MALDI Imaging and Structural Analysis of Rat Brain Lipid Negative

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MALDI Imaging and Structural Analysis of Rat Brain Lipid Negative Ions with 9-Aminoacridine Matrix Christopher D. Cerruti,† Farida Benabdellah,† Olivier Laprévote,†,‡ David Touboul,*,† and Alain Brunelle† †

Centre de Recherche de Gif, Institut de Chimie des Substances Naturelles, CNRS, Avenue de la Terrasse, 91198 Gif-sur-Yvette Cedex, France ‡ Chimie Toxicologie Analytique et Cellulaire, EA4463, Faculté des Sciences Pharmaceutiques et Biologiques, Université Paris Descartes, Sorbonne Paris Cité, 4 avenue de l’Observatoire, 75006 Paris, France S Supporting Information *

ABSTRACT: Mass spectrometry imaging is of growing interest for chemical mapping of lipids at the surface of tissue sections. Many efforts have been devoted to optimize matrix choice and deposition technique for positive ion mode analyses. The identification of lipid species desorbed from tissue sections in the negative mode can be significantly improved by using 9-aminoacridine together with a robust deposition method, yielding a superior signal-to-noise ratio and thus a better contrast for the ion images in comparison to classical matrices such as α-cyano-4-hydroxycinnamic acid, 2,5dihydroxybenzoic acid, or 2,4,6-trihydroxyacetophenone. Twenty-eight different lipid species (phosphatidic acids, phosphatidylethanolamines, phosphatidylserines, phosphatidylglycerols, phosphatidylinositols, phosphatidylinositol-phosphates, and sulfatides) were scrutinized on rat brain tissue sections, and systematic MS/MS studies were conducted. It was possible to identify isobaric species differing by their fatty acid chains thanks to the improved sensitivity.

L

during CID experiments.16 In the negative ion mode the spectrum analysis is facilitated by the exclusive presence of [M-H]− ion signals formed from a large variety of ionized lipids, such as phosphatidic acids (PA), phosphatidylglycerols (PG),17 phosphatidylethanolamines (PE), phosphatidylserines (PS),18 phosphatidylinositols (PI),19 phosphatidylinositolphosphates (PIP),20 and sulfatides (ST).21,22 Moreover MS/MS experiments on such lipids generally lead to characteristic fragment ion signals corresponding to the carboxylates of the fatty acyl chains. The main difficulty of the identification of lipids directly on a tissue section comes from the large range of concentrations and from the very different ionization efficiencies of each species. It means that it is absolutely necessary to choose a matrix which induces the best signal-to-noise ratio for a maximum of lipid classes independently on their distinct physicochemical properties. The two most popular matrices for MALDI imaging in the negative ion mode are 2,5-dihydroxyacetophenone (DHA)23,24 and 2,5-dihydroxybenzoic acid (DHB).25,26 However, DHB tends to form large crystals which could cause molecular delocalization and poor spot-to-spot reproducibility whereas DHA sublimates under high-vacuum conditions, preventing detection after a short period of time.27−32 4-Paranitroaniline (PNA) was also used for MALDI imaging, but it presents the same drawback as DHA, i.e. a high vapor pressure.33−35 2,4,6-Trihydroxyacetophenone (THAP) is a recommended matrix for the analysis of

ipidomics has potentially an important role in the discovery of biomarkers, signaling pathway relationships and disease mechanisms. Cellular lipids are composed of hundreds of distinct chemical species that can be categorized based upon their class (polar headgroup), subclass (acyl, alkyl or alkenyl), and aliphatic chain components. Lipids can be localized on tissue sections using various chemical imaging methods, such as histochemistry,1 infrared,2 or fluorescence spectroscopies.3,4 Nevertheless, the chemical information deduced from these experiments is rather poor. Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS)5−7 has proven to be one of the most powerful ionization techniques for mass spectrometric analysis of biomolecules and in particular lipids.8−11 Direct MALDI analysis of tissue sections enables the acquisition of cellular expression profiles while maintaining the tissue integrity. By acquiring MALDI spectra at regularly spaced positions, it is possible to reconstruct the ion density map of any ion detected over the sample surface.12−15 Ideally, during the acquisition of a MALDI spectrum in the positive ion mode, one single ion peak for each molecular species should be obtained. Tissues are naturally rich in alkali metal ions, such as sodium and potassium, leading to unspecific cationization during MALDI process. The presence of multiple adducts of each lipid molecular species also markedly complicates the mass spectra and their interpretation. We recently demonstrated that the addition of lithium salts to the matrix solution allowed the detection of each lipid as a single intense [M+Li]+ ion peak and improved the structural identification © 2012 American Chemical Society

