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• W2585341696 abstract "1-O-acylceramide is a new class of epidermal cer­amide (Cer) found in humans and mice. Here, we report an ESI linear ion-trap (LIT) multiple-stage MS (MSn) approach with high resolution toward structural characterization of this lipid family isolated from mice. Molecular species desorbed as the [M + H]+ ions were subjected to LIT MS2 to yield predominately the [M + H − H2O]+ ions, followed by MS3 to cleave the 1-O-acyl residue to yield the [M + H − H2O − (1-O-FA)]+ ions. The structures of the N-acyl chain and long-chain base (LCB) of the molecule were determined by MS4 on [M + H − H2O − (1-O-FA)]+ ions that yielded multiple sets of specific ions. Using this approach, isomers varied in the 1-O-acyl (from 14:0- to 30:0-O-acyl) and N-acyl chains (from 14:0- to 34:1-N-acyl) with 18:1-sphingosine as the major LCB were found for the entire family. Minor isomers consisting of 16:1-, 17:1-, 18:2-, and 19:1-sphingosine LCBs with odd fatty acyl chain or with monounsaturated N- or O-fatty acyl substituents were also identified. An estimation of more than 700 1-O-acylceramide species, largely isobaric isomers, are present, underscoring the complexity of this Cer family. 1-O-acylceramide is a new class of epidermal cer­amide (Cer) found in humans and mice. Here, we report an ESI linear ion-trap (LIT) multiple-stage MS (MSn) approach with high resolution toward structural characterization of this lipid family isolated from mice. Molecular species desorbed as the [M + H]+ ions were subjected to LIT MS2 to yield predominately the [M + H − H2O]+ ions, followed by MS3 to cleave the 1-O-acyl residue to yield the [M + H − H2O − (1-O-FA)]+ ions. The structures of the N-acyl chain and long-chain base (LCB) of the molecule were determined by MS4 on [M + H − H2O − (1-O-FA)]+ ions that yielded multiple sets of specific ions. Using this approach, isomers varied in the 1-O-acyl (from 14:0- to 30:0-O-acyl) and N-acyl chains (from 14:0- to 34:1-N-acyl) with 18:1-sphingosine as the major LCB were found for the entire family. Minor isomers consisting of 16:1-, 17:1-, 18:2-, and 19:1-sphingosine LCBs with odd fatty acyl chain or with monounsaturated N- or O-fatty acyl substituents were also identified. An estimation of more than 700 1-O-acylceramide species, largely isobaric isomers, are present, underscoring the complexity of this Cer family. The outermost layer of the epidermis in mammals is rich in ceramides (Cers), which provide a physical barrier to water loss and proper skin functions (1Siegenthaler U. Laine A. Polak L. Studies on contact sensitivity to chromium in the guinea pig. The role of valence in the formation of the antigenic determinant.J. Invest. Dermatol. 1983; 80: 44-47Abstract Full Text PDF PubMed Scopus (26) Google Scholar, 2Imokawa G. Akasaki S. Hattori M. Yoshizuka N. Selective recovery of deranged water-holding properties by stratum corneum lipids.J. Invest. Dermatol. 1986; 87: 758-761Abstract Full Text PDF PubMed Scopus (187) Google Scholar, 3Imokawa G. Akasaki S. Minematsu Y. Kawai M. Importance of intercellular lipids in water-retention properties of the stratum corneum: induction and recovery study of surfactant dry skin.Arch. Dermatol. Res. 1989; 281: 45-51Crossref PubMed Scopus (218) Google Scholar). Epidermal Cers are mainly found in extracellular lipid lamellae and contain various classes of Cers, including those having unusual long-chain amide-linked ω-hydroxyacids, to which the ω-hydroxy group is esterified with an additional long-chain FA, primarily a linoleic acid (4Wertz P.W. Downing D.T. Ceramides of pig epidermis: structure determination.J. Lipid Res. 1983; 24: 759-765Abstract Full Text PDF PubMed Google Scholar, 5Ponec M. Weerheim A. Lankhorst P. Wertz P. New acylceramide in native and reconstructed epidermis.J. Invest. Dermatol. 