Resolving Modifications on Sphingoid Base and N-Acyl Chain of Sphingomyelin Lipids in Complex Lipid Extracts

Abstract

Sphingomyelins (SMs) are an essential class of lipids widely existing in different organisms. The sphingoid base and N-acyl chain are two building blocks which can undergo different types of modifications during lipogenesis, including desaturation, hydroxylation, and methyl branching. Current lipidomic analysis methods cannot provide detailed information on these structural motifs. Herein, we developed a tandem mass spectrometric method by harnessing radical-directed dissociation (RDD) from collision-induced dissociation (CID) of the bicarbonate anion adduct of SM ([M + HCO3]−). A major RDD channel produced a high-abundance fragment carrying the intact N-acyl chain, termed as “N-acyl fragment”, allowing the assignment of the sphingoid base/N-acyl composition and relative quantitation of compositional isomers of SM at high sensitivity. RDD also produced intrachain fragments in lower abundances, which helped localization of methyl branching and hydroxylation in SM. The acetone Paternò–Büchi (PB) reaction was found to be capable of derivatizing the Δ4 carbon–carbon double bond (CC) in sphingosine (SPH) base and producing CC diagnostic ions upon CID, albeit at much lower efficiencies than those of the isolated CC in alkyl chains. A liquid chromatography–mass spectrometry workflow was developed by incorporating MS2 CID of SM via [M + HCO3]− and PB-MS2 CID. The capability of profiling SM with detailed structural information was demonstrated by analyzing complex lipid extracts from porcine brain and Caenorhabditis elegans. These results provided visualization of the sphingoid base/N-acyl compositional isomers of SM lipids and revealed large structural diversity from each sample. These included identification of the sphingadiene base [d18:1­(Δ4,14)], CC location isomers in N-acyls, C-2 hydroxylation of N-acyls, and iso-methyl branched SPH base.

Deep-lipidotyping by mass spectrometry: recent technical advances and applications

“…By integrating on an LC-MS system, the iso- and anteiso-branched LPC 17:0 isomers have been identified and relatively quantified ( 101 ). In another report, C-2 hydroxylation of N-acyls and iso-methyl branched sphingosine base were identified from SMs of C. elegans ( 112 ). The intrachain fragments generated from these methods are of relatively low ion abundances, thus limiting the sensitivity of this method.…”

Computational mass spectrometry accelerates C=C position-resolved untargeted lipidomics using oxygen attachment dissociation

“…Our results indicated that the Δ4 sphingobase 18:1(Δ4),2O is the major component of sphingolipids, which are biosynthesized by dihydroceramide Δ4-desaturase during the ceramide synthesis pathway converting ceramide non-hydroxy fatty acid-dihydrosphingosine (Cer-NDS) into Cer-NS 52 . The C=C positions in the sphingadienine moiety have recently been reported as 18:2(Δ4,14);2O in several studies 20,57,58 . Our platform characterized 18:2(Δ4,14);2O in various tissues containing mouse brain, eye, liver, and human plasma.…”

“…Paterno-Buchi (PB) photochemical reaction [18][19][20][21][22] and mCPBA epoxidation for lipid double-bond identi cation (MELDI) 23,24 are popular techniques that have advantages in a higher signal-to-noise ratio without the need for special MS instruments. Another uses a different principle of mass fragmentation, which includes ultraviolet photodissociation (UVPD) [25][26][27][28] , ozone-induced dissociation (OzID) [29][30][31][32][33] , and electron impact excitation of ions from organics (EIEIO) [34][35][36][37] .…”

Advanced tandem mass spectrometry in metabolomics and lipidomics—methods and applications

“…7, lower). PB reactions with acpy and other carbonyl compounds have been used not only to analyze standards but also to investigate complex lipid extracts from body fluids, cells, or tissues revealing DB positions and sometimes sn-isomers for CEs [116,130], FAs [116], GPs [121], and SLs [131]. Recently, PB methods have been extended to also target small FA metabolites [117][118][119] or have been adapted to benefit other advanced tandem MS tools such as UVPD [115] or ion/ion reactions [122].…”