Anal. Chem. 2007, 79, 8170-8175
MALDI-Linear Ion Trap Microprobe MS/MS Studies of the Effects of Dichloroacetate on Lipid Content of Nerve Tissue Rachelle R. Landgraf,† Timothy J. Garrett,‡ Nigel A. Calcutt,§ Peter W. Stacpoole,‡ and Richard A. Yost*,†
Departments of Chemistry and Medicine, University of Florida, Gainesville, Florida 32611, and Department of Pathology, University of California, San Diego, La Jolla, California 92093
The direct analysis of tissue from both the central and peripheral nervous systems of control rats and those administered the potential neurotoxin dichloroacetate (DCA) was investigated using an intermediate-pressure matrix-assisted laser desorption/ionization (IP-MALDI) source coupled to a linear ion trap (LIT) mass spectrometer. The matrix, 2,5-dihydroxybenzoic acid, was applied to the tissue using a novel automated inkjet printer system. The MSn capabilities of the LIT allowed identification of lipids desorbed directly from tissue. A marked decrease is observed in the intensity of lipid ions in spinal cord and sciatic nerve tissues from rats exposed to DCA. The results also demonstrate the rapid, sensitive, and semiquantitative capabilities of this method. The direct analysis of tissue sections by microprobe mass spectrometry provides an opportunity to identify changes in the spatial distribution of molecular species present within the tissue and the possibility of correlating these data to those obtained using histological techniques.1 When employed in conjunction with tandem mass spectrometry,2 superior specificity and structural elucidation can be achieved through the reduction of matrix background interferences and isolation of isobaric species. Spatial resolution and background reduction are of great importance when analyzing complex sample matrixes such as nerve tissue, where it has been shown that the lipid content of white and gray matter differ significantly.3 Although the interest of microprobe mass spectrometry is currently focused on imaging specific compounds in tissue,4 here we demonstrate the capabilities for rapid, sensitive, and semiquantitative analysis of tissue samples less than 70 µg. * Corresponding author. † Department of Chemistry, University of Florida. ‡ Department of Medicine, University of Florida. § University of California, San Diego. (1) Chaurand, P.; Schwartz, S. A.; Billheimer, D.; Xu, B. J.; Crecelius, A.; Caprioli, R. M. Anal. Chem. 2004, 76, 1145. (2) Garrett, T. J.; Yost, R. A. Anal. Chem. 2006, 78, 2465-2469. (3) 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. (4) Caldwell, R. L.; Caprioli, R. M. Mol. Cell. Proteomics 2005, 4, 394-401.
8170 Analytical Chemistry, Vol. 79, No. 21, November 1, 2007
Lipid composition greatly affects the functionality of neural membranes.5 Alterations in the head group, length of the fatty acid chains, and the degree of saturation are important factors of the physicochemical properties of membranes. Maintenance of biologically significant processes, including apoptosis and variance of activities of transporters and membrane-bound enzymes, is regulated by membrane lipids. In addition, lipids also serve as reservoirs for secondary messengers. The varying developmental patterns of lipids in differing regions of the nervous system give rise to a diverse ratio of major lipid components (sphingolipids and phospholipids).6 Marked changes in this lipid composition have been reported to occur in neurological disorders and can be manifested as changes in membrane fluidity and permeability.5 Therefore, analysis of the lipid profile of neural tissue is important to understand the mechanisms involved in neurological disorders. Of particular interest here is a neurological disorder