Received: August 25, 2011 Accepted: January 18, 2012 Published: January 18, 2012 2164

dx.doi.org/10.1021/ac2025317 | Anal. Chem. 2012, 84, 2164−2171

Analytical Chemistry

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collected and used as a matrix solution. Rat brain sections were homogeneously covered by the matrix solution using a TMSprayer (HTX Technologies, Carrboro, NC, USA) in a single coating step. This robot is equipped with a heated nozzle making a narrow aerosol of matrix droplets. The sample stage is moved below the thermal spray, allowing a regular matrix coating of a rat brain in less than 2 min. Experimental parameters were optimized in order to reach the best sensitivity and reproducibility. This system is coupled to an isocratic pump which allows a constant flow rate of 240 μL.min−1. The sample plate to receive the matrix is anchored on a stage moving in both x and y directions at a linear velocity of 120 cm.min−1. The spray is finally heated to 80 °C. MSI was performed using a 4800 MALDI TOF/TOF mass spectrometer from AB Sciex (Les Ulis, France) equipped with a 200 Hz tripled-frequency Nd/YAG pulsed laser (355 nm) and an electrostatic mirror, leading to a routine mass resolution of about 10 000 in the MS reflectron mode. The data were acquired in the negative ion reflectron mode at an accelerating potential of 20 kV and a delayed extraction time of 450 ns. The number of laser shots per pixel was set at 200, and the distance between two adjacent pixels was fixed to 50 μm, which roughly corresponds to the diameter of the crater formed onto the tissue surface. Mass spectra were recorded between m/z 200 and m/z 2000. External mass calibration was achieved using standard solutions of peptides (Pepmix 5, LaserBio Laboratories, Sophia Antipolis, France). MS images were recorded using 4000 Series Imaging software (www.maldi-msi.org, M. Stoeckli, Novartis Pharma, Basel, Switzerland) and processed using TissueView software (AB Sciex, Les Ulis, France). No isotopic correction is employed for the ion image calculation, and such an algorithm is still under development. For acquisitions of negative ion MS/ MS spectra onto the tissue surface, 1000 laser shots per pixel were needed, with a collision energy fixed at 2 keV. Air or argon was employed as collision gas at a pressure of 3 × 10−6 hPa.

various lipid classes including neutral storage lipids (triacylglycerols), polar membrane lipids (phospho-, sphingolipids, and glycosphingolipids), but it leads to the formation of large crystals which are not compatible with MALDI imaging.36 2-Mercaptobenzothiazole (MBT), which is a matrix already reported to be efficient to desorb and ionize large proteins,37 allowed the detection and identification of a wide variety of lipids in the negative mode.38 MBT offers a good S/N ratio and a high reproducibility from spot to spot, due in part, to its crystallization in very small and homogeneous crystals. Ion images recorded from a rat brain using MBT as a matrix exhibit fine anatomical structures,39 but the number of well-identified ion peaks between m/z 780 and m/z 880 is rather limited. 9-Aminoacrine (9-AA) was first introduced in 2002 by Vermillion-Salsbury and Hercules for protic analytes, and it has been mainly used for the detection of low molecular weight metabolites, such as nucleotides or phospholipids.38,40−46 Only one article reports the use of 9-AA for MALDI imaging of complex sulfatides SM3 and SB1 in mouse kidney.47 As far as we know, no extensive study of in situ lipid detection and identification was achieved yet. This makes incomplete the demonstration of the superiority of the 9-AA over the classical matrices. In this present work, 9-aminoacridine was tested in the negative ion mode as a matrix for the in situ detection of lipid species in rat brain sections. MALDI-MSI experiments were performed in order to exhibit the localization of these molecular species in tissue sections. Finally, structural analyses of the lipids were extensively carried out by tandem mass spectrometry (MS/MS) experiments, especially to differentiate isobaric species.

