Structure Elucidation of Retinoids in Biological Samples Using

for matching retention time with a commercially available or synthetic retinoid. ... of retinoids and didehydroretinoids in rainbow trout Oncorhyn...
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Anal. Chem. 2006, 78, 5719-5728

Structure Elucidation of Retinoids in Biological Samples Using Postsource Decay Laser Desorption/Ionization Mass Spectrometry after High-Performance Liquid Chromatography Separation Moo-Jin Suh, Xiao-Han Tang, and Lorraine J. Gudas*

Department of Pharmacology, Weill Medical College of Cornell University, 1300 York Avenue, New York, New York 10021

Retinoids [retinol (vitamin A) and its metabolites] function in the visual cycle, embryonic development, cellular differentiation, and tissue homeostasis. Notwithstanding pivotal roles of retinoids in mammals, the limited number of commercially available retinoid standards is a major roadblock to identifying and studying retinoids in biological samples. Therefore, a need exists for improved methods to identify retinoid metabolites. We analyzed polar and nonpolar retinoids, including retinoic acid, retinol, retinyl acetate, and other retinyl esters, using postsource decay laser desorption/ionization mass spectrometry (PSD-LDI MS). PSD analysis was employed to examine the PSD fragmentation patterns of retinoids, as these patterns can be used for the characterization of retinoids from biological samples without the need for matching retention time with a commercially available or synthetic retinoid. Mechanisms for the formation of these PSD fragment ions are proposed. The feasibility of employing PSD after HPLC separation was demonstrated by characterizing the endogenous retinoids in canine kidney epithelial cell extracts and in mouse lung. We show that the PSD-LDI MS approach described here can facilitate the identification and characterization of retinoids from mammalian cells and tissues. Vitamin A (retinol) and its natural analogues, known as retinoids, share a common molecular skeleton composed of a nonaromatic six-carbon ring structure with a polyprenoid side chain. Retinoids function in the visual cycle, embryonic development, cellular differentiation, and tissue homeostasis.1-3 The mechanisms by which retinoids regulate growth and differentiation of cells have not been fully elucidated, and bioactive retinoids continue to be discovered.4-7 For example, a metabolite of vitamin A, 11-cis-retinaldehyde, plays a role in the visual cycle as a * To whom correspondence should be addressed. Phone: (212) 746-6250. Fax: (212) 746-8835. E-mail: [email protected]. (1) Gudas, L. J. J. Biol. Chem. 1994, 269, 15399-15402. (2) Means, A. L.; Gudas, L. J. Annu. Rev. Biochem. 1995, 64, 201-233. (3) Mark, M.; Ghyselinck, N. B.; Chambon, P. Annu Rev. Pharmacol. Toxicol. 2006, 46, 451-480. (4) Moise, A. R.; Kuksa, V.; Blaner, W. S.; Baehr, W.; Palczewski, K. J. Biol. Chem. 2005, 280, 27815-27825. 10.1021/ac060492j CCC: $33.50 Published on Web 07/20/2006

© 2006 American Chemical Society

chromophore of visual pigments,8 whereas 4-oxo-retinol is a novel signaling molecule and regulator of cell differentiation.9 Retinol can be metabolized to various vitamin A metabolites including retinyl esters, which are a major storage form of vitamin A.10-12 In various epithelial tissues, retinyl esters account for the majority of the retinoids present.13 Recently, a retinol metabolite, 13,14dihydroretinol, produced by retinol saturase was identified.14 It has been shown that 13,14-dihydroretinoic acid plays a role as a potential ligand for nuclear receptors.4 High-performance liquid chromatography (HPLC) coupled with UV/visible spectrometry has been used for the identification of bioactive retinoids.15-17 Disadvantages of this approach in the identification of retinoids are the limited number of commercially available retinoid standards for the matching of retention time with a known compound. Also, because of their similar molecular skeletons, some retinoids may coelute during the HPLC separation, resulting in equivocal identification. Recently, HPLC has been combined with mass spectrometric approaches to provide both molecular mass information and fragment ion information for structural elucidation through MS/MS.18,19 (5) Golczak, M.; Kuksa, V.; Maeda, T.; Moise, A. R.; Palczewski, K. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 8162-8167. (6) Kagechika, H.; Shudo, K. J. Med. Chem. 2005, 48, 5875-5883. (7) Schmidt, C. K.; Volland, J.; Hamscher, G.; Nau, H. Biochim. Biophys. Acta 2002, 1583, 237-151. (8) Wald, G. Science 1962, 162, 230-239. (9) Achkar, C. C.; Derguini, F.; Blumberg, B.; Langston, A.; Levin, A. A.; Speck, J.; Evans, R. M.; Bolado, J., Jr.; Nakanishi, K.; Buck, J.; Gudas, L. J. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 4879-4884. (10) O’Byrne, S. M.; Wongsiriroj, N.; Libien, J.; Vogel, S.; Goldberg, I. J.; Baehr, W.; Palczewski, K.; Blaner, W. S. J. Biol. Chem. 2005, 280, 35647-35657. (11) MacDonald, P. N.; Ong, D. E. Biochem. Biophy. Res. Commun. 1988, 156, 157-163. (12) Liu, L.; Gudas, L. J. J. Biol. Chem. 2005, 280, 40226-40234. (13) Baron, J. M.; Heise, R.; Blaner, W. S.; Neis, M.; Joussen, S.; Dreuw, A.; Marquardt, Y.; Saurat, J. H.; Merk, H. F.; Bickers, D. R.; Jugert, F. K. J. Invest. Dermatol. 2005, 125, 143-153. (14) Moise, A. R.; Kuksa, V.; Imanishi, Y.; Palczewski, K. J. Biol. Chem. 2004, 279, 50230-50242. (15) Gundersen, T. E.; Blomhoff, R. J. Chromatogr., A 2001, 935, 13-43. (16) Guo, X.; Gudas, L. J. Cancer Res. 1998, 58, 166-176. (17) Randolph, R.; Simon, M. J. Biol. Chem. 1993, 268, 9198-9205. (18) Yu, K.; Little, D.; Plumb, R.; Smith, B. Rapid Commun. Mass Spectrom. 2006, 20, 544-552. (19) Langlois, I.; Oehme, M. Rapid Commun. Mass Spectrom. 2006, 20, 844850.