2003; 120: 581-588Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar, 6Rabionet M. Gorgas K. Sandhoff R. Ceramide synthesis in the epidermis.Biochim. Biophys. Acta. 2014; 1841: 422-434Crossref PubMed Scopus (165) Google Scholar). In addition to the esterified ω-hydroxy FA-containing Cer class, Cers consisting of long to very long N- and 1-O-linked acyl chains were recently identified in humans and mice. This new class of epidermal 1-O-acylceramide derives from classical group I amide-linked nonhydroxy sphingosine Cers and makes up 5% of all esterified Cers. They are hydrophobic and were thought to contribute to the water barrier homeostasis (7Rabionet M. Bayerle A. Marsching C. Jennemann R. Gröne H-J. Yildiz Y. Wachten D. Shaw W. Shayman J.A. Sandhoff R. 1-O-acylceramides are natural components of human and mouse epidermis.J. Lipid Res. 2013; 54: 3312-3321Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). A large body of literature on the quantitative and qualitative analysis of Cers using mass spectrometric techniques, including GC/MS (8Raith K. Darius J. Neubert R.H.H. Ceramide analysis utilizing gas chromatography–mass spectrometry.J. Chromatogr. A. 2000; 876: 229-233Crossref PubMed Scopus (34) Google Scholar, 9Polito A.J. Akita T. Sweeley C.C. Gas chromatography and mass spectrometry of sphingolipid bases. Characterization of sphinga-4,14-dienine from plasma sphingomyelin.Biochemistry. 1968; 7: 2609-2614Crossref PubMed Scopus (42) Google Scholar) and ESI tandem MS with or without online HPLC (10Gu M. Kerwin J.L. Watts J.D. Aebersold R. Ceramide profiling of complex lipid mixtures by electrospray ionization mass spectrometry.Anal. Biochem. 1997; 244: 347-356Crossref PubMed Scopus (126) Google Scholar, 11Hsu F-F. Turk J. Characterization of ceramides by low energy collisional-activated dissociation tandem mass spectrometry with negative-ion electrospray ionization.J. Am. Soc. Mass Spectrom. 2002; 13: 558-570Crossref PubMed Scopus (86) Google Scholar, 12Hsu F.F. Turk J. Stewart M.E. Downing D.T. Structural studies on ceramides as lithiated adducts by low energy collisional-activated dissociation tandem mass spectrometry with electrospray ionization.J. Am. Soc. Mass Spectrom. 2002; 13: 680-695Crossref PubMed Scopus (79) Google Scholar, 13Sullards M.C. Merrill Jr, A.H. Analysis of sphingosine 1-phosphate, ceramides, and other bioactive sphingolipids by high-performance liquid chromatography-tandem mass spectrometry.Sci. STKE. 2001; 2001: pl1PubMed Google Scholar, 14Han X. Characterization and direct quantitation of ceramide molecular species from lipid extracts of biological samples by electrospray ionization tandem mass spectrometry.Anal. Biochem. 2002; 302: 199-212Crossref PubMed Scopus (147) Google Scholar, 15Masukawa Y. Narita H. Shimizu E. Kondo N. Sugai Y. Oba T. Homma R. Ishikawa J. Takagi Y. Kitahara T. Characterization of overall ceramide species in human stratum corneum.J. Lipid Res. 2008; 49: 1466-1476Abstract Full Text Full Text PDF PubMed Scopus (263) Google Scholar, 16t'Kindt R. Jorge L. Dumont E. Couturon P. David F. Sandra P. Sandra K. Profiling and characterizing skin ceramides using reversed-phase liquid chromatography-quadrupole time-of-flight mass spectrometry.Anal. Chem. 2012; 84: 403-411Crossref PubMed Scopus (149) Google Scholar, 17Liebisch G. Drobnik W. Reil M. Trümbach B. Arnecke R. Olgemöller B. Roscher A. Schmitz G. Quantitative measurement of different ceramide species from crude cellular extracts by electrospray ionization tandem mass spectrometry (ESI-MS/MS).J. Lipid Res. 1999; 40: 1539-1546Abstract Full Text Full