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MATERIAL AND METHODS Matrixes (CHCA, DHB, THAP, and 9AA) and solvents were purchased from Sigma-Aldrich (Saint-Quentin-Fallavier, France). All experiments on rat brains were performed in accordance with the protocols approved by the National Commission on animal experimentation and by the recommendations of the European commission DGXI. Male Wistar rats (IBAIC, Orsay, France) between 300 and 420 g were euthanized by an intraperitoneal injection of sodium pentobarbital (>65 mg.kg−1). The trimmed tissue blocks were immediately frozen in dry ice to prevent crack formation during freezing and stored at −80 °C prior to MS experiments. The frozen intact rat brain was cut at −20 °C with a cryostat (model CM3050-S; Leica Microsystems SA, Nanterre, France). Serial tissue sections of 12 μm thickness were immediately deposited onto a stainless steel plate and controlled before and after matrix deposition with an optical microscope (Olympus BX 51 fitted with x1.25 to x50 lenses, Olympus France SAS, Rungis, France) equipped with a Color View I camera, monitored by CellB software (Soft Imaging System GmbH, Münster, Germany). Before matrix application, tissue sections were dried under vacuum at a pressure of a few hPa during 30 min. Although the tissue blocks were held by an optimum cutting temperature (OCT) polymer, they were not embedded into it since any residual polymer might degrade the tissue surface.48 For MSI experiments, matrix solution (9AA) was prepared at 10 mg.mL−1 in ethanol/water (70/30, ν/ν). As the matrix is not completely soluble in this solvent mixture a centrifugation step at 14 000 rpm for 5 min is necessary. The supernatant is

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RESULTS AND DISCUSSION Sample Preparation. The crystallization quality and the signal-to-noise ratio of the m/z 888.8 negative ion peak (this peak is dominating the lipid mass range between m/z 700 to 1500) were first compared for three different common matrices and 9-AA, all manually spotted onto rat brain sections. This ion signal was previously attributed to a deprotonated sulfatide ST d18:1/24:1.21 Figure 1A to J shows, with two different microscope magnifications, the matrix aspect obtained with THAP at a concentration of 10 mg.mL−1 (Figure 1A and B), DHB at a concentration of 20 mg.mL−1 (Figure 1C and D), CHCA at a concentration of 20 mg.mL−1 (Figure 1E and F), 9-AA at a concentration of 10 mg.mL−1 (Figure 1G and H), all in methanol, and finally 9-AA at a concentration of 10 mg.mL−1 in ethanol (Figure 1I and J). The corresponding spectra are shown in Figure 1K to O, with the intensity of the sulfatide ion peak (ST) written in each spectrum. The THAP and DHB droplets exhibit the same morphology, i.e. a quite complete covering of the surface with individual crystals which are elongated aggregates (500 μm long) of small crystals (50 μm diameter). Such morphology is presumed to be responsible for the analyte delocalization on the tissue surface. Moreover, the corresponding recorded spectra (Figure 1K and L) exhibit only low intensity peaks in the lipid mass range, whereas matrix ion peaks are of high intensity. CHCA (Figure 1E and F) induces the formation of compact structures which necessitate a very 2165



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(m/z 255.2), C18:0 (m/z 283.2), C18:1 (m/z 281.2), C20:4 (m/z 303.2), and C22:6 (m/z 327.2) were also detected. This matrix preparation was thus privileged for the following MSI experiments. For a sensitive analysis, a compromise is to be found between a wet deposition, which is needed for an efficient extraction and mixing between the sample compounds and the matrix solution, and a dry deposition, which is needed to avoid delocalization. The morphology of the crystals which depends on the analyte/matrix cocrystallization conditions is known to strongly influence the MALDI-MS spectrum quality.49−52 DHB, CHCA, THAP, and 9-AA are soluble in water/methanol and water/ ethanol mixtures, but 9-AA precipitates with a solvent having a high water content (>50% water). Various tests were performed in order to find the best conditions, compatible with the robotic sprayer, and the ethanol/water mixture (70/30, v,v) finally appeared to be the best compromise for an homogeneous matrix deposition. MS Spectra of Different Brain Areas. Figure 2 gathers negative ion MALDI-MS spectra, with 9-aminoacridine

Figure 1. Pictures of matrix solution droplets deposited on tissue sections and corresponding negative ion MALDI mass spectra: (A-B) THAP (10 mg/mL mixed with 100% methanol); (C-D) DHB (20 mg/mL mixed with 100% methanol); (E-F) CHCA (20 mg/mL mixed with 100% methanol); (G-H) 9AA (10 mg/mL mixed with 100% methanol); (I-J) 9AA (10 mg/mL mixed with 70/30 ethanol/water (v/v)). K, L, M, O: corresponding negative ion MALDI-MS spectra. Intensity values annotated in the spectra (ST) correspond to the m/z 888.6 ion (sulfatide d18:1/24:1).