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Various ionization techniques have been employed for analysis of retinoids by MS. These include electron impact,4,20 atmospheric pressure chemical ionization,21-23 laser desorption/ionization (LDI),24,25 and electrospray ionization (ESI).26,27 Van Breenmen and Huang28 used ESI mass spectrometry to identify retinoids, including retinol, retinal, retinoic acid, and retinyl acetate. However, because these retinoids could not be ionized under the same ESI conditions, different polarities for the detection of polar and nonpolar retinoids were necessary. In addition, this ionization technique led to the formation of a protonated species with loss of a functional group of retinyl acetate, resulting in a lack of molecular mass information. Wingerath et al.24 demonstrated the use of LDI MS to identify retinyl esters in mouse liver. However, it is impossible to distinguish novel and known retinoids on the basis of their molecular masses alone because some retinyl esters such as retinyl palmitoleate (m/z 522) and 3,4-didehydroretinyl palmitate (m/z 522) are isobaric. After that work, Wingerath et al.25 used matrix-assisted laser desorption/ionization (MALDI) MS to analyze cyclic and acyclic analogues of retinoids, although the use of MALDI MS is severely handicapped by the strong interference of matrix ions in the detection of retinoids and other low molecular mass compounds. For confident characterization of retinoids, structural analysis is essential. Therefore, the MS/MS techniques, utilizing soft ionization sources, are highlighted for the structural elucidation of thermally labile biomolecules. These techniques result in improved characterization of retinoids.29-31 Mass spectrometry equipped with reflectron time-of-flight (TOF) is also able to analyze metastable fragment ions produced from a precursor ion and that allows for structural information about the precursor ion using postsource decay (PSD).32 PSD MS has been used to study synthetic polymers33,34 and biomolecules,35-38 but the characterization of retinoids by PSD MS has not been reported to date. (20) Furr, H. C.; Clifford, A. J.; Jones, D. A. Methods Enzymol. 1992, 213, 281290. (21) Li, H.; Tyndale, S. T.; Heath, D. D.; Letcher, R. J. J. Chromatogr., B 2005, 816, 49-56. (22) Ulven, S. M.; Gundersen, T. E.; Weedon, M. S.; Landaas, V. O.; Sakhi, A. K.; Fromm, S. H.; Geronimo, B. A.; Moskaug, J. O.; Blomhoff, R. Dev. Biol. 2000, 220, 379-391. (23) Winkler, P. C.; Perkins, D. D.; Williams, W. K.; Browner, R. F. Anal. Chem. 1988, 60, 489-493. (24) Wingerath, T.; Kirsch, D.; Spengler, B.; Kaufmann, R.; Stahl, W. Anal. Chem. 1997, 69, 3855-3860. (25) Wingerath, T.; Kirsch, D.; Spengler, B.; Stahl, W. Anal. Biochem. 1999, 272, 232-242. (26) McCaffery, P.; Evans, J.; Koul, O.; Volpert, A.; Reid, K.; Ullman, M. D. J. Lipid Res. 2002, 43, 1143-1149. (27) Wang, Y.; Xu, X.; van Lieshout, M.; West, C. E.; Lugtenburg, J.; Verhoeven, M. A.; Creemers, A. F. L.; Muhilal; van Breemen, R. B. Anal. Chem. 2000, 72, 4999-5003. (28) Van Breemen, R.; Huang, C. FASEB J. 1996, 10, 1098-1101. (29) Chithalen, J. V.; Luu, L.; Petkovich, M.; Jones, G. J. Lipid Res. 2002, 43, 1133-1142. (30) Kane, M. A.; Chen, N.; Sparks, S.; Napoli, J. L. Biochem. J. 2005, 388, 363369. (31) Ruhl, R.; Hamscher, G.; Garcia, A. L.; Nau, H.; Schweigert, F. J. Life Sci. 2005, 76, 1613-1622. (32) Spengler, B. J. Mass Spectrom. 1999, 32, 1019-1036. (33) Fournier, I.; Marie, A.; Lesage, D.; Bolbach, G.; Fournier, F.; Tabet, J. C. Rapid Commun. Mass Spectrom. 2002, 16, 696-704. (34) Rizzarelli, P.; Puglisi, C.; Montaudo, G. Rapid Commun. Mass Spectrom. 2005, 19, 2407-2418. (35) Al-Saad, K. A.; Siems, W. F.; Hill, H. H.; Zabrouskov, V.; Knowles, N. R. J. Am. Soc. Mass Spectrom. 2003, 14, 373-382. (36) Berhane, B. T.; Limbach, P. A. J. Mass Spectrom. 2003, 38, 872-878.

