MS

Food Chemistry 166 (2015) 473–478

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Analytical Methods

Molecular species analysis of monosialogangliosides from sea urchin Strongylocentrotus nudus by RPLC-ESI-MS/MS Pei-Xu Cong a,1, Rui-chang Gao b,1, Chang-Hu Xue a, Zhao-Jie Li a, Hong-Wei Zhang c, Muhammad Naseem Khan a, Yong Xue a, Tatsuya Sugawara d, Jie Xu a,⇑ a

College of Food Science and Engineering, Ocean University of China, Qingdao, Shandong Province 266003, PR China School of Food Biological and Engineering, JiangSu University, Zhenjiang, Jiangsu Province 212013, PR China TechnicalCenter of Entry-Exit Inspection and Quarantine, Shandong Entry-Exit Inspection and Quarantine Bureau, Qingdao, Shandong Province 266002, PR China d Division of Applied Biosciences, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan b c

a r t i c l e

i n f o

Article history: Received 19 June 2013 Received in revised form 23 October 2013 Accepted 8 June 2014 Available online 14 June 2014 Keywords: Ganglioside PRLC-MS/MS Fragmentation Ceramide moiety Sialic acid Molecular species

a b s t r a c t Sea urchin gangliosides have been proved to contain neuritogenic activities, which related to their molecular compositions. This study reports a method utilizing reversed-phase chromatography coupled to mass spectrometry for structure investigation and molecular species determination of the monosialogangliosides from sea urchin Strongylocentrotus nudus. Two types of sulfated and nonsulfated monosialogangliosides were isolated from the sea urchin ovary. In MS2 spectra of both nonsulfated monosialoganglioside and sulfated monosialoganglioside, 2-6 linked sialic acids were identified by the characteristic fragments of 0,4A2-CO2 and 0,2A1. Fragment ions at m/z 139.1 and m/z 169.1 of nonsulfated monosialoganglioside might be characteristic for 8-sulfated sialic acid residue. Retention time of the molecules was effectively used in the characterization of unknown molecules, and molecules that differ in mass by only 0.04 Da were easily differentiated. Ó 2014 Elsevier Ltd. All rights reserved.

1. Introduction Gangliosides are a family of sialic acid (Sia) containing glycosphingolipids. The glycosphingolipids are involved in a variety of biological processes, such as cell to cell recognition and cell signalling. Disruptions in the expression and metabolism of glycosphingolipids, particularly gangliosides, could affect the brain functions, which cause various diseases (Yu, Nakatani, & Yanagisawa, 2009). When administered exogenously to neurons in vitro and in vivo, gangliosides exert two principal effects, namely neuronotrophic and neuritogenic activities. Moreover, gangliosides influence neuronal plasticity during development, adulthood and aging (Ledeen, 1984; Mocchetti, 2005). Interestingly, apart from vertebrates, gangliosides have also been found in sea urchins (Ijuin et al., 1996; Kubo, Irie, Inagaki, & Hoshi, 1990; Yamada et al., 2008) and proved to possess ⇑ Corresponding authors. Address: College of Food Science and Engineering, Ocean University of China, No. 5, Yu Shan Road, Qingdao, Shandong Province 266003, PR China. Tel./fax: +86 532 82031908 (J. Xu). E-mail addresses: [email protected] (P.-X. Cong), [email protected] (R.-c. Gao), [email protected] (C.-H. Xue), [email protected] (Z.-J. Li), light04@126. com (H.-W. Zhang), [email protected] (M.N. Khan), [email protected] (Y. Xue), [email protected] (T. Sugawara), [email protected] (J. Xu). 1 Pei-Xu Cong and Rui-Chang Gao contributed equally to this work. http://dx.doi.org/10.1016/j.foodchem.2014.06.028 0308-8146/Ó 2014 Elsevier Ltd. All rights reserved.