Figure 2. Negative ion MALDI mass spectra (matrix 9-aminoacridine) acquired from different areas of a rat brain section: A: corpus callosum; B: medulla oblongata; C: white matter part of cerebellum; D: gray matter part of cerebellum.

high laser intensity to acquire a mass spectrum (Figure 1M). Compared to the two previous matrices, signals in the lipid range were more intense, whereas the number of peaks remained low. It must be noted that the diameter of the ablated material over the surface is increasing when increasing the number of laser shots and the laser intensity, leading to a degradation of the spatial resolution. For 9-aminoacridine in methanol, the spectrum exhibits numerous signals with substantial signal-to-noise ratios in the lipid mass range (Figure 1N). Nevertheless, irregular elongated crystals (Figure 1G and H) were formed onto the sample surface, presumably leading to spatial delocalization. In order to overcome this limitation without degrading the mass spectrum quality, different solvent mixtures were compared. The crystallization of 9-AA in ethanol/water (70/30, v,v) shows small diameter crystals (10−100 μm) which can be compatible with MSI experiments at 50 μm spatial resolution (Figure 1I and J), and several tens of ion peaks in the lipid range with signal-to-noise ratios higher than 10 are observed in the corresponding spectrum (Figure 1O). It must also be noted that the fatty acid carboxylates C16:0

deposited by the robotic sprayer, from different anatomical areas of a rat brain section: corpus callosum (Figure 2A), medulla oblongata (Figure 2B), white matter of cerebellum (Figure 2C), and gray matter of cerebellum (Figure 2D). Under the experimental conditions already described, no signal corresponding to sulfated or phosphorylated glycosphingolipids was recorded above m/z 1000. This is because the ethanol/water mixture (70/30, v/v) of the solvent system was not compatible with glycolipids. Ammonium sulfate buffer and heptafluorobutyric acid (HFBA) would be needed for their detection, as described by Colsch et al.,23 but this leads to a poor sensitivity for the detection of species belonging to other lipid classes. In the m/z range between 700 and 1000, nearly thirty different ion peaks were detected in the corpus callosum. A similar spectrum was recorded in the medulla oblongata with only slight intensity changes, whereas ion peaks between m/z 850 and m/z 950 were 20 times overexpressed in the white matter. To the 2166