involving the disturbance of mitochondrial function. The ability of mitochondria to efficiently convert substrate fuels into energy is a requisite for life among eukaryotic organisms, and interruption of this process can lead to several clinical complications, including lactate. Congenital forms of lactic acidosis arise from loss-offunction mutations in genes coding for respiratory chain and other mitochondrial enzymes.7-10 This results in an accumulation of lactate and hydrogen ions in blood, urine, and cerebrospinal fluid. Highly oxidative tissues, such as the nervous system, are particularly vulnerable to this perturbed state, and congenital lactic acidosis (CLA) is typically associated with progressive neurological and neuromuscular deterioration and early death. The investigational drug dichloroacetate (DCA) has been used in the chronic treatment of CLA, because of its lactate-lowering capabilities and (5) Farooqui, A. A.; Horrocks, L. A.; Farooqui, T. Chem. Phys. Lipids 2000, 106, 1-29. (6) Ishibe, T.; Yamamoto, A. J. Neurochem. 1978, 32, 1665-1670. (7) Robinson, B. H. Lactic Academia: Disorders of Pyruvate Carboxylase and Pyruvate Dehydrogenase. In The Metabolic and Molecular Basis of Inherited Disease, 9th ed.; Scriver, C. R., Beaudet, A. L., Sly, W. S., Valle, D., Eds.; McGraw-Hill: New York, 2001; Chapter 100, pp 2275-2296. (8) Stacpoole, P. W.; Gilbert, L. R. Pyruvate Dehydrogenasae Complex Deficiency. In Clinical Studies in Medical Biochemistry, 3rd ed.; Glew, R. H., Rosenthal, M. D., Eds.; Oxford University Press: New York, 2007. (9) Munnich, A.; Ro¨tig, A.; Cormier-Daire, V.; Rustin, P. Clinical Presentation of Respiratory Chain Deficiency. In The Metabolic and Molecular Basis of Inherited Disease, 9th ed.; Scriver, C. R., Beaudet, A. L., Sly, W. S., Valle, D., Eds.; McGraw-Hill: New York, 2001; Chapter 99, pp 2261-2274. (10) DiMauro, S.; Schon, E. A. N. Engl. J. Med. 2003, 348, 2656-2668. 10.1021/ac0713555 CCC: $37.00
© 2007 American Chemical Society Published on Web 09/26/2007
potential to increase cellular energetics.11-13 These effects are mediated by the interaction of DCA with the pyruvate dehydrogenase complex (PDC), which is located in the mitochondria. PDC catalyzes the rate-limiting step in the aerobic oxidation of glucose, pyruvate, and lactate. The regulation of PDC is in part controlled by reversible phosphorylation. DCA inhibits the kinase involved in phosphorylation and locks PDC in its unphosphorylated, active form.14 However, the use of DCA has been mitigated in some patients due to reversible peripheral neuropathy,12,13 which has also been demonstrated in dosed animals at exposure levels g50 mg/kg/day for several weeks or months.15 We hypothesized that DCA-associated neuropathy could be due in part to a change in lipids in both the central and peripheral nervous systems where lipid content can be as high as 70-85% by weight.9 Traditional analytical methods used for the analysis of lipids have lacked both speed and efficiency. These conventional techniques, which include thin-layer16 and gas17 chromatography, involve multistep sample preparation procedures that are laborintensive and time-consuming. High-performance liquid chromatography has gained popularity in lipid analysis due to the decreased need for sample preparation but still suffers from long elution times and complicated solvent systems.18,19 In this study, we demonstrate rapid lipid analysis using a novel matrix-assisted laser desorption/linear ion trap (MALDI-LIT) microprobe tandem mass spectrometer system and an automated MALDI matrix application procedure. Tandem MS capabilities (MS2 and MSn) allow lipid identification without complicated sample preparation on samples 70 µg or