neuritogenic activity. As compared with mammalian gangliosides, the basic sugar moiety of the sea urchin gangliosides is the simple disaccharide, Sia2-6Glc (Higuchi, Inagaki, Yamada, & Miyamoto, 2007). Furthermore to be hydroxylated at C-11 position, the sialic acid residues of sea urchin gangliosides were possibly methylated (Yamada et al., 2008) and sulfated at C8 position (Ijuin et al., 1996; Kubo et al., 1990). The ceramide moieties of the sea urchin gangliosides are typically comprised of both non-hydroxy and a-hydroxy fatty acids (FA), and phytosphingosine-type long-chain bases (LCB). The FA chain lengths vary a lot and many of them are containing more than 20 carbons. The variations in ceramide moiety result in a complex molecular composition of sea urchin gangliosides therefore may affect their bioactivities, which were revealed in the experiments toward rat pheochromocytoma cells (PC12 cells) (Kaneko, Yamada, Miyamoto, Inagaki, & Higuchi, 2007) of sea cucumber gangliosides SJG-1and HLG-1 (Kaneko, Kisa, Yamada, Miyamoto, & Higuchi, 1999; Yamada, Matsubara, Kaneko, Miyamoto, & Higuchi, 2001). Electrospray ionization (ESI) mass spectrometric using collision-induced dissociation has provided valuable tools for investigating the linkages of complex sialylated gangliosides (Meisen, Peter-Katalinic, & Muthing, 2003) and the ceramide moieties of glycosphingolipids (Colsch et al., 2004). High heterogeneity of ganglioside extracts result in the difficulty in identifying each of the

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molecules by direct infusion ESI-MS methods. Coupling of liquid chromatography (LC) with MS via ESI provides an improved sensitivity, selectivity and the possibility for high-throughput experiments (Sisu, Flangea, Serb, Rizzi, & Zamfir, 2011). In general, sphingolipids in biological samples are usually quantified by running a triple quadrupole mass spectrometer in multiple reactions monitoring (MRM) mode (Shaner et al., 2009). However, MRM requires prior knowledge about which compounds are present in the given sample. Besides, as a given mass can often be assigned to two or more biologically relevant structures, assigning a molecular structure based only on precursor and product masses is not always feasible. In theory, the retention time of an LC column provides biochemical information of given molecule. Ikeda and coworkers reported a method that using reversed-phase (RP) column to separate mouse brain ganglioside molecular species in same class (Ikeda, Shimizu, & Taguchi, 2008). On the RP column, either the sphingoid chain or the fatty-acyl chains influence the elution sequence of the gangliosides, and therefore, molecular species in a ganglioside class will likely separated from each other. The aim of this research project was to develop a RPLC-MS based profiling approach for the determination of sea urchin gangliosides, in which the gangliosides are separated and identified on the basis of hydrophobicity and mass spectrometry.

Clara). Both positive and negative modes were employed. Conditions for ESI-MS were as follows: ESI voltage, 3750 V in negative ionization mode and 4000 V in positive ionization mode; Vaporizing gas flow, 6 L/min, at a temperature of 300 °C; Nebulizer pressure, 25 psi; MS2 spectra were obtained from collision induced dissociation (CID) using nitrogen as the collision gas at a pressure of 0.2 MPa. The collision energy was adjusted from 30 eV to 60 eV. Supplementary MS3 experiments were performed on an LTQ-Orbitrap XL (Thermo Electron Corporation, Waltham, MA) that was coupled with a syringe pump (Harvard Apparatus, Holliston, MA). 2.4. Reversed phase liquid chromatography The liquid chromatographic system was an Agilent 1260 series system consisting of equipped with a binary pump. Chromatographic separations were conducted on an YMC-Pack Pro C8 column (2.0  100 mm, 3 lm, YMC Corporation, Tokyo, Japan) at 25 °C. Sea urchin gangliosides with different ceramides were eluted with a mobile phase prepared by mixing acetonitrile and 20 mM ammonium acetate at a ratio of 75:25 (v/v). Sample solutions were prepared by dissolving 2 mg purified sea urchin gangliosides into 1 mL of pure water. Typically, the inject volume was 5 lL and the column was eluted with mobile phase at a flow rate of 0.2 mL/min. Each run was completed in 50 min.