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and the pituitary can be distinguished at the level of the medulla oblongata according to a line-shaped signal on all images, whereas the fourth ventricle can be localized between the cerebellum and the medulla oblongata (Supporting Information, Figure S1). Near the corpus callosum a comma-shaped signal appears corresponding to the lambdoid septal zone. Below the corpus callosum, the stria medullaris of the thalamus is enlightened. To the right of the stria medullaris, two regions of high intensity in all images correspond to the fasciculus retrof lexus (top) and to the mammillothalamic tract (down). The images of the ions at m/z 699.5, 700.5, 701.5, 728.6, 744.6, 806.6, 821.6, 852.6, 862.6, 888.6, 904.6, and 935.6 indicate a colocalization in the brain, i.e. in the white matter part of the rat cerebellum, corpus callosum, and medulla oblongata (Figure 3B to I, K, and L to O). On the other side, the ions at m/z 885.6 and m/z 890.6 were found in the gray matter part of the cerebellum and in the striatum (Figure 3J and L). No major delocalization is noticed at the anatomical level from these images. Nevertheless it must be noted that an intense signal of 2−3 pixels large is detected at the edge of the tissue section in all images due to an edge effect. This effect is usually correlated to a small retraction of the tissue during its dehydration under vacuum leading to the deposition of a lipid fingerprint on the sample plate. Contrary to the positive ion mode where lipids can be protonated ([M+H]+) or cationized by sodium ([M+Na]+) or potassium ([M+K]+), the negative ion mode leads to the detection of only deprotonated [M-H]− ion species, allowing an easier attribution using lipid databases.46 For a preliminary structure determination, the LIPID MAPS Structure Database (LMSD) was first used.53 This tool, which is available for free on Internet, allows identifying possible lipid classes for each m/z value. The ions at m/z 699.5 and m/z 701.5 are thus belonging to the class of phosphatidic acids (PA). The ion at m/z 744.6 corresponds to a phosphatidylethanolamine (PE) or a plasmalogen phosphatidylserine (pPS), whereas the ions at m/z 700.5 and m/z 728.6 can be attributed to plasmalogen phosphatidylethanolamines (pPE). Phosphatidylserines (PS) are represented by the ions at m/z 806.6, m/z 852.6, and m/z 862.6. Signals at m/z 821.6, m/z 885.6, and m/z 935.6 can be assigned to phosphatidylglycerol (PG), phosphatidylinositol (PI), and phosphatidylinositolphosphate (PIP) species, respectively. The ion peaks at m/z 888.6 and m/z 904.6 exhibit a mass difference of 16 amu which can be attributed to the absence or the presence of a hydroxyl group. The first ion at m/z 888.6 is a sulfatide (ST), and the latter at m/z 904.16 is a hydroxylsulfatide (ST−OH). The total number of carbon atoms and insaturations can thus be simply given by the exact m/z value. Nevertheless, in order to determine the fatty acid chain lengths and confirm the lipid class, MS/MS experiments are required. In Situ Structural Characterization of Lipid Ions by Tandem Mass Spectrometry. When acquiring MS/MS spectra with a MALDI TOF/TOF, the precursor ion needs to be carefully selected because of the limited mass resolution of the electrostatic ion gate used for this purpose.54 Various lipid ion species have very close mass-to-charge ratio making them extremely difficult to be selected as a single precursor ion. Overlapping of isotopic patterns of lipids is frequently observed, leading to mix MS/MS spectra, which cannot be easily interpreted. A reduction to less than 1 amu for the precursor ion window selection greatly reduces the sensitivity, with the consequence that no MS/MS spectrum can be acquired for low intensity ions. For example, Woods and co-workers acquired MS/MS spectra of lipids in the negative ion mode using a 4700

opposite, the spectrum recorded in the gray matter of cerebellum led to low intensity ion peaks, and an over representation of signals at m/z 762.6, m/z 790.7, m/z 857.7, and m/z 885.7 was observed. It is thus clear that representative negative ion spectra of different anatomical areas of a rat brain can be recorded with excellent signal-to-noise ratios using 9-aminoacridine. Imaging. Figure 3 shows a microscope picture of a rat brain sagittal section (Figure 3A) from which negative ion images

Figure 3. Optical image and MALDI-TOF negative ion images (50 μm lateral resolution) of a sagittal rat brain section: A: Optical ion image of the sagittal rat brain section; B: m/z 699.5 ([PA36:2-H]−); C: m/z 701.5 ([PA36:1-H]−); D: m/z 744.6 ([PE36:1-H]−); E: m/z 700.6 ([Alkenyl-PE34:2-H]−); F: m/z 728.6 ([PE p36:2-H]−); G: m/z 806.6 ([PS38:6-H]−); H: m/z 852.6 ([PS42:11-H]−); I: m/z 862.6 ([PS42:6-H]−); J: m/z 890.6 ([PS-O38:7-H]−); K: m/z 821.6 ([PG40:6-H]−); L: m/z 885.6 ([PI38:4-H]−); M: m/z 935.6 ([PIP36:5-H]−); N: m/z 888.6 ([STd18:1/24:1-H]−); O: m/z 904.6 ([STd18:1/h24:1-H]−). Intensity (I) range is given for each ion image.

were recorded by MALDI-MSI (Figure 3B to O). The different distributions of lipid species allowed us to characterize some anatomical areas within the brain section. The medial lemniscus 2167