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We demonstrate herein that PSD-LDI MS is a highly effective method for structural elucidation of polar and nonpolar retinoids, including retinoic acid, retinol, retinyl acetate, and other retinyl esters. To test the suitability of the approach for the characterization of retinoids in biological samples, PSD-LDI MS was employed to characterize the endogenous retinoids extracted from canine kidney epithelial cells in culture and from mouse tissues. Retinoids were identified without the need for matching the retention time with commercially available retinoids or synthesizing retinoids. Thus, these results indicate that PSD-LDI MS should facilitate the identification and characterization of novel and known retinoid metabolites in mammalian cells and tissues. MATERIALS AND METHODS Materials. All chemicals, including 2,5-dihydroxybenzoic acid (DHA) and R-cyano-4-hydroxycinnamic acid (CHCA), were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO). HPLC grade or better solvents used for sample extraction and chromatography were obtained from Fisher Scientific (Fairlawn, NJ). Retinoid standards, including all-trans-retinol (ROH), all-transretinoic acid (atRA), retinyl acetate, and retinyl palmitate, were purchased from Sigma Chemical Co. Cell Culture. Madin-Darby canine kidney (MDCK) type II cells, a generous gift from Dr. Gravotta (Weill Medical College of Cornell University), were maintained in Dulbecco’s minimal essential Eagle’s medium supplemented with L-glutamine, penicillin-streptomycin, and 10% fetal bovine serum. Cells were plated on 150-mm dishes and cultured to 60% confluence prior to retinol treatment. A stock solution of retinol at a concentration of 1 mM was prepared in 100% ethanol and diluted 1:500 to a final concentration of 2 µM into the culture medium for cell treatments. At 60% confluence, cells were treated with retinol for 24 h and harvested after washing with ice-cold phosphate-buffered saline (PBS). Control cultures received ethanol vehicle alone. Extraction of Retinoids from MDCK cells and Mouse Tissues. Retinoid extraction from MDCK cells was performed as described previously under dim light at room temperature.16 Briefly, 350 µL of acetonitrile/butanol (50:50, v/v) and 0.01% butylated hydroxytoluene were added to 500 µL of MDCK cell suspension in ice-cold PBS. After vortexing, addition of 300 µL of a saturated (1.3 kg/L) K2HPO4 solution followed. The samples were then centrifuged at 12000g for 10 min at room temperature to separate the phases. The supernatants were collected and transferred to an injector vial for HPLC analysis. A male mouse (8 weeks old) was sacrificed, and tissue samples were harvested in the dark and stored at -70 °C until the retinoids were extracted. The frozen lung sample (∼20 mg) from the mouse was weighed and homogenized in 40 µL of ice-cold PBS with a homogenizer (Ika-works Inc., Cincinnati, OH) and then transferred into Eppendorf tubes. The volume was adjusted to 500 µL with ice-cold PBS. The retinoid extraction from the tissue was performed as described above. HPLC Analysis of Retinoids. For HPLC analysis, a Waters Millennium system (Waters Corp., Milford, MA) equipped with a photodiode array detector was used to separate the various (37) Khan, M. A.; Wang, Y.; Heidelberger, S.; Alvelius, G.; Liu, S.; Sjovall, J.; Griffiths, W. J. Steroids 2006, 71, 42-53. (38) Lattova, E.; Perreault, H.; Krokhin, O. J. Am. Soc. Mass Spectrom. 2004, 15, 725-735.

