Complete structural characterization of ceramides as [M−H]− ions by multiple-stage linear ion trap mass spectrometry

Abstract

Ceramide is a huge lipid family consisting of diversified structures including various modifications in the fatty acyl chain and the long chain base (LCB). In this contribution, negative-ion ESI linear ion-trap multiple-stage mass spectrometric method (LIT MSn) towards complete structural determination of ceramides in ten major families characterized as the [M – H]− ions is described. Multiple sets of fragment ions reflecting the fatty acyl chain and LCB were observed in the CID MS2 spectrum, while the sequential MS3 and MS4 spectra contain structural information for locating the double bond and the functional groups, permitting realization of the fragmentation processes. Thereby, differentiation of ceramide molecules varied by chain length, the LCB (sphingosine, phytosphigosine, 6-hydroxy-sphingosine), and by the modification (α-hydroxy-, β-hydroxy-, ω-hydroxy-FA) can be achieved; and many isomeric structures in the biological specimen can be revealed in detail.

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

“…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).…”

Glycosphingolipidomics of donkey milk by hydrophilic interaction liquid chromatography coupled to ESI and multistage MS

“…The low intensity peak at m/z 199.1 agrees to the fatty acid carboxylate anion C12:0. As previously reported for CID–MS/MS of ceramides, this product ion is arising from an exclusive rearrangement of the acyl chain and provides direct identification of the fatty acid substituent . In negative ion mode the MS/MS fragmentation pattern should, in theory, afford information about both the headgroups and types of SBs and fatty acids; unfortunately, diagnostic ions exhibited very low intensities even upon injection of pure standards so, most likely, they would be hardly detectable in sample extracts.…”

“…Although experimental and theoretical data on the fragmentation of ceramides upon ESI in negative ion mode are available , no results are reported on the characterization of neutral GSLs by MS/MS in CID experiments. To rationalize a plausible fragmentation pattern for HexCers, tandem MS analyses were initially performed on standard nonendogenous GSLs in a low collisional energy regime in both negative and positive ion modes (Figure ).…”

“…To obtain a more refined structural characterization on SB, CID‐MS 3 spectra of ions detected at m/z 480.4 and 464.3 were acquired, in negative and positive ion mode, respectively. To deal with the complexity of these compounds and related fragments, a systematic nomenclature has been established which describes the fingerprint fragmentations . The product ions rising from multiple losses of water, HCHO, combined H 2 O and HCHO are common ions for Cer so they are nominated as “c” ions; in the m/z descending order they become c 1 , c 2 , c 3 , c 4 , and so on.…”

“…Alternatively, fast‐atom bombardment , MALDI , and ESI in combination with MS/MS have been successfully used to this aim. Among them, very recently, it has been reported that linear ion trap (LIT) with MS/MS analyses in negative ion mode allowed the complete structural characterization of ten ceramide subfamilies including sphingosine, dihydrosphingosine, phytosphingosine, and 6‐hydroxysphingosine with hydroxylated and nonhydroxylated fatty acids . Conversely, limited structural information is available on their glyco‐derivatives being the loss of sugar moiety the most important recorded fragment ion .…”