Text PDF PubMed Google Scholar, 18van Smeden J. Hoppel L. van der Heijden R. Hankemeier T. Vreeken R.J. Bouwstra J.A. LC/MS analysis of stratum corneum lipids: ceramide profiling and discovery.J. Lipid Res. 2011; 52: 1211-1221Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar), has been published; however, very few studies have focused on the structural characterization of complex skin Cers. Among them, Masukawa et al. (15Masukawa Y. Narita H. Shimizu E. Kondo N. Sugai Y. Oba T. Homma R. Ishikawa J. Takagi Y. Kitahara T. Characterization of overall ceramide species in human stratum corneum.J. Lipid Res. 2008; 49: 1466-1476Abstract Full Text Full Text PDF PubMed Scopus (263) Google Scholar) and t'Kindt et al. (16t'Kindt R. Jorge L. Dumont E. Couturon P. David F. Sandra P. Sandra K. Profiling and characterizing skin ceramides using reversed-phase liquid chromatography-quadrupole time-of-flight mass spectrometry.Anal. Chem. 2012; 84: 403-411Crossref PubMed Scopus (149) Google Scholar) conducted the most extensive LC/MS/MS analysis of Cers in human stratum corneum, and revealed hundreds of Cer species in various classes, including many isobaric isomers, but species in the 1-O-acylceramide family were not reported. Rabionet et al. (7Rabionet M. Bayerle A. Marsching C. Jennemann R. Gröne H-J. Yildiz Y. Wachten D. Shaw W. Shayman J.A. Sandhoff R. 1-O-acylceramides are natural components of human and mouse epidermis.J. Lipid Res. 2013; 54: 3312-3321Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar) reported the presence of 1-O-acylceramides and a minor 1-O-acylceramide subfamily comprising the N-α-hydroxyacyl group in human and mouse epidermis using UPLC-ESI-tandem quadrupole MS and several isobaric isomers were found. Here, we apply linear ion-trap (LIT) multiple-stage MS (MSn) in combination with high-resolution accurate mass measurements to unveil the structural details of the 1-O-acylceramides isolated from mouse epidermis. Our results reveal the complexity of this 1-O-acylceramide family, which consists of C18-sphingosine as the major long-chain base (LCB) together with minor C16-, C17-, C18-, C19-, and C20-sphingosine LCBs and a C18-dehydrosphingosine LCB to which a whole array of amide-linked fatty acyl substituents (ranging from C14 to C32 with zero to one double bond) and 1-O-linked acyl groups (ranging from C14 to C32 with zero to one double bond) are attached. All chemicals and solvents were purchased from Fisher Scientific (Waltham, MA). Standard 1-O-oleoyl-N-heptadecanoyl-D-erythro-sphingosine (18:1-d18:1/17:0-Cer; please refer to the Nomenclature section for an explanation of abbreviations such as this) was purchased from Avanti Polar Lipids, Inc. (Alabaster, AL). An AMP+ MS kit (50 test) containing N-(4-aminomethylphenyl) pyridinium (AMPP) derivatizing reagent, n-butanol (HOBt), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, and acetonitrile/DMF solution was purchased from Cayman Chemical (Ann Arbor, MI). To the lyophilized 10 mg epidermis sample from newborn mice in a centrifuge tube, 0.8 ml water was added. The sample was soaked for 5 min at room temperature and 3 ml chloroform/methanol (1:2, v:v) were added. Following 30 s vortexing and 2 h shaking at room temperature, an additional 1 ml chloroform and 1 ml water were added. The extraction tube was vortexed for another 30 s, centrifuged at 1750 × g at room temperature for 5 min, and the organic layer was transferred to another tube, dried under nitrogen, and stored at −20°C until use. To fractionate, the crude lipids redissolved in 500 ul CHCl3 were loaded to a 3 ml/500 mg Macherey-Nagel (Duren, Germany) amino Chromabond Sep-Pak column, which was prewashed with 2× 3 ml hexane. The column was first eluted with 