retinoids. A 4.6 × 250 mm C18 column (5-µm particle size, Vydac, Hesperia, CA) was used at a flow rate of 1.5 mL/min using a gradient solvent system. A solution of 15 mM ammonium acetate at pH 6.5 (mobile phase A), a mixture of acetonitrile and ddH2O (85:15, v/v, mobile phase B), and a mixture of acetonitrile and dichloromethane (80:20, v/v, mobile phase C) were used for separation. In chromatograms, retinoids were identified by HPLC based on retention time, UV spectrum, and coelution with synthetic reference compounds. Retinyl esters were quantified using retinyl palmitate standard. The limit of detection was 200 pmol of retinyl palmitate/mL. Saponification of the retinoid extract was performed as previously described 39 with a slight modification. Briefly, saponification was carried out by mixing 100 µL of the isolated supernatant with 10 µL of 30% methanolic potassium hydroxide for 10 min under dim light at room temperature. Mass Spectrometric Analysis of Retinoids. Stock solutions of standard retinoids were prepared at a concentration of 1 mM in ethanol. Stock solutions of standard retinoids were diluted with ethanol for mass spectrometric analysis. For MS analysis of retinoids, the individual peaks on an HPLC chromatogram were collected and dried by a SpeedVac SC 100 (Savant, Farmingdale, NY). The dried samples were resuspended in 5 µL of ethanol. Aliquots (0.5-1.0 µL; corresponding to 10-50 pmol of sample) of the suspended solution were applied into a stainless steel target and allowed to air-dry before mass spectrometry analysis. Mass spectrometric experiments were performed on an Applied Biosystems (Foster City, CA) Voyager DE-PRO reflectron TOF mass spectrometer equipped with a nitrogen laser emitting at 337 nm. The instrument was run in the positive ion mode by accumulating 300 laser shots, and measurements were performed in the linear mode as well as in the reflector mode. The potential applied to the target was 20 kV, and the focusing guide wire was held at a potential of 0.05% of acceleration voltage. A delay time of 50 ns between laser pulse and initiation of the acceleration voltage enabled optimal resolution. Laser power was adjusted to slightly above the threshold to obtain optimal resolution and signal-to-noise ratios. Instrument calibration was performed using retinol and retinyl palmitate standards. No matrix was used in this mass spectrometric analysis. In the PSD experiments for structural characterization, the precursor ions of interest were isolated using a timed ion selector, and PSD fragment ion spectra were recorded through stitching mass windows generated by sequential reduction of the mirror ratio. In each spectral window, 200 single-shot spectra were averaged and the PSD mirror ratio varied in seven steps from 1 to 0.237 to obtain fragment ions. The laser power was adjusted for each segment to maximize the number of fragment ions. Instrument calibration for the PSD experiments was performed using fragment ions of a standard peptide, angiotensin II. The accuracy and precision of PSD mass assignments reached (0.3 Da in the mass range of m/z 0-700. Data acquisition and data processing were performed using the Voyager software version 5.1 and the Data Explorer software version 4.0 (Applied Biosystems). (39) Wingerath, T.; Stahl, W.; Sies, H. Arch. Biochem. Biophy. 1995, 324, 385390.

Figure 1. Chemical structures (A) and reflectron LDI mass spectrum (B) of a mixture of standard retinoids investigated in this study. The LDI mass spectrum was obtained from the positive ion mode.

RESULTS AND DISCUSSION Although approaches for identification of retinoids based upon HPLC and UV spectra, HPLC and MS, or a combination of the three methods are commonly used and effective, such approaches have been used only to a limited extent for the structural elucidation of retinoids. For the structural elucidation of novel or known retinoids, which have a similar structure or an identical molecular mass, in biological mixtures using MS, the molecular mass determination of both parent and fragmentation ions is essential. For identification of thermally labile retinoids and to avoid the loss of intact molecular mass information when analyzing polar and nonpolar retinoids, a mass spectrometric approach employing a soft ionization source is desirable, i.e., LDI. Thus, we tested the utility of PSD-LDI MS for identifying structures of polar and nonpolar retinoids, as well as its applicability for identifying and characterizing retinoids in complex biological samples. Both the molecular and characteristic fragment ion information for specific retinoids can potentially be obtained for an individual fraction from an HPLC chromatogram, even though this fraction may contain other coeluting biological compounds (e.g., phospholipids, sphingolipids, glycerides, and fatty acids). Analysis of Retinoid Standards. In an initial test of the utility of LDI-TOF MS for retinoid characterization, commercially available retinoid standards were selected (Figure 1A) and analyzed in a positive ion mode. All retinoid standards generated radical molecular ions [M]+• without the loss of intact molecular mass information, as well as fragment and oxidized ions, and showed well-resolved monoisotopic peaks (Figure 1B). Retinoids are Analytical Chemistry, Vol. 78, No. 16, August 15, 2006