2. Materials and methods 2.5. MRM analysis 2.1. Materials The sea urchin Strongylocentrotus nudus were purchased from a local market. The ovaries of the sea urchins were separated from the guts and shells then lyophilized. Chromatographic grade acetonitrile was purchased from Mallinckrodt Baker (Phillipsburg, NJ). Acetic acid and ammonium acetate were purchased from Sigma– Aldrich (St. Louis, MO). Pure water was obtained from a Milli-Q water system (Millipore, Millipore, MA). Other chemical reagents used for extraction and isolation were from local commercial sources. 2.2. Extraction and isolation of sea urchin gangliosides The sea urchin gangliosides were extracted according to a method reported by Svennerholm and Fredman (1980). Briefly, the lyophilized powder of the sea urchin ovaries was extracted twice with three volumes of chloroform–methanol–water (4:8:3, v/v/v). The mixture was then stirred for 30 min at room temperature and then centrifuged at 2000g for 30 min. The supernatant was collected, filtered and pooled. Water was added to the supernatant to give a final chloroform–methanol–water ratio of 4:8:5.6 (v/v/v). After vortex mixing and centrifugation, the resulted upper phases were directly passed through a Daisogel-SP120-C8 column (3.5  20 cm, 40–60 lm, Daiso Chemical, Osaka, Japan) to clean and desalt. Recovered gangliosides were redissolved in chloroform–methanol–water (30:60:8, v/v/v) and applied to a DEAESephadex A25 column (1.7  45 cm, 40–120 lm, GE Healthcare Bio-sciences AB, Sweden). After washing the column with 350 mL solvent A (chloroform–methanol–water 30:60:8, v/v/v) to clean up neutral lipids, a linear gradient from solvent A to solvent B (200 mM ammonium acetate dissolved in solvent A) was proceeded. Total elution volume was 1000 mL and 10 mL of each fraction was collected. 2.3. Mass spectrometry for investigation of cleavage pathways Mass spectrometry experiments for investigation of cleavage pathway of sea urchin gangliosides were performed on an Agilent G6410B triple quadrupole instrument (Agilent Technologies, Santa

To profile the ceramide structure and the relative content of each molecule in sea urchin gangliosides, MRM analyses were performed by monitoring sialic acid ions in negative ion modes and LCB ions in positive ion mode. Nitrogen severed as vaporizing gas at a flow rate of 8 L/min, the nebulizer pressure was 30 psi, and dwell time for each transition was 50 ms. Fragmentor values were 160 V for NMG and 260 V for SMG in negative ionization mode, and 280 V for NMG and 290 V for SMG in positive ionization mode respectively. The precursor ions to sialic acid ions in negative ionization mode and precursor ions to LCB ions in positive ionization mode were monitored during the MRM analyses. 3. Results 3.1. Mass spectrometry Two types of gangliosides with disaccharide, Sia2-6Glc, from the sea urchins S. nudus were isolated and simply classified as nonsulfated-monosialogangliosides (NMGs, eluted at 0.09–0.14 M ammonium acetate) and sulfated-monosialoganglioside (SMGs, eluted at 0.16–0.19 M ammonium acetate). Molecular weights of NGMs and SMGs were inferred from their deprotonated molecular ions. In negative ion mode, NMG was detected as the singly charged state ion ([M–H]), while SMG was detected as both singly and doubly charged state ions ([M–H] and [M–2H]2) (Fig. 1). SMGs transformed from mainly doubly charged ions to singly charged ions as the fragmentor value was increased to 260 V. The considerable heterogeneity observed in the spectrum was supposed mainly attribute to the variations in the ceramides and sialic acids. MS2 analyses performed on the [M–H] ions of NMGs showed that they were possessing terminal sialic acid residue (either Neu5Gc: N-hydroxyacetylneuraminic acid or Neu5Ac: N-acetylneuraminic acid). In MS2 spectra of precursor ion at m/z 1121.7, characteristic fragment ions (Meisen et al., 2003; Wheeler & Harvey, 2000) of 6-sialyated hexose as 0,4A2-CO2 ions (m/z 322.1), 0,2 A1-H2O of the Neu5Gc (m/z 218.1) and 0,2X1 ions were identified (Fig. 2). Consistent with the disaccharide core (Sia2-6Glc), a C2 fragment ion (m/z 486.1) was identified. Negative CID-MS2

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Fig. 1. Negative MS1 spectra of sea urchin gangliosides from Strongylocentrotus nudus, NMG (A); SMG (B).

conducted on the [M–2H]2 ions of SMGs showed the tendency of the molecules to lose terminal sialic acid residue. However, the sulfated sialic acid ion (B1 ion) underwent cleavage and produced a series of dehydrated or de-N-acetylated fragments (Fig. 3A). Among these fragments at m/z 169.1 and m/z 138.9 were supposed to be characteristic ions of 8-O-sulfated sialic acids with respect to the probably existence of 4-O-sulfated sialic acids in echinoderms (Yamada et al., 1998). The proposed fragmentation pattern of sulfated sialic acids was shown in Fig. 3B. The pattern was confirmed by conducting MS3 experiments on the B1 fragment, and the same dehydrated or de-N-acetylated fragments were observed (data not shown). Similarly, 0,4A2-CO2 fragment at m/z 386.1 (HSO3-8Neu5Ac) that characteristic for the 6-sialyated hexose was obtained in the MS2 spectrum. In negative ionization mode, the FA and LCB fragment ions of the NMG and SMG molecules were detected in low abundance, which making the ceramide structures of the minor molecules difficult to identify. Therefore, structure identification of the ceramide moieties were switched to positive ionization mode (Colsch et al., 2004). Both NMG and SMG produced ceramide ions with improved sensitivity at a fragmentor value of 280 V. Along with CID, the LCB and FA fragments from the amide bond cleavage were observed, and the most abundant ion was [LCB-2H2O]+. 3.2. Detailed molecular species analyses Because gangliosides are amphiphilic molecules, they can be separated either on normal-phase columns or reversed-phase