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deprotonated species. The ions at m/z 259 and m/z 257 are characteristic of the galactose 3-sulfate moiety and of a galactono-1,5-lactone 3-sulfate anion (Supporting Information, Figure S2), respectively. The ion at m/z 241 results from a successive loss of H2O from the m/z 259 ion leading to an anhydrogalactosylpyranose 3-sulfate. This fragmentation pattern allowed us to assign the m/z 888.6 ion to a sulfatide ST d18:1/24:1. The fragment ion peak at m/z 300 ion can be attributed to a 1-O-2′-aminoethenylgalactosyl 3-sulfate ion, thus confirming the presence of a sulfated sugar unit. A series of ions between m/z 580 and m/z 816 with an increment of 14 amu was also clearly detected, corresponding to charge remote fragmentation processes. The series was partially interrupted between m/z 748 and m/z 774 (Δm/z = 26) indicating that the double-bond on the 24:1-fatty acyl substituent is probably located at C-15, counting from the carbonyl carbon of the fatty acid.22 A similar fragmentation pattern was obtained when selecting the m/z 862.6 ion, and each attribution is reported in Table 1. The level of information obtained with our 4800 MALDI TOF/TOF mass spectrometer is thus clearly higher than the one obtained with a former 4700 MALDI TOF/TOF system. Figure 4B shows the fragment ion spectrum of the ion at m/z 904.6. The characteristic fragment ions at m/z 259, m/z 257, and m/z 241 lead to assign this ion to a hydroxysulfatide ST− OH d18:1/h24:1.21 This was confirmed by the detection of an ion at m/z 300 and of a group of five ions at m/z 507, m/z 522, m/z 540, m/z 550, m/z 568, corresponding to successive fragmentations after the loss of the fatty acid chain. Finally, a series of ions between m/z 652 and m/z 848 with a 14 amu increment is detected with a significant decrease of intensity for the ion at m/z 778 indicating the possible presence of a doublebond at C-15 position. Similar fragmentation patterns were obtained for m/z 822.6, m/z 850.6, m/z 878.6, m/z 906.6, m/z 932.6, and m/z 934.6 ions (data not shown). Figure 4C shows the MS/MS fragment ion spectrum of the m/z 885.6 precursor ion, which has already been assigned to a PI C18:0/C20:4 species.19 The m/z 241 fragment ion corresponds to the inositol phosphate headgroup, whereas the m/z 283 and m/z 303 fragment ions correspond to the carboxylates of stearic acid (C18:0) and of arachidonic acid (C20:4), respectively. The assignment of the fatty acid chain lengths is confirmed by the signals at m/z 581 and m/z 601 which are characteristic of the loss of the arachidonyl and stearyl chains as ketenes. Finally, fragment ions at m/z 419 and at m/z 439 are related to the loss of the inositol headgroup (−162 a.m.u.) plus the fatty acid chain. The same fragmentation pattern is obtained from the m/z 857.6 ion (data not shown). Figure 4D shows the MS/MS fragment ion spectrum of the m/z 834.6 precursor ion (PS C18:0/C22:6).18 The signal at m/z 747 is arising from the loss of 87 amu which indicates the presence of a serine headgroup, whereas signals at m/z 283 and m/z 327 are characteristic of carboxylates of stearic acid (C18:0) and of docosahexaenoic acid (C22:6). The other major fragment ions are formed from the successive losses of the serine headgroup and of the acyl chains as carboxylic acids (m/z 419 and m/z 463) or as ketene (m/z 437). The same fragmentation pattern is obtained for the m/z 762.6, m/z 786.6, m/z 788.6, and m/z 810.6 ions (data not shown). Compared to previous studies, signal-to-noise ratios of the fragment ion signals are greatly enhanced for PI and PS by the combination of 9-aminoacridine and a 4800 MALDI TOF/TOF instrument.

MALDI TOF/TOF mass spectrometer with a collision energy fixed at 1 keV, and air pressure was used in the collision cell.17 The obtained spectra displayed low signal-to-noise ratio signals, and the fragment ion peak characteristic of the headgroup was not detected below m/z 200. 9-Aminoacrine as a matrix together with a 4800 MALDI TOF/TOF enabled to systematically perform CID experiments on negative lipid ions leading to a signal-to-noise ratio large enough for their structure elucidation. Collision-induced dissociation (CID) of phospholipid ions at 2 keV collision energy (Figure 4A to D) systematically led to

Figure 4. in situ product-ion spectra of the [M-H]− following ions, recorded on a rat brain section: A: m/z 888.6; B: m/z 904.6; C: m/z 885.6; D: m/z 834.6; E: m/z 728.6; F: m/z 744.6; G: m/z 716.6; H: m/z 774.6. Insert in part G corresponds to the theoretical isotopic distribution of convoluted C18H33O2− (90%) and C18H35O2− (10%). Collision energy was set at 2 keV and air or argon was used as collision gas.