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Figure 2. Reversed-phase HPLC chromatogram (A) and UV spectra (B) of a mixture of standard retinoids. Retinoic acid (RA) at 24.7 min; retinol (ROH) at 35.2 min; retinyl acetate (RAce) at 41.8 min; retinyl palmitate (RP) at 57.0 min. The elution profile was monitored at 325 nm for the appearance of all retinoids. This experiment was performed in duplicate.

thermolabile, photosensitive, and easily attacked by oxidants due to their electron-rich polyene chain.15,40 Additionally, a loss of CO2 (-44 Da) from retinyl esters through a cyclic transition state 41 was observed, and this gave a unique characteristic for retinyl esters in LDI MS, which is consistent with previous observations.24 In LDI MS, a fragment ion was observed at m/z 255, resulting from a loss of CH2OH (-31 Da) in retinol and a loss of CO2H (-45 Da) in atRA. Allyl cleavage and elimination of a fatty acyl chain from retinyl esters formed fragment ions at m/z 255 and 269, respectively. The characteristic fragment ions at m/z 269 and the [M - 44]+• satellite ion from the loss of CO2 may be used as a diagnostic indicator of the presence of retinyl esters in the complex mass spectra obtained from a mixture of retinoids in biological samples. Various matrixes, including DHA and CHCA, were tested for the analysis of retinoids using a MALDI approach. They produced matrix cluster ions within a low-mass range, interfering with the detection of retinoid ions. Therefore, these matrixes were not suitable for the identification of retinoids in biological samples. In addition, these matrix peaks suppressed the signals of the retinoids in MALDI mass spectra (data not shown). Standard mixtures of retinol, atRA, retinyl acetate, and retinyl palmitate were resolved by gradient reversed-phase HPLC (Figure 2A), and UV spectra were characterized (Figure 2B). All retinoid standards investigated were isolated with baseline resolution in our HPLC system. Using a reversed-phase C18 column, polar retinoids were eluted first, followed by nonpolar retinoids, according to hydrophobicity. Minor peaks on the chromatogram most likely arise from thermal isomerization of retinoid standards (40) Ake, M.; Fabre, H.; Malan, A. K.; Mandrou, B. J. Chromatogr., A 1998, 826, 183-189. (41) Bowie, J. H.; Williams, D. H.; Madsen, P.; Schroll, G.; Lawesson, S.-O. Tetrahedron 1967, 23, 305-320.

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during the separation or as residual impurities from retinoid synthetic reactions. atRA exhibits a maximum absorbance at 343 nm, and the other retinoids investigated show a similar maximum absorbance. Because retinoids have similar structural and physical properties, it is impossible to distinguish all natural and synthetic retinoids based on UV/visible spectrometry alone. In addition, comparison of HPLC retention times of putative retinoid peaks with those of authentic retinoid standards was limited by the few authentic retinoid standards that are commercially available. Therefore, HPLC in combination with UV/visible spectrometry holds limited utility for the identification of many of the retinoids in biological samples. Structural Information on Retinoid Standards Using PSD Mass Spectrometry. Retinoid standards, collected after HPLC separation (Figure 2A) were used to establish the optimal conditions for PSD-LDI MS analysis. In positive ion mode, the predominant radical molecular ions were selected to determine characteristic fragmentation patterns of the retinoids. Six or seven segments of PSD data were acquired for each ion of interest and were then stitched together. The polar retinoids atRA and retinol gave rise to precursor ions [M]+• at m/z 300 and 286 and consequent PSD fragment ions as depicted in Figure 3A and B. PSD fragment ions of atRA were detected along with the many fragment ions in the low molecular weight regions, at m/z 255 and 285. These peaks corresponded to the loss of CO2H (-45 Da) and CH3 (-15 Da), respectively, whereas the loss of CH2OH (-31 Da) from the molecular ion of retinol generated a PSD fragment ion at m/z 255. Other common PSD fragment ions were assigned based on the retinyl moiety generated from retinyl palmitate (See Scheme 1).

Figure 3. PSD mass spectra of standard retinoids collected from HPLC. The PSD fragmentation patterns of standard retinoids show that they generate molecular ions as well as characteristic PSD fragment ions. The PSD mass spectra were obtained from the positive ion mode.