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columns. However, it is preferable to separate the molecules using a reversed-phase column due to complexity of ceramide moieties of NMGs and SMGs. For detailed molecular species determination, molecules detected by MS1 scan, at a range between 500 and 1300, were further analyzed by MRM. Sialic acid types were determined by detecting m/z 290.1 for Neu5Ac, m/z 306.1 for Neu5Gc, m/z 370.1 for HSO3-8Neu5Ac and m/z 386.1 for HSO3-8Neu5Gc in negative ionization mode. The ceramide structures were identified by monitoring precursor ceramide ions to the product ions specific to LCB, i.e., m/z 254.1 for t16:0, m/z 268.1 for t17:0, m/z 282.1 for t18:0 and m/z 296.1 for t19:0. In the RPLC system employed, with the carbon number of the ceramide increased the retention time of the molecules increased while substitutions of the hydroxyl groups and double bonds decreased the retention (Fig. 4). Moreover isobaric precursors with difference in ceramide carbon numbers were easily differentiated by their hydrophobicity. Structure isomers containing equal ceramide carbon number (e.g. t16:0-C22:1h and t18:0-C20:1h, t16:0C22:1h and t18:0-C20:1h) coeluted in the RPLC system, but molecules possessing longer N-acyl chain were retained slightly longer with addition of acetic acid to the solvent (Supplemental Fig. 1). Using this method, over 50 molecules with detailed structure were identified (Supplemental Table 1). The identified molecules were classified into groups according to their sialic acid and fatty acid types. The retention time versus ceramide carbon numbers for molecules within the same group were fitted to exponential equations (Table 1). After operating the equations on the assumption that they have equal retention time (i.e., equal y value), linear equations concerning relationships among the number of ceramide carbons (x values) were obtained, e.g., x2 = 0.982x1 + 2.235, x3 = 1.015x2, x2 = 1.002x4, and x8 = 1.078x2  0.441. These derived equations state the elimination of the double bond on N-acyl chain produces an equal effect as the elongation of the ceramide moiety by approximately 1.5 carbons; elimination of the hydroxyl group on N-acyl chain equals to elongation of the ceramide chain by approximately 0.5 carbon; elimination of the hydroxyl group on the acetyl group of Neu5Gc is equivalent to the elongation of the ceramide moiety by approximately 0.07 carbon; and ceramide moiety elongates approximately 2.4 carbons while the sulfuric group of SMG is eliminated.

4. Discussion In this study, for the first time we used ESI-MS2 to analyze sea urchin gangliosides and proposed their fragmentation pattern. The characteristic ions, 0,2A1-H2O and 0,4A2-CO2, responsible for 2-6linked sialic acid were found in the negative MS2 spectrum of the sea urchin gangliosides (Meisen et al., 2003). These ions are helpful to identify the molecules from the 3-sialyted GM4 which naturally

Fig. 2. Negative MS2 mass spectrum of NMG (t18:0-C22:1) and fragmentation scheme. The corresponding m/z values of fragment ions are assigned in the spectrum.

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Fig. 3. (A) Negative MS2 mass spectra of double charged SMG possessing HSO3-8Neu5Ac (t18:0-C22:1) and (B) proposed fragmentation pathway of sulfated sialic acid cleaved from non-reducing end. The corresponding m/z values of fragment ions are assigned in the spectrum.