smaller. EXPERIMENTAL SECTION Instrumentation. Studies were carried out on a linear ion trap fitted with a MALDI ion source that operates at intermediate pressure (Finnigan LTQ with vMALDI, Thermo Fisher Corporation, San Jose, CA), as described previously.3 Briefly, a N2 laser with a wavelength of 337 nm is directed to the source by means of a fiber optic cable. Optics external to the vacuum chamber allow laser spot diameters in the range of 80-120 µm that are focused at an incident angle of 32° onto the sample plate. The source operates at a pressure of 0.17 Torr, which is ∼105 times higher pressure than a traditional high-vacuum MALDI source, but ∼104 times below that of an atmospheric pressure (AP) MALDI source. This intermediate pressure reduces the amount of in-source fragmentation, compared to traditional vacuum MALDI, and yields (11) Stacpoole, P. W.; Barnes, C. L.; Hurbanis, M. D.; Cannon, S. L.; Kerr, D. S. Arch. Dis. Child. 1997, 77, 535-541. (12) Kaufmann, P.; Engelstad, K.; Wei, Y.; Jhung, S.; Sano, M. C.; Shungu, D. C.; Millar, W. S.; Hong, X.; Gooch, C. L.; Mao, X.; Pascual, J. M.; Hirano, M.; Stacpoole, P. W.; DiMauro, S.; De Vivo, D. C. Neurology 2006, 66, 32430. (13) Stacpoole, P. W.; Kerr, D. S.; Barnes, C.; Bunch, S. T.; Carney, P. R.; Fennell, E. M.; Felitsyn, N. M.; Gilmore, R. L.; Greer, M.; Henderson, G. N.; Hutson, A. D.; Neiberger, R. E.; O’Brien, R. G.; Perkins, L. E.; Quisling, R. G.; Shroads, A. L.; Shuster, J. J.; Silverstein, J. H.; Theriaque, D. W.; Valenstein, E. Pediatrics 2006, 117, 1519-1531. (14) Stacpoole, P. W. Metabolism 1997, 46, 306-321. (15) Stacpoole, P. W.; Henderson, G. N.; Yan, Z.; Cornett, R.; James, M. O. Drug Metab. Rev. 1998, 30, 499-539. (16) Macala, L. J.; Yu, R. K.; Ando, S. J. Lipid Res. 1983, 24, 1243-1250. (17) Kuksis, A.; Stachnyk, O.; Holub, B. J. J. Lipid Research 1969, 10, 660-667. (18) Kim, H. Y.; Wang, T. C. L.; Ma, Y. C. Anal. Chem. 1994, 66, 3977-3982. (19) Taguchi, R.; Hayakaws, J.; Takeuchi, Y.; Tshida, M. J. Mass Spectrom. 2000, 35, 953-966.
a higher signal than that produced by AP MALDI. Two vacuumrated stepper motors control the two-dimensional movement of the sample plate. A set of modified quadrupole rods, q00, at the front of the multipole assembly permits entry of the laser beam and camera access to view the sample plate. The rest of the system is identical to that of a typical Finnigan LTQ. Animal Treatment and Tissue Preparation. All animal studies were approved by the local IACUC at UCSD and were performed in an AAALAC-approved vivarium. Adult (250-300 g body weight) female Sprague-Dawley rats (Harlan, San Diego, CA) were housed 3 per cage under a 12 h light-dark cycle with free access to food (Harlan Teklad 7001) and water. Animals that received DCA (sodium salt, TCI America, Portland, OR) were given a daily dose of 500 mg/kg by gavage for eight weeks after they achieved a weight of 250 g and were sacrificed 1 day after the final dose. Excised tissue was immediately flash frozen in liquid nitrogen and stored at -80 °C until shipped on dry ice in plastic tubes to the University of Florida, where it was again stored at -80 °C until processed. Frozen tissue was sectioned to a thickness of 10 µm at a temperature of -20 °C using a Leica CM1850 cryotome (Houston, TX). The tissue was attached to the cryotome sample stage using water or a water/ice slush to avoid the mass spectral interferences caused by traditional techniques that use polymers to affix the tissue to the sample stage. Tissue sections were allowed to thaw on glass microscope slides and then stored at -80 °C. Before matrix application, the tissue was allowed to dry for 30 min in a vacuum desiccator. Matrix was applied to the tissue using an Epson Stylus