Linear ion-trap MSn with high-resolution MS reveals structural diversity of 1-O-acylceramide family in mouse epidermis[S]

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.

family, which consists of C 18 -sphingosine as the major longchain base (LCB) together with minor C 16 -, C 17 -, C 18 -, C 19 -, and C 20 -sphingosine LCBs and a C 18 -dehydrosphingosine LCB to which a whole array of amide-linked fatty acyl substituents (ranging from C 14 to C 32 with zero to one double bond) and 1-O-linked acyl groups (ranging from C 14 to C 32 with zero to one double bond) are attached.

Preparation of FA-AMPP derivative for locating the double bonds on the FA chain
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 (19). 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 (20).

MS
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 l/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 × 10 4 , 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 MS n 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 MS n spectra (n = 2-4).

The fragmentation pathways of 1-O-acylceramide
When subjected to ESI in the positive-ion mode, Cers were mainly seen as the [M + H  H 2 O] + ions, due to facile loss of a water molecule (10). 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-Ooleoyl group as an acid, and of m/z 264 (e 3b″ ), arising from further cleavage of the N-heptaoctanoyl substituent as a ketene (Scheme 1). This fragmentation process was supported by the MS 4 spectrum of the ion of m/z 516 (816 → 798 → 516; Fig. 1C), which contained prominent ions of m/z 264 (e 3b″ ) (Scheme 1) that are unique to Cers consisting of C 18sphingosine LCB (10). 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 C 18 H 30 residue (Scheme 1). This latter fragmentation process also led to the ions of m/z 247, representing the highly conjugated triene cations. The . These MS 2 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.

Characterization of 1-O-acylceramide isolated from mouse epidermis
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 C n H 2n2 O 4 N 1 (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 C n H 2n4 O 4 N 1 (n = 53-72) was also observed, indicating the presence of the minor species with an additional double bond ( Table 1)  (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 C n H 2n4 O 5 N 1 were not observed, excluding the presence of the subfamily of 1-O-acylceramides with the N-hydroxyacyl group previously reported by Rabionet and colleagues (6, 7) The LIT MS n mass spectrometric approaches together with high-resolution mass measurements toward identification of these mouse epidermis 1-O-acylceramdes are described below.

Revelation of isobaric isomers in the molecular species
Multiple isobaric isomers were observed for all the ions that appeared in the ESI-MS spectrum (Table 1) Table 2).

Identification of 1-O-acylceramides with unsaturated bonds
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) Table  S1). The combined information arising from MS 2 , MS 3 , and MS 4 revealed the presence of 24 isobaric structures (see supplemental Table S1).

Identification of minor 1-O-acylceramide species in the mixture
The number of the isobaric isomers appears to grow even bigger for the minor ion species, similar to those seen for other lipid classes (21). For example, the MS    Table S2). The combined structural information from MS n (n = 2-4) reveals 26 isobaric isomers (supplemental Table S2 Table 3. In total, an estimate of 710 species were found in this lipid family.

