized on human lens tissue using desorption electrospray ionization (DESI) on an LTQ-Orbitrap instrument.50 These compounds showed similar distribution patterns between bovine and human species. A mammalian mature lens is an avascular tissue predominantly composed of lens fiber cells, and these lens fiber cells originate from a single layer of epithelial cells that migrate toward the center of a lens to form the nucleus during cell differentiation and lens development. These oldest cells in the nucleus region are compactly wrapped by the younger fiber cells, which form the cortex. Within a mammalian lens, there is little translocation of biological molecules, as cells are being compressed toward the nucleus throughout life. As has been previously reported, ocular lens is an organ that contains abundant lipids, primarily consisting of PCs, SMs (including dihydrosphingomyelins, DHSMs), and cholesterol,51 and these lipids play an important role in maintaining the integrity of the compact lens structure.51,52 The distribution patterns of the lipid entities, whose compositions have been shown to change in the older tissues of a porcine lens,47 may be relevant for lens opacification and cataractogenesis. Among the various lens lipids, cholesterol has been reported to show increased abundance as lenses age, while the total lipid content shows the opposite trend, and lower phospholipid levels are found in the older cells than in younger cells.49,52,53 The increase in the ratio of cholesterol to phospholipid as one moves toward the lens nucleus has been reported in both bovine and human species.52 In this present study, most of the lipid entities detected were found at higher concentrations in the outer cortex of the bovine calf lenses, but with some exceptions. CerPs, and SMs, especially the DHSMs, showed increased abundances in the middle to inner cortex. Parts j and k of Figure 2 show the distribution patterns of CerP(d18:0/ 24:1) and SM(18:1.24:0) detected in their potassium adduct form. These distribution patterns are in agreement with the results from previous DESI54 and MALDI-MSI55 experiments on human lenses. The compounds that were assigned as oxidized cholesterol derivatives appeared to be at higher concentrations in the nuclear region in the bovine calf lenses. Figure 2m shows the spatial distribution pattern of one of these compounds, 7-ketocholesterol or it isomer(s), across the lens tissue. 7-Ketocholesterol and other oxidized cholesterol derivatives have been linked to cataractogenesis, and their

matrix coating was used. Therefore, although the quality of the MALDI mass spectra and the number of the detected endogenous lipids actually improved slightly with the addition of ammonium hydroxide, a high-pH matrix solution was not evaluated further. Significant differences in the number of detected lipids were not observed with different concentrations of TFA or formic acid in acetonitrileapproximately 40 lipid entities being observed in all cases. Because of the hydrophobicity of DT and its limited solubility in acetonitrile and methanol, we next evaluated a lipophilic organic solvent. Because the primary focus of this experiment was the analysis of lipids, we used a mixture of chloroform/methanol (2:1, v/v) with 1% formic acid, which is a typical organic solvent for lipid extraction.41 This solvent system produced stronger lipid signals with low background levels, and a total of >70 lipid entities were detected from the same lens tissue. All of the 46 lipid entities which were observed with the acetonitrile solvents were also detected using chloroform/methanol as the solvent. The increased signal intensities may be due to better cocrystallization of these lipids with DT when the chloroform/methanol solvent is used. As has been observed for analytes in liquid extraction surface analysis mass spectrometry (LESA-MS),42 the lipophilic nature of the solvent may also help to prevent the solubilization of other biological components such as peptides and proteins which could affect the cocrystallization of the lipids with DT and the subsequent MALDI process. On the basis of these observations, this matrix−solvent system provided the best performance for MALDI detection of endogenous lipids in our experiments and was used for all of the subsequent imaging experiments. DT for Imaging Bovine Lens Lipids. Using a saturated solution of DT in chloroform/methanol (2:1, v/v), containing 1% formic acid in the final mixture as the matrix solution, 64 endogenous lipid entities were successfully detected and 57 of them were imaged in bovine lens tissues, within the mass range from m/z 200 to 1400 by positive-ion MALDI-FTICR MS. Figure 2a shows a zoomed-in region of the mass spectrum where multiple lipids entities were observed. Figure 2b shows a zoomed-in region of the mass spectrum showing the detection of two lipid entities with a ca. 50 ppm m/z difference. The distinctive ion maps generated from each of these two lipids can be seen in Figure 2c. The ion maps for the 57 lipid entities that were imaged at the equatorial plane clearly show their spatial distribution patterns. Metabolite database searches against METLIN and/or HMDB using the measured accurate masses, followed by MS/MS experiments, and the comparison of the MS/MS spectra with previously published data43−47 were performed to identify these lipid entities. These were assigned as PCs, PEs, SMs, PSs, PGs, PAs, ceramide phosphates (CerPs), sterol lipids, acyl carnitines, and glycerides. As expected, most of the observed lipids were found to be polar phospholipids, particularly PCs. The MS/MS fragmentation of PCs has been shown to produce a prominent peak at m/z 184.073 attributed to the polar PC headgroup, phosphocholine,48 as well as additional structurally important information which allowed the unambiguous identification of the molecules. In addition, 16 lipid entities were assigned as sterol lipids, though most of them (with the exception of cholesterol) could not be unambiguously assigned to unique identities due to difficulties in differentiating among their individual structural isomers, even with MS/MS data. All the 64 identified lipid entities are summarized in Supporting Information Table 2. Parts d−m of Figure 2 show the optical images of an imaged 8395