Photo R220 inkjet printer.20 Parameters within the Epson CD printing software were adjusted to give an even coat of matrix across the entire tissue section. Evenness was monitored visually by means of a dissecting microscope with transmitted illumination through a blue filter. The microscope slides were placed in a modified CD tray holder and passed through the printer 10 times. The matrix used was 2,5-dihydroxybenzoic acid (DHB) at a concentration of 40 mg/ mL in 70/30 methanol/water with an added 10 mM of sodium acetate in order to promote cationized lipids. Tissue Analysis. Microscope slides affixed with coated tissue were adhered to a modified MALDI sample plate using doublesided tape. The laser raster size was set to 100 µm, which equals the laser spot diameter. All experiments were performed with automatic gain control turned off, and the signal was optimized by manually adjusting the laser power and the number of laser shots fired on each tissue specimen. Full scan MS experiments were typically run at a relative laser power of 30 (arbitrary units) with 10 laser shots per step. MS2 and MS3 experiments usually required a relative laser power around 40 with 12 laser shots per step. Three serial sections of spinal cord from one control and three DCA-treated animals were each analyzed three times. Eight serial sections of sciatic nerve from three control and three DCAtreated animals were also each analyzed three times. Data were averaged over an entire tissue section with care taken to exclude signal from areas off of the tissue. Qualitative data were processed using standard Xcalibur software, and semiquantitative results were processed using Microsoft Excel. (20) Baluya D. L.; Garrett, T. J.; Yost, R. A. Anal. Chem. 2007, 79, 6862-6867.
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Table 1. Identification of Eight Abundant Lipid Ions Present in the Spinal Cord and Sciatic Nerve Tissues by MS2 and MS3, Showing the Major Neutral Losses (NLs) Observed and the Lipid Assignment Based on MS2 and MS3 Data MS2
MS3
m/z
observed major NLs
m/z transition
observed major NLs
peak assignmenta
753.4 756.4 782.4 798.3 810.4 835.5 837.5 850.3
59, 183 59, 183, 205 59, 183 59 59, 183, 205 59, 183 59, 183 18, 59, 87, 162, 180, 338, 366
753.4 f 694.5 756.4 f 697.4 782.4 f 723.4 798.3 f 739.3 810.4 f 751.4 835.5 f 776.5 837.5 f 778.5 850.3 f 791.4 850.3 f 763.3 850.3 f 688.4 850.3 f 670.4 850.3 f 512.3 850.3 f 484.4
124 124, 146, 256 124, 146, 256, 282 124, 162 124, 146 124 124, 146 124 282, 284 18, 366 18, 366 18, 162, 180 17, 162
[SPM(18:0) + Na]+ [PC(16:0,16:0) + Na]+ [PC(16:0,18:1) + Na]+ [PC(16:0,18:1) + K]+ [PC(18:0,18:1) + Na]+ [SPM(24:1) + Na]+ [SPM(24:0) + Na]+ [PC(36:1) + Na + K]+ [PS(18:0,18:1) + Na + K]+ [Cer(24:0hb) + Na]+ [Cer(24:0h) + Na]+ [Cer(24:0h) + Na]+ [Cer(24:0h) + Na]+
a Varying fatty acid tail: (x:y) x, number of carbons; y, number of double bonds. SPM, sphingomyelin; PC, phosphatidylcholine; PS, phosphatidylserine; Cer, cerebroside. b h denotes a fatty acid tail hydroxylated at the C2 carbon
Figure 1. (A) MS2 spectrum of the m/z 850.3 ion from the control, rat spinal cord tissue, and corresponding neutral losses. The losses indicate the presence of at least three isobaric lipids that have been identified as a phosphatidylcholine (PC), a phosphatidylserine (PS), and a cerebroside (Cer). (B) MS3 spectrum of m/z transition 850.3 f 763.3. The MS2 neutral loss of 87 is indicative of the loss of the serine head group (C3H6NO2) from a phosphatidylserine (PS). The MS3 major losses correspond to stearic acid (NL 284, C18:0) and oleic acid (NL 282, C18:1). This fragmentation allows identification of this ion as [PS(18:0,18:1) + Na + K]+ (structure shown as inset above).