Fig. 4. Elution order of the sea urchin gangliosides on an octyl column. (A) The retention time increased as the carbon number of the ceramide increased; (B) substitutions of the hydroxyl groups on ceramide or sialic acid moiety, double bonds on fatty chain decreased the retention.

existed in animal tissues. The charge site may significantly affect the cleavage pattern of SMG in negative ionization mode, as neither cross ring fragments show in MS2 spectrum of deprotonated ions (Supplemental Fig. 2A) nor in MS3 spectrum of the desulfated ions produced in the MS2 (Supplemental Fig. 2B). Possible combinations of head groups, fatty acids, and sphingoid base backbones direct to the high degree of variety of sphingolipids (Merrill, Sullards, Allegood, Kelly, & Wang, 2005). Since most of ganglioside extracts exhibit high heterogeneity, it requires separation prior to MS analysis (Sisu et al., 2011). Normal-phase chromatography and hydrophilic interaction chromatography (HILIC) are preferred strategies to separate gangliosides essentially by classes on the basis of that sugar chain polarity. Typically,

monosialogangliosides elute first, followed by disialogangliosides and trisialogangliosides. Using HILIC methods, regioisomers of gangliosides, such as GD1a, GD1b and GT1a, GT1b can be individually separated from one another (Fong, Norris, Lowe, & McJarrow, 2009). From our experience, HILIC methods on NH2 or diol columns were capable to separate sea urchin gangliosides into classes as NMG and SMG, but difficult to separate molecules within the same structural class. HILIC methods are unable to clearly resolve ions within same class that have an almost identical mass e.g. Neu5Gc-t18:0-C20:0 and Neu5Gc-t16:0-C21:1h. Besides, quantification of minor molecules such as m/z 1107.7 is likely to be interfered by isotope ion of m/z 1105.7. Therefore, the lipid profiling approach using RPLC column makes a significant improvement

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P.-X. Cong et al. / Food Chemistry 166 (2015) 473–478 Table 1 Classification of molecules in NMG and SMG according to sialic acid and fatty acid types. Group number

Sia typea

Fatty acid Double bond number

Hydroxyl number

1 2

G G

0 1

0 0

3

G

1

1

4 5 6 7 8

A AS AS AS GS

1 0 1 0 1

0 0 0 1 0

[M–H] ions

Exponential curve

995.7, 1023.7, 1037.7, 1051.7, 1079.7 (32-38 ceramide carbons) 1049.7, 1063.7, 1077.7, 1091.7, 1105.7, 1119.7, 1133.7 (36-42 ceramide carbons) 1065.7, 1079.7, 1093.7, 1107.7, 1121.7, 1135.7, 1149.7 (36-42ceramide carbons) 1061.7, 1075.7, 1089.7, 1117.7 (38-42 ceramide carbons) 1059.7, 1087.7, 1115.7, 1129.7 (32-39 ceramide carbons) 1113.7, 1127.7, 1141.7, 1155.7, 1169.7, 1183.7 (36-41 ceramide carbons) 1075.7, 1103.7, 1131.7, 1173.7 (32-41 ceramide carbons) 1129.7, 1143.7, 1157.7, 1185.7 (36-40 ceramide carbons)

y1 ¼ 0:0016e0:2528x1 y2 ¼ 0:0009e0:2574x2 y3 ¼ 0:0009e0:2534x3 y4 y5 y6 y7 y8

¼ 0:0009e0:2579x4 ¼ 0:0072e0:1932x5 ¼ 0:0012e0:2339x6 ¼ 0:0047e0:2036x7 ¼ 0:0010e0:2387x8

a The sialic acids are abbreviated as A for Neu5Ac, and G for Neu5Gc; AS for HSO3-8Neu5Ac, and GS for HSO3-8Neu5Gc; The correlation coefficient r for every curve was P 0.99.