3 ml hexane:diethyl ether (90:10, v:v) (fraction 1), followed by 3 ml hexane:ethyl acetate (75:25, v:v) (fraction 2), 3 ml chloroform:methanol (15:1, v:v) (fraction 3), 2× 3 ml diisopropyl ether:acetic acid (98:5, v:v) (fractions 4 and 5), and finally eluted with 3 ml acetone:methanol (9:1.4, v:v) (fraction 6) by gravity. The eluants containing 1-O-acylceramide (fraction 2) and minor 1-O-acyl-N-α-OH-acylceramide (fraction 3) lipids were dried under a stream of nitrogen. The dried samples were redissolved in CHCl3:CH3OH (1:2, v:v) before ESI-MS analysis. To locate the double bond along the 1-O-acyl and N-acyl chains, 1-O-acylceramide was hydrolyzed in 1 ml of solution prepared from 8.6 ml of concentrated HCl and 9.6 ml of water, diluted to 100 ml with methanol as previously described (19Robson K.J. Stewart M.E. Michelsen S. Lazo N.D. Downing D.T. 6-Hydroxy-4-sphingenine in human epidermal ceramides.J. Lipid Res. 1994; 35: 2060-2068Abstract Full Text PDF PubMed Google Scholar). After heating at 80°C for 18 h, the hydrolysate was evaporated to dryness with a stream of nitrogen, and FA-AMPP derivative was prepared with the AMP+ MS kit according to the manufacturer's instructions, as described previously (20Hsu F-F. Characterization of hydroxyphthioceranoic and phthioceranoic acids by charge-switch derivatization and CID tandem mass spectrometry.J. Am. Soc. Mass Spectrom. 2016; 27: 622-632Crossref PubMed Scopus (16) Google Scholar). Both high-resolution (R = 100,000 at m/z 400) higher collision energy dissociation (HCD) and low-energy collision-induced dissociation (CID) tandem MS experiments were conducted on a Thermo Scientific (San Jose, CA) LTQ Orbitrap Velos mass spectrometer with an Xcalibur operating system. Lipid extracts in chloroform/methanol (2/1) were infused (1.5 μl/min) to the ESI source, where the skimmer was set at ground potential, the electrospray needle was set at 4.0 kV, and temperature of the heated capillary was 300°C. The automatic gain control of the ion trap was set to 5 × 104, with a maximum injection time of 50 ms. Helium was used as the buffer and collision gas at a pressure of 1 × 10−3 mbar (0.75 mTorr). The MSn experiments were carried out with an optimized relative collision energy ranging from 30 to 45% with an activation q value at 0.25 and the activation time at 10 ms to leave a minimal residual abundance of precursor ion (around 20%). The mass selection window for the precursor ions was set at 1 Da wide to admit the monoisotopic ion to the ion-trap for CID for unit resolution detection in the ion-trap or high-resolution accurate mass detection in the Orbitrap mass analyzer. Mass spectra were accumulated in the profile mode, typically for 2–10 min for MSn spectra (n = 2–4). For simplicity, 1-O-oleoyl-N-heptadecanoyl-D-erythro-sphingosine is abbreviated as 18:1-d18:1/17:0-Cer to reflect that an oleoyl (18:1) group is attached to the 1-hydroxy LCB of d18:1/17:0-Cer by an ester bond. Likewise, Cers such as 1-O-tetraeicosanoyl-N-tetraeicosenoyl-D-erythro-sphingosine and 1-O-tetraeicosanoyl-N-tetraeicosanoyl-D-erythro-dehydrosphingosine are abbreviated as 24:0-d18:1/24:1-Cer and 24:0-d18:2/24:0-Cer, respectively. The designation of the fragment ions is according to that previously described (12Hsu F.F. Turk J. Stewart M.E. Downing D.T. Structural studies on ceramides as lithiated adducts by low energy collisional-activated dissociation tandem mass spectrometry with electrospray ionization.J. Am. Soc. Mass Spectrom. 2002; 13: 680-695Crossref PubMed Scopus (79) Google Scholar). When subjected to ESI in the positive-ion mode, Cers were mainly seen as the [M + H − H2O]+ ions, due to facile loss of a water molecule (10Gu M. Kerwin J.L. Watts J.D. Aebersold R. Ceramide profiling of complex lipid mixtures by electrospray ionization mass spectrometry.Anal. Biochem. 