Scheme 1. Proposed Mechanisms of PSD Fragment Ions Generated from Retinyl Palmitate

As nonpolar retinoids, retinyl acetate and retinyl palmitate have similar PSD fragment ions in their PSD mass spectra. A detailed explanation is provided from consideration of the PSD mass spectrum of retinyl palmitate. In its PSD mass spectrum, the retinyl palmitate ion at m/z 524 generated characteristic fragment

ions at m/z 43, 57, 69, 93, 123, 145, 175, 197, 211, 253, 255, and 268 in positive ion mode. The dominant PSD fragment ion was identified at m/z 268, resulting from the elimination of palmitic acid (C16H32O2) as a neutral loss (See Scheme 1). From this information, we infer the formation on a fatty acid from retinyl Analytical Chemistry, Vol. 78, No. 16, August 15, 2006

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Figure 4. HPLC chromatogram obtained before (A) and after (C) KOH saponification of retinoid extracted from MDCK cells in culture. (B) UV spectra of peaks eluting with retention times between 50 and 65 min. The elution profile was monitored at 325 nm for the appearance of all retinoids. This experiment was performed in duplicate.

esters. This was confirmed by the detection of palmitate anion [C16H31O2]- at m/z 255 in negative ion mode (data not shown). Furthermore, PSD fragment ions at m/z 93, 123, 145, and 175 were generated from the breaking of alternative polyene bonds in the retinyl moiety. The elimination of a fragment by ring opening generated PSD fragment ions at m/z 57, 69, 197, and 211. Ions at m/z 43 and 83 appear to be formed by a different mechanism (See Scheme 1.). The radical ion generated from the retinyl moiety is stabilized by resonance and provides the driving force for the reaction. Many fragment ions may result from the radical ion, which has considerable energy, sufficient to cleave CdC or CsH bonds. Although we could not assign all of the fragment ions observed on the PSD mass spectra due to the conjugated double bond system of the retinoid, the characteristic PSD fragment ions at m/z 43, 69, 83, 93, and 119 can provide unique structural information for potentially characterizing novel retinoids. Application of PSD Mass Spectrometry to Biological Samples from MDCK Cells and Mouse Lung Extracts. To evaluate the effectiveness of PSD-LID MS for characterizing retinoids in biological samples, MDCK cells were cultured in the 5724

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Scheme 2. Saponification Reaction of Retinyl Ester under Basic Conditions

presence of retinol as described in the Materials and Methods. The esterification of retinol by the enzyme lecithin-retinol acyltransferase results in retinyl esters, which are a major storage form of vitamin A.10,11 After culturing MDCK cells in the presence of 2 µM retinol for 24 h, retinoids were extracted and separated by reversed-phase HPLC and identified based on UV absorbance spectra of the main peaks, as previously reported 16 (Figure 4A and B). Notably, in the absence of exogenously added retinol, there were no retinyl esters detected in the MDCK cells (data not shown). Based on the retention times and UV absorbance spectra of available retinoid standards, only two peaks on the chromatogram

Figure 5. (Left) Linear LDI mass spectra of each fraction; (35.2 min), B (52.7 min), and C (53.8 min), collected from HPLC chromatogram (Figure 4A) of retinoids extracted from MDCK cells in culture. Linear LDI mass spectra were obtained from the positive ion mode. (Right) PSD mass spectra of each parent ion (marked with a solid circle) in LDI mass spectra. The selected molecular ions (solid circles) were further investigated to obtain fragment ions in the PSD mode. The PSD mass spectra were obtained from the positive ion mode.

could be assigned: retinol at 35.2 min and retinyl palmitate at 57.2 min. The other peaks that eluted after 50 min are most likely retinyl esters, based on UV absorbance and previous reports,16,17 but identification of these peaks by HPLC alone is impossible

without reference standards. Based on the fact that the retention indices of retinyl esters in reversed phase increase linearly with increasing fatty acyl chain length and decrease with increasing numbers of double bonds in the fatty acyl chain, we predict that Analytical Chemistry, Vol. 78, No. 16, August 15, 2006

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Table 1. Peak Assignment of Retinoids from MDCK Cell Extracts by LDI and PSD MS retention time (min)

a

positive LDI MS (m/z)

negative LDI MS (m/z)

35.2

255.3, 286.3

naa

52.7

269.2, 478.3, 522.5

253.2

53.8

269.3, 506.6, 550.7

281.3

57.2

269.3, 524.7, 524.7

255.3

63.1

269.3, 508.6, 552.6

283.3

positive PSD MS (m/z)

no. of C (fatty acid)

no. of CdC

42.9, 57.0, 69.0, 83.0, 94.8, 109.1, 119.1, 255.1, 286.1 42.9, 57.1, 69.0, 123.1, 144.1, 175.2, 197.1, 253.3, 268.2, 522.6 43.0, 57.1, 69.1, 105.1, 119.1, 123.1, 144.1, 175.1, 197.1, 211.2, 253.2, 268.3, 550.8 57.1, 69.1, 83.1, 93.1, 105.1, 119.1, 123.1, 132.1, 144.1, 175.2, 197.1, 211.2, 268.2, 524.6 42.9, 57.0, 69.1, 119.1, 144.1, 268.2, 552.7

na

na

16

1

retinyl palmitoleate

18

1

retinyl oleate

16

0

retinyl palmitate

18

0

retinyl stearate

assignment retinol

na, not available.