RESULTS AND DISCUSSION Lipid Identification. Lipid ions produced by MALDI-MS are detected as singly charged ions in the region around mass-tocharge (m/z) 700-900.2 The ions chosen for analysis were identified using tandem mass spectrometry, and the major product ions are presented in Table 1 (for basic lipid structure see 8172 Analytical Chemistry, Vol. 79, No. 21, November 1, 2007
reference 18, Scheme 1). Ions of m/z 753.4, 756.4, 782.4, 798.3, 810.4, 835.5, and 837.5 predominately exhibit neutral losses (NLs) of 59, 183, and 205. A NL of 59 is consistent with the loss of trimethylamine from a phophatidylcholine (PC) or sphingomyelin (SPM) that has been cationized.2 With the use of the nitrogen rule, an ion can be identified as a PC or SPM based on its nominal
Figure 2. Mass spectra of the lipid region averaged over a section of sciatic nerve tissue collected from (A) control and (B) DCA-treated rats.
mass. Protonation and cationization of odd numbered PCs and even numbered SPMs result in the detection of even m/z values for PCs and odd m/z values for SPMs. NLs of 183 and 205 correspond to the loss of the phosphocholine head group without sodium and with sodium, respectively. Product ions resulting from the MS2 analysis of m/z 798.3 and 810.4 also include NLs equivalent to the losses of palmitic acid (NL 256, C16:0) and oleic acid (NL 282, C18:1) for m/z 798.3 and the losses of stearic acid (NL 284, C18:0) and oleic acid (NL 282, C18:1) for m/z 810.4. Comparison of intensities of the product ions allows for the assignment of the fatty acid tails. The more abundant product ion is assigned to the sn-1 carbon and the less abundant to the sn-2 carbon.21 While the abundance of these ions is less than 1% of the most abundant ion, the wide dynamic range of the linear ion trap22 affords correct structural identification of these low abundance ions. MS3 was performed on each MS2 product ion corresponding to the NL of 59 in order to identify fatty acid composition. NLs of 124, 146, and 162 correspond to the loss of ethyl phosphate without a cation, with sodium, and with potassium, respectively. Product ions arising from the transitions m/z 756.4 f 697.4 and 782.4 f 723.4 yield MS3 product ions matching the losses of palmitic acid (NL 256, C16:0) for both m/z transitions and oleic acid (NL 282, C18:1) for m/z 723.4. MS4 was performed on the MS3 product ions identified as SPMs; however, no further structural information was obtained. Therefore, identification of these ions was made using a database developed by us that contains a list of the (21) Han, X.; Gross, R. W. Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 10635-10639. (22) Schwartz, J. C.; Senko, M. W.; Syka, J. E. P. J. Am. Soc. Mass Spectrom. 2002, 13, 659-669.