1997; 244: 347-356Crossref PubMed Scopus (126) Google Scholar). By contrast, 1-O-acylceramide formed [M + H]+ ions, attributable to the notion that the attachment of the 1-O-acyl group may have deterred the water loss process. For example, the synthetic 18:1-d18:1/17:0-Cer standard was seen at m/z 816.7, corresponding to the [M + H]+ ions, which nevertheless formed the prominent ion of m/z 798.7 (Fig. 1A) by loss of water, when subjected to CID in an ion-trap. The water loss most likely involved the participation of the 3-hydroxy group of LCB (Scheme 1). The speculation was established by the findings that further dissociation of the ion of m/z 798 (816 → 798; Fig. 1B) yielded ions of m/z 516, arising from elimination of the 1-O-oleoyl group as an acid, and of m/z 264 (e3b″), arising from further cleavage of the N-heptaoctanoyl substituent as a ketene (Scheme 1). This fragmentation process was supported by the MS4 spectrum of the ion of m/z 516 (816 → 798 → 516; Fig. 1C), which contained prominent ions of m/z 264 (e3b″) (Scheme 1) that are unique to Cers consisting of C18-sphingosine LCB (10Gu M. Kerwin J.L. Watts J.D. Aebersold R. Ceramide profiling of complex lipid mixtures by electrospray ionization mass spectrometry.Anal. Biochem. 1997; 244: 347-356Crossref PubMed Scopus (126) Google Scholar). The spectrum also contained the ions of m/z 294, likely arising from cleavage of the LCB to eliminate a terminally conjugated 1,3-hexadecadiene, and of m/z 270, originated from the highly conjugated ions of m/z 516 that eliminate a C18H30 residue (Scheme 1). This latter fragmentation process also led to the ions of m/z 247, representing the highly conjugated triene cations. The ions of m/z 294 and 270 are equivalent to the c2a (m/z 294) and d1a (m/z 270) ions, as previously described (12Hsu F.F. Turk J. Stewart M.E. Downing D.T. Structural studies on ceramides as lithiated adducts by low energy collisional-activated dissociation tandem mass spectrometry with electrospray ionization.J. Am. Soc. Mass Spectrom. 2002; 13: 680-695Crossref PubMed Scopus (79) Google Scholar). The confirmation of the suggested structures is further supported by the elemental composition extracted by high-resolution MS (data not shown). The observation of the ions of m/z 264 reflecting the presence of d18:1-LCB and the 294/270 ion pair reflecting the N-heptadecanoyl substituents plus the MS3 spectrum of m/z 798 (Fig. 1B) that defines the 1-O-oleoyl group (via loss of oleic acid) led to complete assignment of the 18:1-d18:1/17:0-Cer structure.Scheme 1The fragmentation pathways proposed for the [M + H]+ ions of 1-O-oleoyl-d18:1/17:0-Cer at m/z 816.7.View Large Image Figure ViewerDownload Hi-res image Download (PPT) In the negative-ion mode in the presence of Cl− or HCO2−, however, ions at m/z 850 and 860 corresponding to the [M + Cl]− and [M + HCO2]− ions, respectively, were observed (data not shown), similar to that previously reported for Cers (11Hsu F-F. Turk J. Characterization of ceramides by low energy collisional-activated dissociation tandem mass spectrometry with negative-ion electrospray ionization.J. Am. Soc. Mass Spectrom. 