a peak with a retention time of 63.1 min has a longer fatty acyl chain than retinyl palmitate, which elutes at 57.2 min. Peaks eluting at 52.7 and 53.8 min, respectively, would be predicted to have a shorter chain length or more double bonds than retinyl palmitate. To determine whether the tentatively assigned peaks were retinyl esters, saponification was carried out (See Scheme 2.) and resulted in an increase in free retinol (retention time at 35.2 min). This indicates that a retinol moiety was liberated from the tentatively assigned retinyl esters. Peaks eluting with retention times between 50 and 65 min were not detected after saponification (Figure 4C). In addition, the cis isomer of retinol was resolved (small peak eluting shortly before all-trans-retinol) from the trans isomer with the HPLC gradient we employ. The tentatively assigned retinyl esters were quantified, using retinyl palmitate as a standard, and ranged from 24 to 85 pmol in 6.5 × 106 MDCK cells. For further characterization of each peak, especially the tentatively assigned retinyl esters, the fractions corresponding to each peak on the chromatogram were analyzed by LDI and PSD MS. As seen in Figure 5A-C (left panels), each fraction of the sample gave rise to many parent ions than the retinoid standard mass spectrum because the fractions from the HPLC separation have many coeluting compounds. To demonstrate that the LDI and PSD approaches are suitable for the identification of retinoids in biological samples, retinol and retinyl palmitate, known retinoids, with retention times of 35.2 and 57.2 min, respectively, were analyzed. The molecular ion and characteristic fragment ion of retinol were observed at m/z 286 and 255. Although the molecular ion of retinol was observed on the LDI mass spectrum, the precursor ion at m/z 286 was selected for further structural analysis, and a PSD mass spectrum was obtained (Figure 5A, right panel). The PSD fragmentation patterns were found to be identical to those of retinol standard (Figure 3B). In addition, the peak corresponding to 57.2 min was identified as retinyl palmitate, which has a molecular ion at m/z 524, a satellite ion at m/z 480, and a characteristic fragment ion at m/z 269. Thus, the PSD fragmentation patterns of the retinyl palmitate standard and the ion putatively ascribed to retinyl palmitate in MDCK cells were identical (data not shown). In a similar fashion, LDI mass spectra were obtained from peaks with retention times of 52.7 and 53.8 min, respectively. As 5726

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seen in the mass spectrum of the standard retinyl palmitate, the characteristic fragment ion at m/z 269 was observed on LDI mass spectra (Figure 5B and C, left panels), indicating the presence of a retinyl ester. The molecular ion of a retinyl ester was assigned on the LDI mass spectrum based on the presence or absence of an [M - 44] +• satellite ion that results from loss of CO2. The molecular ions with a satellite ion were selected at m/z 522 (satellite ion at m/z 478) and 550 (satellite ion at m/z 506) on each LDI mass spectrum and then further analyzed by PSD to validate the assignment of the retinoid identity and to characterize the structures of additional retinyl esters. PSD mass spectra obtained from the selected molecular ions (solid circles) are shown (Figure 5B and C, right panels). Based on the fact that the mass difference between a molecular ion and the characteristic fragment ion at m/z 268 indicates the reports on the fatty acid structure in a retinyl ester, we deduced that the 254 Da from the PSD mass spectrum of Figure 5B and the 282 Da from Figure 5C indicate a palmitoleic acid and an oleic acid, respectively. Retinyl palmitoleate and 3,4-didehydroretinyl palmitate have an identical molecular mass at m/z 522, but their characteristic fragment ions from PSD MS may be different (268 Da from the retinyl moiety of retinyl palmitoleate and 266 Da from the 3,4-didehydroretinyl moiety of 3,4-didehydroretinyl palmitate or 254 Da from the palmitoleic acid moiety of retinyl palmitoleate and 256 Da from the palmitic acid moiety of 3,4-didehydroretinyl palmitate). Therefore, the peaks at 52.7 and 53.8 min were assigned as retinyl palmitoleate and retinyl oleate, respectively. The peak with a retention time of 63.1 min was also analyzed in the same fashion and was assigned as retinyl stearate. The information obtained by both LDI and PSD MS is summarized in Table 1. The information on retinyl esters obtained by HPLC in combination with mass spectrometric analysis is consistent with the fact that the fatty acids, including palmitoleate, oleate, palmitate, and stearate, are contained in the most abundant retinyl esters in epidermal keratinocytes.17 Based upon the strategy outlined above, retinoid identification and characterization were extended to mouse tissues, including lung. The HPLC chromatogram profile of endogenous retinoids extracted from the mouse lung was similar to that of the retinoids extracted from MDCK cells (data not shown). However, the peak eluting at 52.1 min on the HPLC chromatogram of the retinoids extracted from the mouse lung contained two different retinyl