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Figure 3. MALDI tissue images of palmitoylcarnitine and oleoylcarnitine and their confirmation by LC−MS and LC−MS/MS. Panels a and c are the MALDI tissue images of palmitoylcarnitine and oleoylcarnitine, respectively. Panels b and d are the extracted ion chromatograms (XICs) of m/z 400.342 (palmitoylcarnitine) and m/z 426.358 (oleoylcarnitine), respectively, by LC−MS. (e) MS/MS spectrum of palmitoylcarnitine. The inset is a standard MS/MS spectrum of palmitoylcarnitine retrieved from the HMDB database (ref 32) with permission.

higher abundances in the nucleus may be related to this process.56 MALDI-MSI has been used for tissue imaging of ocular lenses in the mammalian species in order to determine the spatial distribution patterns of lens structural proteins57 and integral membrane proteins.58 Recently, there have been two papers published that used MALDI-FTICR MS for the localization of sphingomyelins,47 and PEs, PCs, and PAs46 in porcine lens, using CHCA as the MALDI matrix. Both papers use data from the same imaging experiment, which is one of only a few published experiments using MALDI-FTICR MS instruments for MSI of endogenous lipids. When CHCA was used, clear images could be produced for only a few highly abundant lipids.46 In contrast, when DT was used as a MALDI matrix, abundant signals were generated with significantly fewer summed spectra and a shorter acquisition time than when CHCA was used. Unique Distribution Patterns of Acyl Carnitines in Bovine Calf Lens. In addition to the above-mentioned phospholipids and sterol lipids, there were two imaged ions (at m/z 400.34201 and 426.35780) which showed consistently unique distribution patterns across the tissue sections imaged.

The distribution patterns for these two ions are displayed in Figure 3, parts a and c. These ions appeared to be exclusively localized to a thin sharply defined region in the outer cortex at the equatorial lens sections from bovine calves. Chemical formulas generated for these two peaks corresponded to two unique elemental compositions, C23H45NO4 and C25H47NO4, respectively, within a 1 ppm mass error and allowing an unlimited number of C, H, O, and N, and a maximum of 2 S and 2 P. Database searching against both the HMDB and the METLIN databases yielded palmitoylcarnitine as the only lipid candidate for m/z 400.34201 and oleoylcarnitine (or its isomers) as the only candidate for m/z 426.35780. The lowmass cutoff on the FTICR system used in this study is ca. 130 Da. This made it difficult to observe the characteristic fragment ion (m/z 85) for acyl carnitines by MS/MS. To confirm the distribution patterns as well as the identities of these two mass-matched acyl carnitines using a complementary technique, quadrupole time-of-flight (Q-TOF) LC− MS/MS experiments were conducted on lipid extracts from four tissue samples that had been manually dissected from the outer, middle, and inner cortex, and the nucleus regions of the same bovine calf lenses that had been previously imaged. Parts 8396