molecular weights of major phospholipids with varying fatty acid composition and ionization states ([M + H]+, [M + Na]+, and [M + K]+). This classification is simplified because SPMs typically have only one varying fatty acid tail. When analyzing small molecules in a complex sample such as nerve tissue, the likelihood of detecting isobaric species is extremely high. One such example can be seen by performing tandem MS on m/z 850.3 from any one of the tissue samples used in this study. The MS2 spectrum of this ion taken from a control section of spinal cord is illustrated in Figure 1A. A NL of 18 is indicative of a loss of water, which is observed for both lipid ions and matrix ions. The NLs of 59, 87, and 162 suggest the presence of a PC, a phosphatidylserine (PS), and a cerebroside (Cer). The classification of these ions is described below. First, the NL of 59 (detected at m/z 791.4) is again attributed to the loss of trimethylamine, and MS3 of m/z 850.3 f 791.4 confirms the loss of ethyl phosphate producing a major ion at m/z 667.4. While no ions relating to the fatty acid composition were obtained from the MS3 spectrum, an expansion in the m/z region 530-600 of the 850.3 MS2 spectrum provides ions for possible identification. The product ions at m/z 594.6, 568.2, and 540.3 from the MS2 spectrum correspond to losses of palmitic acid (NL 256, C16:0), oleic acid (NL 282, C18:1), and gadoleic acid (NL 310, C20:1), respectively. With knowledge of these and the molecular weight, possible identification could include either [PC(18:0, 18:1) + Na + K]+ or [PC(16:0, 20:1) + Na + K]+. Once more, because no fatty acid composition was obtained from the isolation of m/z 791.4, this PC can be classified in general as [PC(36:1) + Na + K]+. Analytical Chemistry, Vol. 79, No. 21, November 1, 2007
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Figure 3. Ion intensity of eight abundant lipid ions found in (A) spinal cord tissue of one control (C7) and three DCA-treated (DCA1, DCA4, DCA5) rats and (B) sciatic nerve tissue of three control (C4, C5, C9) and three DCA-treated (DCA1, DCA4, DCA5) animals. The error bars are (1 standard error. Identification of each ion is the following (left to right): [SPM(18:0) + Na]+; [PC(16:0,16:0) + Na]+; [PC(16:0,18:1) + Na]+; [PC(16:0,18:1) + K]+; [PC(18:0,18:1) + Na]+; [SPM(24:1) + Na]+; [SPM(24:0) + Na]+; and isobaric lipids.
The second isobaric species exhibits a NL of 87 (detected at m/z 763.4), which represents the loss of the serine head group (C3H6NO2) from a PS. Fatty acid assignment is readily obtained from the MS3 transition of 850.3 f 763.3 (Figure 1B). The product ions at m/z 481.3 and 479.2 are a result of the losses of oleic (C18: 1) acid and stearic acid (C18:0), respectively. This ion is thus identified as [PS(18:0, 18:1) + Na + K]+. The final species identified at m/z 850.3 produces fragment ions that correspond to NLs of 162 and 180. These NLs indicate the losses of C6H10O5 from a galactose sugar and the entire galactose unit (C6H12O6) from a cerebroside. MS3 performed on the m/z transitions of both 850.3 f 688.4 f 322.3 and 850.3 f 670.4 f 304.3 exhibit a NL of 366, which also appears in the MS2 spectrum (850.3 f 484.3). The product ions at m/z 484.3 and 512.3 are typical fragments produced by a significant number of cerebrosides23 and result from the loss of the fatty acid tail from (23) Cha, S.; Yeung, E. S. Anal. Chem. 2007, 79, 2373-2385.
8174 Analytical Chemistry, Vol. 79, No. 21, November 1, 2007
the sphingosine base. The peak detected at m/z 512.3 in the MS2 spectrum is observed when fragmentation occurs after the carbonyl carbon on the amide linked fatty acid, and m/z 484.4 is a result of the loss of the entire fatty acid tail. It is likely that the NL of 366 is attributed to the loss of hydroxylated lignoceric acid (C24:0h) due to the elevated abundance of hydroxylated cerebrosides in neuronal tissue.24 This ion is therefore identified as [Cer(24:0h) + Na]+. Tissue Comparison. Figure 2 shows mass spectra (signal intensity from the first analysis of each section) averaged over the entire section of sciatic nerve from (A) control and (B) DCAtreated animals. Although the relative abundance of the lipid ions in tissue from both animal groups is quite similar, there is a 2.5fold decrease in signal in tissue exposed to DCA, compared to control tissue. The abundant ions in the m/z 700-900 region (24) Eckhardt, M.; Yaghootfam, A.; Fewou, S. N.; Zoller, I.; Gieselmann, V. Biochem. J. 2005, 388, 245-254.