HCD tandem mass spectrometric analysis of FA-AMPP derivative for locating the double bond(s) on the fatty acyl chain
High-resolution mass measurements on the hydrolysate-AMPP reaction products revealed that the presence of the FA-AMPP ion series ranged from C14 to C32:0 with zero or one double bond (supplemental Table S3) in which the molecules with the saturated fatty acyl chain were the major species; while minor species with unsaturated bond and with odd chain acyl substituents were also present (supplemental Table S1). HCD MS 2 on the M + ions of the FA-AMPP derivatives indicated that the double bond position of the FA substituents were all located at n-9 (-9).   (6,7). Another important finding in this study is that the abundant species, such as the ions of m/z 888.9, 916.9, 972.9, and 1,000.1, all contain d18:1/24:0-Cer as the major core structure, and the isobaric isomers are seen by the variation of the 1-O-acyl chain, rather than by variation of the Cer core. More than 20 isobaric isomers are present in more than half of the molecular species, and more than 700 structures (Table 1), including a minor subfamily consisting of dehydrosphingosine LCB, are present. Rabionet and colleagues (6,7) reported the presence of the 1-O-acyl N--hydroxyacylceramide subfamily in mouse and human epidermis. Interestingly, this Cer subfamily was found as a very minor species, and was eluted separately (fraction 3) from the major 1-O-acyl N-acylceramides (fraction 2). The presence of the vast number of structures in the 1-O-acylceramide family, as seen in this study, underscores the complexity of epidermal Cers, which consist of more than 10 Cer families (15,16,18,24). Thus, this study highlights the utility of highresolution LIT MS in the structural elucidation of complex lipid structures and the application of this technique in the complete characterization and differentiation of various Cer classes, including the location of the double bond(s) and hydroxyl group(s) on the fatty acyl chain and LCB has been reported recently (25).
There is a stark difference in the profiles of the MS 4 spectra of m/z 612 (Fig. 3C), of which the fatty acyl chain contains a double bond, and of m/z 614 (Fig. 2B), whose fatty acyl chain is saturated. The water, in which the first water loss may involve the participation of the 1-hydroxy group (supplemental Scheme S1). However, whether this difference in the sequence of the elimination processes leading to these precursor ions (i.e., the third generation ions) contributes to the differential cleavage of fatty acyl groups to form the ions of m/z 264 upon being further subjected to CID is also unclear. The prominence of the ions of m/z 264, as seen in Fig. 1C, may also reflect the fact that loss of a shorter 17:0-fatty acyl chain as a ketene in 18:1-d18:1/17:0-Cer is probably more facile than the analogous loss of the longer 26:0-fatty acyl chain in 22:0-d18:1/26:0-Cer, as supported by the notion that ions of m/z 264 in the MS 4 spectrum of m/z 642 (Fig. 2c) is less prominent (than m/z 420).   The observation of the 1-O-acyl and N-acyl substituents ranging from C14 to C32 with zero or one double bond in this Cer family (Table 1, 3) is consistent with the results from the high-resolution mass measurement of the AMPP derivatives of the acid hydrolysate that yielded the M + ion series ranging from C14 to C32 with zero or one double bond (supplemental Table S3). There are several minor species possessing two double bonds in the 1-O-acyl (e.g., 24:2 and 26:2; Table 1) or N-acyl (e.g., 24:2, 26:2, and 28:2; Table 3) chain, and the very long N-34:1-fatty acyl chaincontaining species are also seen (Table 3). However, the corresponding FA species were not observed in the hydrolysate (supplemental Table S3). These FAs may be lost or too low to be detected after hydrolysis and derivatization steps. Nevertheless, the realization of the position of the double bond of the unsaturated FA substituents at n-9 for all the FA substituents, excepting 16:1-FA of which the double bond appears to be located at C6, is consistent with the notion of, for example, the presence of sphingolipids with d18:1/ 15 24:1 (n-9) core structure as reported previously (26). The 16:1-FA appears to be a  6 16:1-FA, which is a sapienic acid exclusively found in sebum (27).
Results from our preliminary study indicate that there is a significantly higher abundance (greater than three times) of the 1-O-acylceramide family in the epidermis of FA transport protein 4 (FATP4)-deficient newborn mice (Fatp4 / mice) compared with heterozygous control newborn mice (Fatp +/ ), while the level of the 1-O-acylceramide family is restored in the rescued null Fatp4 newborn mice (Fatp4 / ; Tg(IVL-Fatp1) (28) (data not shown). Mutations in SLC27A4, the gene encoding FATP4, are known to cause ichthyosis prematurity syndrome in humans (29,30). FATP4 is critical for development of fetal skin (31) and it has been hypothesized that lack of FATP4 in epidermal keratinocytes induces abnormal lipid metabolism in the epidermis that initiates the observed alterations in epidermal signaling pathways (32,33). It is still unclear whether the FATP4 gene plays a role in the synthesis of this complex Cer family. Studies toward understanding the mechanism underlying the increase of this 1-O-acylceramide family in Fatp4 mutant fetal epidermis are currently in progress in our laboratory.