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imaging by MALDI-MS, and our work has demonstrated the usefulness of this new matrix. The distribution of acylcarnitines is a unique biological finding determined using this matrix and was confirmed using manual tissue dissection LC−MS/MS. This distribution pattern determined using this matrix has not previously been shown using any other method. This distribution pattern showed less palmitoylcarnitine and olenylcarnitine in the middle and inner cortex and the nucleus regions (where the older lens fiber cells are located). It also showed that these two acyl carnitines are localized outside a postulated extracellular diffusion barrier region within the ocular lens. Experiments to improve the matrix coating and sample preparation procedures in order to extend the use of DT as a general matrix for MS tissue imaging are currently underway.

b and d of Figure 3 show the overlaid extracted ion chromatograms (XICs) for these two compounds detected from the above respective tissue regions. As can be seen in these figures, the XICs for these ions show the same abundance distribution patterns as those from MALDI imaging (Figure 3, parts a and b). In the LC−MS chromatograms, the compound at m/z 400.342 showed the same retention time as that of the standard palmitoylcarnitine compound (data not shown). Comparison of the acquired MS/ MS spectrum for this compound (Figure 3e) with the standard MS/MS spectrum from the HMDB database32 (Figure 3e, inset) also confirmed the identity of m/z 400.342 as palmitoylcarnitine. Although no standard compound or reference MS/MS spectrum was available for oleoylcarnitine, the Q-TOF MS/MS spectrum of the chromatographic peak for m/z 426.358 showed fragment ions at m/z 357.1, 265.3, 144.1, and 85.0. Fragment ions at m/z 85.0 and m/z 144.1 are characteristic of acyl carnitines, while fragment ions at m/z 357.1 and 265.3 correspond to the loss of trimethylamine and free carnitine from oleoylcarnitine. On the basis of this MS/MS information, we feel that the ion at m/z 426.35780 most likely corresponds to oleoylcarnitine. To our knowledge, this is the first time that this unique distribution pattern of long-chain acyl carnitines in mammalian lens has been shown. Acyl carnitines are a group of intermediate lipids involved in mitochondrial fatty acid oxidation and transport of long-chain acyl groups from cytoplasm into mitochondria. This transport is opposed by carnitine which moves in the opposite direction across the mitochondrial membranes through an enzyme system composed of mitochondrial carnitine acylcarnitine translocase and carnitine palmitoyltransferases I and II.59 During lens development and aging, mature fiber cells lose their organelles including their mitochondria60 and the endoplasmic reticulum,61 which may be the reason for the lower concentrations of palmitoylcarnitine and acylcarnitine found in the middle and inner cortex and the nucleus regions, where older lens fiber cells are located. It has been reported that there is an extracellular diffusion barrier region around the middle cortex region of mammalian lens, and this barrier region restricts the movement of small molecules in and out of the lens core.62 Our data shows that the two acyl carnitines are distributed outside this barrier region, where the young lens fiber cells are located. It is also noted that the distribution patterns of the two observed acylcarnitines are very similar to those palmitoylated and oleoylated aquapirin-0, the most abundant integral membrane protein in mammalian lens,63 whose abundance decreases with lens fiber cell age.

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ASSOCIATED CONTENT

S Supporting Information *

Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Phone: 250-483-3221. Fax: 250-483-3238. E-mail: christoph@ proteincentre.com. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Genome Canada and Genome British Columbia for platform funding and support. We also thank Dr. Carol E. Parker for critical review of the manuscript and editing assistance. C.H.L. also thanks the British Columbia Proteomics Network for support.

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REFERENCES

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CONCLUSIONS In this work, DT was examined and compared to two commonly used MALDI matrixes as a potential new MALDI matrix for tissue imaging of small-molecule endogenous lipids, particularly polar lipids, by FTICR MS in the positive-ion mode. DT allowed for the detection of more lipids than CHCA and DHB as lipid entities detected with CHCA and DHB were also detected with DT. Combined with appropriate sample preparation, including formic acid prewetted tissue mounting, the use of a DT matrix on the MALDI quadrupole−FTICR mass spectrometer allowed the successful determination of spatial abundances of endogenous lipids, including two acyl carnitines, within the bovine calf lens. To our knowledge, this is the first time that DT has been used as a matrix for tissue 8397

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Article

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