2002; 13: 558-570Crossref PubMed Scopus (86) Google Scholar). MS2 on the [M + HCO2]− ions of m/z 860 (Fig. 1D) gave prominent ions of m/z 281, corresponding to the 18:1-carboxylate anion arising from cleavage of the 1-O-fatty acyl substituent, together with the ions of m/z 814 (860 − HCO2H) representing the [M – H]− ions arising from loss of HCO2H. The MS2 spectrum of the [M + Cl]− ions at m/z 850 is similar, mainly consisting of the 18:1-carboxylate anion of m/z 281 (not shown). These MS2 spectra readily recognized the 1-O-oleoyl group, but failed to provide further structural information applicable for complete identification of the molecule and, thus, its utility in the structural identification was not investigated further. High-resolution ESI mass spectrometric analysis of the 1-O-acylceramides isolated from mouse epidermis (fraction 2) showed an array of abundant [M + H]+ ions (Table 1) with an elemental composition of CnH2n−2O4N1 (n = 53–72), suggesting that the 1-O-acylceramide family mainly consists of sphingosine LCB. A minor ion series with two fewer hydrogens with an elemental composition of CnH2n−4O4N1 (n = 53–72) was also observed, indicating the presence of the minor species with an additional double bond (Table 1). The observation of the analogous of the [M + Cl]− or [M + HCO2]− (not shown) ions in the negative ion mode is consistent with the presence of this 1-O-acylceramide family. In contrast, ions with an elemental composition of CnH2n−4O5N1 were not observed, excluding the presence of the subfamily of 1-O-acylceramides with the N-α-hydroxyacyl group previously reported by Rabionet and colleagues (6Rabionet M. Gorgas K. Sandhoff R. Ceramide synthesis in the epidermis.Biochim. Biophys. Acta. 2014; 1841: 422-434Crossref PubMed Scopus (165) Google Scholar, 7Rabionet M. Bayerle A. Marsching C. Jennemann R. Gröne H-J. Yildiz Y. Wachten D. Shaw W. Shayman J.A. Sandhoff R. 1-O-acylceramides are natural components of human and mouse epidermis.J. Lipid Res. 2013; 54: 3312-3321Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar) The LIT MSn mass spectrometric approaches together with high-resolution mass measurements toward identification of these mouse epidermis 1-O-acylceramdes are described below.TABLE 1The structures of epidermal 1-O-acylceratnide revealed by LIT MSn and high-resolution MSm/z [M + H]+Theoretical Mass (Da)Deviation (mDa)CompositionRelative Intensity(%)Major Structure1-O-Acyl SubstituentaAbundances in descending order.Isomer NumberMajorMinor816.7804816.78030.05C53 H102 04 N18:1-d18:1/17:0 (synthetic standard)18:1——832.8116832.81160.01C54 H106 04 N2.2318:0-d18:1/18:0; 19:0-d18:1/17:018:0; 19:016:07860.8431860.84290.16C56 H110 04 N4.2416:0-d18:1/22:0; 22:0-d18:1/16:016:0; 22:024:0; 14:0; 15:0; 20:0; 19:0; 23:0; 18:026874.8586874.8586−0.04C57 H112 04 N10.5024:0-d17:1/16:0; 16:0-d17:1/24:024:0; 16:015:0; 23:0; 14:0; 22:0; 18:0; 17:0; 25:0; 21:027886.8585886.8586−0.12C58 H112 04 N2.9524:0-d18:1/16:1; 16:0-d18:1/24:124:0; 16:022:0; 18:0; 23:1; 24:1; 25:0; 20:0; 17:0;16888.8742888.87420.01C58 H114 04 N35.6016:0-d18:1/24:016:0;18:0; 24:0; 22:0 14:0; 20:0; 23:0; 15:0; 17:0; 26:0.26900.8739900.8742−0.33C59 H114 04 N3.3224:0-d17:1/18:124:0;18:1; 16:1; 23:0; 19:0; 25:0; 25:1; 17:1; 15:0; 22:0; 26:0;21902.8899902.88990.03C59 H116 04 N21.8018:0-d18:1/23:0; 18:0-d16:1/25:0; 16:0-d17:1/26:018:0; 16:017:0; 24:0; 15:0; 22:0; 23:0; 25:0; 20:0; 19:0; 21:0; 14:033904.8688bDetected in fraction 3.904.8692−0.39C58 H114 05 Ndetected24:0-d18:1/h16:024:0—1912.8750912.87490.01C60 H114 04 N4.3324:0-d18:1/18:224:0;18:2; 18:1; 24:1; 14:0; 16:1; 25:0; 26:0;15914.8900914.88990.14C60 H116 04 N8.9824:0-d18:1/18:124:0;18:1; 16:1; 16:0; 18:0; 25:0; 26:0; 23:0;17916.9055916.9055−0.04C60 H118 04 N44.7518:0-d18:1/24:0; 16:0-d18:1/26:018:0; 16:024:0; 22:0; 20:0; 26:015928.9058928.90550.30C61 H118 04 N2.0918:1-d17:1/26:018:1;25:0; 24:0; 17:1; 16:0; 16:1; 20:0; 20:1; 18:0; 26:024930.9212930.92120.00C61 H120 04 N17.2118:0-d18:1/25:018:0;24:0; 17:0; 19:0; 20:0; 16:0; 22:0; 23:0; 25:0; 21:0; 15:0; 26:033942.9211942.9212−0.04C62 H120 04 N5.6318:0-d18:1/26:118:0;18:1; 20:0; 20:1; 22:0; 16:0; 24:018944.9368944.9368−0.06C62 H122 04 N35.4018:0-d18:1/26:018:0;20:0; 24:0; 22:0; 16:0; 