over existing methods because the physicochemical characteristics of the ceramide moiety were used to facilitate their identification. The method that we present greatly aids in the identification of molecules within the same ganglioside class. The visualization of data is one of the key issues in Sphingolipidomics systems analysis (Merrill et al., 2009). Actually, the retention time on RPLC column versus m/z data of sphingolipids can be processed for two-dimensional (2D) plots conveniently (Hejazi et al., 2011). With the 2D method, RT could be made independent of N-acyl chain length because lipids clusters with same fatty acid type aligned horizontally. To validate the application of the 2D method in sea urchin ganglioside profiling, data of molecules of NMG with Neu5Gc residue were processed as described by Hejazi. We extracted the m/z and RT bearing monounsaturated and hydroxylated N-acyl chains. The six data points were then fitted to a centered exponential curves (y = 3  10(8)e0.018205x, R2 = 0.9999) and relative RT values were obtained by fitting all observed m/z values to the equation. The difference between theoretical and observed RT was termed ‘relative RT’, and was plotted against m/z (Supplemental Fig. 3). Molecules with an identical number of carbons, but differing degrees of unsaturation such as m/z 1077.7 (38:1) and m/z 1079.7 (38:0) align vertically. And the molecules with identical m/z 1079.7 (38:0 and 37:1h) but differ in their carbon numbers present two distinct peaks. The ganglioside molecules with a hydroxylated fatty acid or normal fatty acid still aligned exponentially but not horizontally. It seems a proper gradient is beneficial for developing the 2D method. The visualization method is very valuable as it simplified the identification procedure and effectively used RT in the characterization of unknown molecules. However, the method is limited in identifying unknown sea urchin gangliosides at present because of the insufficient of commercially available NMG and SMG standards. There are review articles summarized that dietary gangliosides from milk and animal tissues inhibits proinflammatory signalling in the intestine, prevent infection of pathogenic bacteria, and influence neonatal brain development (McJarrow, Schnell, Jumpsen, & Clandinin, 2009; Rueda, 2007). Ganglioside is rich in sea urchin ovary, amounting to approximately 0.42 mg/g of ovary (Yamada et al., 2008). Though the structures are different from mammalian gangliosides, the potential neuritogenic activity of sea urchins gangliosides is suggested by applying them to rat pheochromocytoma cell line (PC12 cells) with presence of nerve growth factor. However, there is no further research focusing on how the gangliosides work as dietary nutrients to date. In conclusion, a RPLC-ESI-MS/MS approach that can identify the individual molecular species has been developed for the targeted analysis of the sea urchin gangliosides. This advancement provided great sensitivity, as low-concentration molecules were detected

effectively. Therefore, the method will be useful for investigations of the bioactivity and metabolism of the sea urchin gangliosides in vivo. Acknowledgements The authors thank Prof. Su-Mei Ren (College of Medicine and Pharmaceutics, Ocean University of China) for generous support with the LTQ-Orbitrap XL mass spectrometer. Financial support for this study was provided by the National Key Technology Research and Development Program of the Ministry of Science and Technology of China (No. 2012BAD33B07), National Natural Science Foundation of China (No. 31201329), and China Public Science and Technology Research Funds Project of Ocean (No. 201105029). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.foodchem.2014. 06.028. References Colsch, B., Afonso, C., Popa, I., Portoukalian, J., Fournier, F., Tabet, J. C., et al. (2004). Characterization of the ceramide moieties of sphingoglycolipids from mouse brain by ESI-MS/MS: Identification of ceramides containing sphingadienine. Journal of Lipid Research, 45(2), 281–286. Fong, B., Norris, C., Lowe, E., & McJarrow, P. (2009). Liquid chromatography-highresolution mass spectrometry for quantitative analysis of gangliosides. Lipids, 44(9), 867–874. Hejazi, L., Wong, J. W., Cheng, D., Proschogo, N., Ebrahimi, D., Garner, B., et al. (2011). Mass and relative elution time profiling: Two-dimensional analysis of sphingolipids in Alzheimer’s disease brains. Biochemical Journal, 438(1), 165–175. Higuchi, R., Inagaki, M., Yamada, K., & Miyamoto, T. (2007). Biologically active gangliosides from echinoderms. Journal of Natural Medicines, 61(4), 367–370. Ijuin, T., Kitajima, K., Song, Y., Kitazume, S., Inoue, S., Haslam, S., et al. (1996). Isolation and identification of novel sulfated and nonsulfated oligosialyl glycosphingolipids from sea urchin sperm. Glycoconjugate Journal, 13(3), 401–413. Ikeda, K., Shimizu, T., & Taguchi, R. (2008). Targeted analysis of ganglioside and sulfatide molecular species by LC/ESI-MS/MS with theoretically expanded multiple reaction monitoring. Journal of Lipid Research, 49(12), 2678–2689. Kaneko, M., Kisa, F., Yamada, K., Miyamoto, T., & Higuchi, R. (1999). Structure of neuritogenic active ganglioside from the sea cucumber Stichopus japonicus. European Journal of Organic Chemistry, 1999(11), 3171–3174. Kaneko, M., Yamada, K., Miyamoto, T., Inagaki, M., & Higuchi, R. (2007). Neuritogenic activity of gangliosides from echinoderms and their structureactivity relationship. Chemical & Pharmaceutical Bulletin (Tokyo), 55(3), 462–463. Kubo, H., Irie, A., Inagaki, F., & Hoshi, M. (1990). Gangliosides from the eggs of the sea urchin, Anthocidaris crassispina. Journal of Biochemistry, 108(2), 185–192. Ledeen, R. W. (1984). Biology of gangliosides: Neuritogenic and neuronotrophic properties. Journal of Neuroscience Research, 12(2–3), 147–159.

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