the detection of fragment ion peaks at m/z 79 ([PO3]−) and m/z 97 ([H2PO4]−), while sulfatide ions gave rise to the fragment ion peaks at m/z 80 ([SO3]−) and m/z 97 ([HSO4]−). Although intense, none of these ion peaks is specific of one single lipid subclass, and they will not be further considered in the following discussion. The product ion spectrum of the precursor ion at m/z 888.6 (Figure 4A) shows four characteristic fragment ion signals at m/z 390, m/z 259, m/z 257, and m/z 241. According to the fragmentation processes described by Hsu and Turk,22 the ion at m/z 390 corresponds to the elimination of the dihydroxy long chain base as an aldehyde (CH3(CH2)7CHCHCHO) or to the loss of (CH3(CH2)7CHCHCHO + H2) from the 2168



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Table 1. Mass Peak Assignments of Lipid Ion Species in Negative Ion MS Modea [M-H](m/z) 699.5 701.5 700.6

class PA Plasmalogen PE

726.6 728.6

716.6

MS/MS identification

36:2 36:1 p34:1

C18:1/C18:1 C18:0/C18:1 p18:1/C16:0 p16:0/C18:1 p18:1/Cl8:1 p18:0/C18:1 p16:0/C20:1 p20:1/C16:0 C18:0/C16:1 C18:1/C16:0 C18:0/C18:1 C16:0/C20:1 C18:0/C16:0 C18:1/C18:1 C18:0/C18:1 C18:0/C2O:4 C18:0/C22:6 PS p18:0/C18:0

p36:2 p36:1

PE

744.6 762.6 786.6 788.6 810.6 834.6 774.6

identification lipid maps

34:1 36:1

PS

Plasmalogen PS +PE

34:0 36:2 36:1 38:4 40:6 p36:1

[M-H](m/z)

localization WM CC WM CC MO WM CC MO

821.5 857.6 885.6 935.5 965.5 860.6 862.6 888.6 890.6 822.6 850.6 878.6 904.6 906.6 932.6 934.6

WM CC MO WM CC MO

WM CC MO WM CC MO WM CC WM CC WM CC WM CC WM CC WMS

MO MO MO MO MO

class

PG PI PIP ST

ST−OH

identification lipid maps

40:6 36:4 38:4 36:5 38:4

MS/MS identification PS o18:0/C18:0 PE p18:0/C22:6 ? C16:0/C20:4 C18:0/C2O:4 C16:1/C20:4 C20:4/C18:O d18:1/22:1 d18:1/22:0 d18:1/24:1 d18:1/24:0 d18:1/h18:0 d18:1/h20:0 d18:1/h22:0 d18:1/h24:1 d18:1/h24:0 d18:1/h26:1 d18:1/h26:0

localization

WM CC GM S GM S WM CC WM CC WM CC WM CC WM CC WM CC WM CC

MO

WM WM WM WM WM

MO MO MO MO MO

CC CC CC CC CC

MO MO MO MO MO MO MO

Table 1. continued a

WM, CC, and MO correspond to the white matter, the corpus callosum, and the medulla oblongata, respectively. PA: phosphatidic acid, PE: phosphatidylethanolamine, PS: phosphatidylserine, PG: phosphatidylglycerol, PI: phosphatidylinositol, PIP: phosphatidylinositolphosphate, and ST: sulfatide (ST). In bold, the lipid structures which are newly described in this article.