14:0; 26:018956.9368956.9368−0.08C63 H122 04 N2.1222:0-d17:1/24:122:0;24:0; 18:1; 24:1; 20:1; 20:0; 21:0; 23:0; 19:1; 25:0; 25:1; 26:1; 26:0; 16:0; 16:140958.9523958.9525−0.15C63 H124 04 N17.2022;0-d18:1/23:022:0;24:0; 20:0; 18:0; 23:0; 21:0;19:0; 16:0; 25:0; 17:0; 26:0; 15:035970.9525970.95250.04C64 H124 04 N6.4022:0-d18:1/24:1; 22:1-d18:1/24:022:0; 22:124:1; 24:0; 18:0; 20:0; 20:1; 16:0; 26:1; 14:0; 30:1; 28:134972.9680972.9681−0.10C64 H126 04 N48.4022:0-d18:1/24:0; 24:0-d18:1/22:022:0; 24:020:0; 18:0; 23:0; 16:015984.9681984.96810.00C65 H126 04 N8.1524:0-d17:1/24:1; 24:1-d17:1/24:024:0; 24:122:0; 25:0; 22:1; 23:1; 25:0; 25:1; 21:0; 26:133986.9838986.98380.03C65 H128 04 N48.4524:0-d17:1/24:024:0;22:0; 23:0; 21:0; 18:0; 25:0; 20:0; 16:0; 26:0; 17:0; 15:030996.9679996.9681−0.19C66 H126 04 N2.2824:0-d18:1/24:2; 24:1-d18:1/24:124:0; 24:124:2; 22:0; 22:1; 22:2; 26:1; 26:2; 20:1; 20:0; 25:024998.9836998.9838−0.14C66 H128 04 N21.0224:0-d18:1/24:1; 24:1-d18:1/24:024:0; 24:122:0; 16:0; 18:0; 26:0; 20:0; 32:0; 30:0; 28:0; 14:0241,000.99941,000.9994−0.07C66 H130 04 N100.0024:0-d18:1/24:024:0;22:0; 23:0; 25:0; 26:0141,012.99931,012.9994−0.11C67 H130 04 N13.2324:0-d18:1/25:1; 24:0-d17:1/26:1; 24:1-d18:1/25:024:0; 24:123:0; 25:1; 25:0; 26:1; 22:0; 18:0; 16:0; 17:0; 15:0; 20:0; 21:0; 19:0331,015.01501,015.0151−0.11C67 H132 04 N58.8324:0-d18:1/25:0; 24:0-d17:1/26:024:0;23:0; 25:0; 22:0; 26:0; 18:0; 16:0; 20:0201,027.01491,027.0151−0.16C68 H132 04 N22.4324:0-d18:1/26:1; 24:1-d18:1/26:024:0; 24:126:1; 18:0; 16:0; 22:0; 20:0151,029.03081,029.03070.06C68 H134 04 N87.0224:0-d18:1/26:024:0;26:0; 21:0; 18:0101,043.04571,043.0457−0.08C69 H136 04 N10.5225:0-d18:1/26:0; 24:0-d18:1/27:0; 26:0-d18:1/25:025:0,24:0, 26:0;23:0; 22:0; 28:0; 27:0201,053.03071,053.0307−0.08C70 H134 04 N2.2926:1-d18:1/26:1; 26:2-d 18:1/26:026:1; 26:224:0; 24:1; 26:0, 28:1; 24:2; 22:0; 28:2; 22:1; 28:0271,055.04641,055.04640.04C70 H136 04 N5.1426:1-d18:1/26:0; 24:0-d18:1/28:126:1; 24:026:0; 28:1; 25:0; 30:1; 27:1211,057.06231,057.06200.23C70 H138 04 N9.1126:0-d18:1/26:0; 24:0-d18:1/28:026:0; 24:022:0; 25:0; 28:0; 27:0; 30:018Total710a Abundances in descending order.b Detected in fraction 3. Open table in a new tab Multiple isobaric isomers were observed for all the ions that appeared in the ESI-MS spectrum (Table 1). For example, the MS2 spectrum of the ions of m/z 1,001.1 is dominated by the ion of m/z 982.9 arising from loss of water (not shown). MS3 on the ions of m/z 982.9 (1,001.1 → 982.9; Fig. 2A) gave rise to the prominent ions of m/z 614 arising from loss of the 1-O-24:0-FA substituent, along with minor ions at m/z 642, 628, 600, and 586 arising from losses of 1-O-22:0-, -23:0-, -25:0-, and -26:0-FA substituents, respectively. The MS4 spectrum of m/z 614 (1,001.1 → 982.9 → 614; Fig. 2B) contained prominent ions at m/z 392 (c2a) and 368 (d1a) arising from cleavages of the LCB, along with the ions of m/z 264 (e3b″), indicating the presence of the major d18:1/24:0-Cer structure (12Hsu F.F. Turk J. Stewart M.E. Downing D.T. Structural studies on ceramides as lithiated adducts by low energy collisional-activated dissociation tandem mass spectrometry with electrospray ionization.J. Am. Soc. Mass Spectrom. 