Figure 6. Reflectron LDI mass spectrum of fraction (52.1 min) collected from HPLC chromatogram of retinoid extracted from mouse lung with positive ion mode (A) and with negative ion mode (B). The peaks at m/z 253 and 279 correspond to palmitoleate (C16:1) and linolate (C18:2) anions, respectively. The selected molecular ions (solid circles) were further investigated to obtain fragment ions in the PSD mode. PSD mass spectra of the marked circles at m/z 522 (C) and at m/z 548 (D), respectively. The PSD mass spectra were obtained from the positive ion mode. Table 2. Peak Assignment of Retinoids from Mouse Lung Extracts by LDI and PSD MS positive LDI MS (m/z)

retention time (min)

negative LDI MS (m/z)

33.0

255.2, 286.2

naa

52.1

269.2, 478.5, 504.5, 522.4, 548.5

253.2 279.2

a

53.3

269.3, 506.6, 550.6

281.3

57.2

269.2, 524.5, 524.5

255.2

64.6

269.3, 508.6, 552.6

283.2

positive PSD MS (m/z)

no. of C (fatty acid)

no. of CdC

42.9, 57.0 69.1, 83.0, 94.8, 109.1, 119.1, 134.9, 145.1, 159.0, 255.1 286.1 38.1, 42.9, 57.0, 69.0, 82.7, 105.1, 119.1, 123.1, 132.1, 144.1, 175.1, 197.1, 253.1, 268.2, 522.6 43.0, 57.0, 69.0, 83.1,95.1, 105.2, 123.1, 132.1, 144.1,159.1, 175.2, 197.1, 225.1, 253.1, 268.2, 548.6 43.0, 57.1, 69.1, 105.1, 119.1, 144.1, 175.1, 197.1, 253.2, 268.3, 550.8 42.9, 57.1, 69.0, 83.1, 93.1, 105.1, 119.1, 123.1,132.0, 145.0, 175.0, 211.1, 253.1, 268.2, 524.6 42.9, 57.0, 69.1, 119.1, 123.1, 144.1, 175.1, 268.2, 552.8

na

na

16

1

retinyl palmitoleate

18

2

retinyl linolate

18

1

retinyl oleate

16

0

retinyl palmitate

18

0

retinyl stearate

assignment retinol

na, not available.

esters, retinyl palmitoleate and linolate, which were identified using both positive and negative ion modes in LDI MS and further characterized by PSD MS (Figure 6). Additional information obtained by both LDI and PSD MS is summarized in Table 2. Although we could not detect all of the peaks observed on the chromatogram due to the detection limits of this mass spectrometric approach, the major peaks eluting with retention times between 50 and 65 min were assigned as retinyl esters by PSD

MS. Thus, PSD MS is suitable for the identification and characterization of retinoids in mammalian cells and tissues. CONCLUSIONS In this study, the PSD-LDI MS approach was employed for the characterization of polar and nonpolar retinoids, revealing the molecular ions of retinoids and their characteristic fragment ions for the structural elucidation. The feasibility of the PSD method Analytical Chemistry, Vol. 78, No. 16, August 15, 2006

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was also demonstrated by characterizing endogenous retinoids extracted from MDCK cells in culture and from a mouse lung, although the retinoids in these samples are primarily retinyl esters. On the basis of an [M - 44] +• satellite ion along with a molecular ion, the targeted PSD can be applied after HPLC separation to distinguish and characterize retinyl esters among complex coeluting compounds from biological samples. To our knowledge, this is the first PSD application for the identification and characterization of retinoids in biological samples without the need for matching the retention time with commercially available retinoids or synthesizing retinoids. Although this research was carried out using off-line PSD-LDI MS after HPLC separation, the coupling of HPLC and PSD-LDI MS or LDI TOF/TOF MS would extend the utility of the approach by saving time and increasing detection sensitivity. With this PSD approach the characterization of novel retinoid metabolites in biological samples is now underway in our laboratory. Our understanding of retinoid metabolism in mammals should be

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improved through the use of this PSD-LDI MS approach, which allows the identification of retinoids and their metabolites in mixtures of biological complexity. ACKNOWLEDGMENT This work was supported by National Institutes of Health grants (R01 DE10389 and R01 CA097543) to L.J.G. and a shared instrument grant from the national centers for research resources (RR19355) to Steven S. Grass. We acknowledge Albert M. Morrishow for technical assistance in mass spectrometry and the Weill Cornell Mass Spectrometry Facility, Karl B. Ecklund for editorial assistance, and Simne Langton, Cristina Fernandez and Dr. Steven S. Gross for critically reading the manuscript.

Received for review March 17, 2006. Accepted June 19, 2006. AC060492J