in order to show that the group of peaks at m/z 281, 282, 283, and 284 are not only corresponding the isotopic pattern of a C18:1 carboxylate but more likely of a mixture of C18:1 carboxylate plus a minor contribution of C18:0 carboxylate (C18H35O2−, m/z 283.2). More problematic is the situation of the ion at m/z 774.6, of which the fragment ion spectrum is shown in Figure 4H. This ion was previously attributed to a PE p18:0/C22:6.17 A careful scrutiny of the published MS/MS spectrum indicates ion peaks between m/z 280 and m/z 290 with a low abundance. Due to its very low intensity, it was very tricky to select the single ion at m/z 774.6 without selecting neighbor peaks at m/z 773.6 and m/z 775.6. The resulting mass spectrum is thus a combination of fragment ion signals from different species of close masses. In fact, the fragments at m/z 281 (C18:1) and m/z 283 (C18:0) can be related to the PS (alkyl18:0/C18:1 or o18:0/ C18:1) and PS (p18:0/C18:0), and the fragment at m/z 327 (C22:6) can be linked to the PE (p18:0/C22:6). The PS species exhibit an exact m/z value at 774.5655, whereas the PE species is at m/z 774.5443. A mass resolution of about 40 000 is therefore necessary to separate these two signals that is incompatible with the performance of our instrument (R∼15 000). Finally the minor fragment ion peaks at m/z 303 (C20:4) and 331 (C22:4) could be attributed to the phosphatidic acids C22:2/C20:4 and C20:2/C22:4 (m/z 773.5). The previous assignment to a PE p18:0/C22:6 is thus incomplete, indicating a complex situation. In such a case, it is clear that a high mass resolution instrument will be of great benefit for the MS and MS/MS mode. Nevertheless neither LTQ-Orbitrap nor FTICR instruments are able to select an ion signal with a window as narrow as 0.02 amu only, thus making impossible the complete structural identification of this lipid species in the MS/MS mode. Finally, ions at m/z 699.5, m/z 701.5, m/z 935.6, and m/z 965.6 were also subjected to MS/MS experiments confirming previous attributions.

More challenging was the MS/MS study of the ion at m/z 728.6 (Figure 4E). Due to a limited sensitivity, only a tentative assignment as PE p18:0/C18:1 was already proposed by Jackson et al. in agreement with the LIPID MAPS database.17 Here it was possible to select this ion and fragment it with a 2 keV collision energy. A signal at m/z 140 indicates the presence of an ethanolamine headgroup, whereas the major fragment ion peak at m/z 281 (oleate) confirms the previous attribution. More interestingly, fragment ions at m/z 255 and m/z 309 were also detected and correspond to fatty acids C16:0 and C20:1, respectively. As previously described by Hsu et Turk, CID fragmentation of deprotonated plasmalogen phospholipids leads to the formation of a single carboxylate ion corresponding to the fatty acid substituent at sn-2 position.55 This clearly indicates that the signal of the ion at m/z 728.6 which was directly desorbed from the tissue section can be attributed to three different isobaric lipid species, PE p18:0/C18:1 as well as PE p16:0/C20:1 and PE p20:1/C16:0. From our knowledge, it is the first time that such isobaric species are described directly from a brain tissue section. This experiment opens the opportunity to increase the number of lipid species directly desorbed from a biological sample thanks to a significant increase of sensitivity. Similar results were also obtained for the ions at m/z 700.6, m/z 726.6, and m/z 754.6, also corresponding to plasmalogens PEs. Similarly, the MS/MS spectrum of the m/z 744.6 ion (Figure 4F) unambiguously indicates the presence of four different fatty acids, in this case C16:0, C18:1, C18:0, and C20:1, leading to the characterization of two isobaric species PE C18:1/C18:0 and PE C16:0/C20:1. The fragment ions at m/z 460/462 and m/z 478/480 corresponding to the loss of C18:0 and C18:1 as carboxylic acids or ketenes, respectively, confirms that the PE (C18:1/C18:0) is the major species. Two other isobaric species, PE C18:0/C16:1 and PE C18:1/C16:0, were also distinguished in the fragment ion spectrum of the m/z 716.6 precursor ion which is shown in Figure 2G. In this case, the isotopic pattern of the C18:1 carboxylate (C18H33O2−, m/z 281.2) was simulated, as shown in the insert of Figure 2G, 2169



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CONCLUSION Mass spectrometry imaging can be used to determine the location of a large variety of molecular lipid species, and to elucidate their structure directly from a tissue section. In this paper, the use of both 9-aminoacridine as matrix and of a recent MALDI-TOF/TOF instrument can be considered as an efficient combination for in situ analysis of phospholipids and sulfatides in the negative ion mode. This approach is complementary to the use of DHA as matrix for specific analysis of gangliosides species. Other platforms based on Orbitrap or FTICR technologies are also of interest for MS imaging due to their high mass resolution (more than 30 000) and mass accuracy (

MALDI Imaging and Structural Analysis of Rat Brain Lipid Negative

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