2002; 13: 680-695Crossref PubMed Scopus (79) Google Scholar) arising from the major 24:0-d18:1/24:0-Cer isomer. The spectrum (Fig. 2B) also contained the homologous ions at m/z 420 (c2a), 396 (d1a), and 236 (e3b″) and at m/z 406 (c2a), 382 (d1a), and 250 (e3b″), indicating the presence of 24:0-d16:1/26:0-Cer and 24:0-d17:1/25:0-Cer minor isomers, respectively. Similarly, the MS4 spectrum of the ions of m/z 642 (1,001.1 → 982.9 → 642; Fig. 2c) is dominated by the ions of m/z 420 (c2a), 396 (d1a), and 264 (e3b″), pointing to the presence of d18:1/26:0 structure, along with a minor ion set of m/z 434 (c2a), 410 (d1a), and 250 (e3b″) that identifies the d17:1/27:0 substituent. The combined structural information from MS3 (Fig. 2B) and MS4 (Fig. 2C) resulted in the assignment of 22:0-d18:1/26:0- and 22:0-d17:1/27:0-Cer minor isomers. The minor isomeric LCB/N-acyl-FAs were also seen by the MS4 spectra of the ions of m/z 628, 600, and 586 (Table 2) that resulted in the identification of 23:0-d17:1/26:0, 23:0-d18:1/25:0, 23:0-d16:1/27:0 (1-O-23:0 series); 25:0-d17:1/24:0, 25:0-d18:1/23:0, 25:0-d16:1/25:0 (1-O-25:0 series); and of 26:0-d16:1/24:0, 26:0-d18:1/22:0, 26:0-d17:1/23:0 (1-O-26:0 series) isobaric structures. These analyses resulted in the assignment of 14 isomers (Table 2).TABLE 2Structures of the isobaric isomers deduced from MSn on the ions of m/z 1001.1MS3 (1,001.1 → 982.99)aIons of m/z 982.99 are due to loss of water from ions of m/z 1,001.1 via MS2.MS4 (1,001.1 → 982.99 → F2)Structural AssignmentFragment Ions (F2)1-O-Acyl Group (from loss of)LCBN-AcylLCB/N-Acyl Moietiese3b″c2ad1a64222:0264420396d18:1/26:022:0-d18:1/26:0250434410d17:1/27:022:0-d17:1/27:062823:0250420396d17:1/26:023:0-d17:1/26:0264406382d18:1/25:023:0-d18:1/25:0278392368d19:1/24:023:0-d19:1/24:0614bMajor isomer.24:0264392368d18:1/24:024:0-d18:1/24:0236420396d16:1/26:024:0-d16:1/26:0250406382d17:1/25:024:0-d17:1/25:060025:0250392368d17:1/24:025:0-d17:1/24:0264378354d18:1/23:025:0-d18:1/23:0236406382d16:1/25:025:0-d16:1/25:058626:0236392368d16:1/24:026:0-d16:1/24:0264364340d18:1/22:026:0-d18:1/22:0250378354d17:1/23:026:0-d17:1/23:0The main isomer is indicated by boldface type.a Ions of m/z 982.99 are due to loss of water from ions of m/z 1,001.1 via MS2.b Major isomer. Open table in a new tab The main isomer is indicated by boldface type. In addition to the major species containing multiple isobaric isomers, minor species consisting of 1-O-acylceramides with additional double bonds located at 1-O-acyl, N-acyl substituent, or LCB are present (Table 1). For example, the MS2 spectrum of the [M + H]+ ions of m/z 998.9 is dominated by the ions of m/z 980.9 (loss of water) (data not shown) as seen earlier. MS3 on the ions of m/z 980.9 (998.9 → 980.9; Fig. 3A) yielded ions at m/z 724, 698, 642, 640, 626, 614, 612, 600, and 586, arising from elimination of 1-O-16:0-, -18:1-, -22:1-, -22:0-, -23:0-, -24:1-, -24:0-, -25:1-, and -26:1-fatty acyl substituents, respectively. The MS4 spectrum of the major ions of m/z 614 (998 → 980 → 614; data not shown) is similar to that shown in Fig. 2C, indicating that the molecules consisted of isomeric d18:1/24:0-, d16:1/26:0-, and d17:1/25:0-Cer substituents, leading to the assignment of 24:1-d18:1/24:0, 24:1-d16:1/26:0, 24:1-d17:1/25:0 isomers. MS4 on the ions of m/z 640 (998→ 980 → 640; Fig. 3B) yielded the predominant ions of m/z 264 (e3b″), suggesting the presence of d18:1-LCB and the 418(c2a)/394(d1a) ion pair that recognize the 26:1-fatty acyl substituent. The results gave assignment of the major 22:0-d18:1/26:1 isomer. The spectrum (Fig. 3B) also contained the minor ions of m/z 262 (e3b″), which signifies the presence of d18:2-LCB, a dehydrosphingosine LCB, and the companion ions of m/z 420(c2a)/396(d1a) that identify the N-26:0 acyl group, indicating that a minor 22:0-d18:2/26:0-Cer isomer is also present. Similar results were also observed for the MS4 spectrum of the ions of m/z 612 (Fig. 3C), which is dominated by the ions of m/z 264 (e3b″) alo" @default.
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• W2585341696 title "Linear ion-trap MSn with high-resolution MS reveals structural diversity of 1-O-acylceramide family in mouse epidermis" @default.
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