correspond to phospholipids, as described above. Many of the abundant ions above m/z 900 originate from DHB matrix clusters (seen both on and off tissue). Ions below m/z 700 can be attributed to DHB matrix ions and possibly lysophospholipids (intermediates involved in biosynthesis and metabolism of phospholipids25). Lysophospholipids detected in this m/z region could be artifacts created during sample preparation; future experiments will be performed to identify ions in this lower m/z region. A comparison of the ion intensities of eight abundant lipid ions from control and DCA-exposed tissues is depicted in Figure 3. Data collected from spinal cord tissue of one control (C7) and three rats administered DCA (DCA1, DCA4, DCA5) are shown in Figure 3A. Three 10 µm serial sections were cut from the spinal cord of each animal and analyzed three times at a laser power of 30 with 10 laser shots per raster step across the entire tissue section. Because of ablation of matrix during MALDI, the signal decreased by an average factor of 1.7 between the first and second analyses and an average of 2.2 between the first and third analyses. Therefore, all intensities were normalized by a factor of 1.7 for analysis 2 and a factor of 2.2 for analysis 3. Data from sciatic nerve tissue of three control (C4, C5, C9) and three DCA-treated (DCA1, DCA4, DCA5) animals are shown in Figure 3B. In this case, eight serial sections of sciatic nerve from each animal were analyzed, and signal from only the first analysis of each tissue section was used for comparison purposes. The ion signal for the major lipids is reproducible ((20% SE) among the control and DCA-exposed tissue for both spinal cord and sciatic nerve. A consistent decrease in ion signal intensity between the control and DCA-exposed tissue is observed for both spinal cord and sciatic nerve (Table 2). The average decrease in the intensity of lipids measured in spinal cord tissue was 4.7, while the average decrease in the lipids measured in sciatic nerve tissue was 6.2. Preliminary studies have shown that a maximum of 80 ng/mg DCA is detected in nerve tissue (unpublished results). Experiments were conducted in which a solution of 80 ppm DCA was mixed with a solution of 10 ppm lipids on a standard MALDI plate, and no decrease in lipid ion signal was observed. This shows that the decrease in signal discussed previously is not due to ion suppression caused by the presence of DCA (data not shown). DCA administration was initially hypothesized to have a greater effect on peripheral nerves due to the manifestation of peripheral neuropathy with prolonged (25) Khaselev, N.; Murphy, R. C. J. Am. Soc. Mass Spectrom. 2000, 11, 283291.
Table 2. Factor Decrease of Ion Intensity of DCA-Treated Tissue Compared to Control Tissue m/z
peak assignmenta
753.4 756.4 782.4 798.3 810.4 835.5 837.5 850.3 average
[SPM(18:0) + Na]+ [PC(16:0,16:0) + Na]+ [PC(16:0,18:1) + Na]+ [PC(16:0,18:1) + K]+ [PC(18:0,18:1) + Na]+ [SPM(24:1) + Na]+ [SPM(24:0) + Na]+ isobaric lipids
spinal cord
sciatic nerve
average
4.5 5.9 5.2 5.2 4.7 4.1 4.1 4.0 4.7
4.7 5.8 7.1 4.8 6.6 7.3 7.9 5.1 6.2
4.6 5.9 6.2 5.0 5.7 5.7 6.0 4.6 5.5
a Varying fatty acid tail: (x:y) x, number of carbons; y, number of double bonds. SPM, sphingomyelin; PC, phosphatidylcholine; PS, phosphatidylserine; Cer, cerebroside.
dosing.12,15 However, these data show no statistical difference (tcalculated < ttable at 95% confidence) between the impact on the central nervous system and peripheral nervous system lipids. Further investigation of the effect of DCA on lipid synthesis in nerve tissue is required to explain this finding. CONCLUSIONS We demonstrated that IP-MALDI/MSn could be employed to analyze neuronal tissue for lipid identification and comparison. The data show the potential of this technique for rapid sample analysis (
