Identification and Quantitation of Fatty Acid Double Bond Positional Isomers: A Shotgun Lipidomics Approach Using Charge-Switch Derivatization

The specific locations of double bonds in mammalian lipids have profound effects on biological membrane structure, dynamics and lipid second messenger production. Herein, we describe a shotgun lipidomics approach that exploits charge-switch derivatization with <i>N</i>-(4-aminomethylphenyl) pyridinium (AMPP) and tandem mass spectrometry for identification and quantification of fatty acid double bond positional isomers. Through charge-switch derivatization of fatty acids followed by positive-ion mode ionization and fragmentation analysis, a marked increase in analytic sensitivity (low fmol/μL) and the identification of double bond positional isomers can be obtained. Specifically, the locations of proximal double bonds in AMPP-derivatized fatty acids are identified by diagnostic fragment ions resulting from the markedly reduced 1,4-hydrogen elimination from the proximal olefinic carbons. Additional fragmentation patterns resulting from allylic cleavages further substantiated the double bond position assignments. Moreover, quantification of fatty acid double bond positional isomers is achieved by the linear relationship of the normalized intensities of characteristic fragment ions vs the isomeric compositions of discrete fatty acid positional isomers. The application of this approach for the analysis of fatty acids in human serum demonstrated the existence of two double bond isomers of linolenic acid (i.e., Δ^6,9,12 18:3, γ-linolenic acid (GLA), and Δ^9,12,15 18:3, α-linolenic acid (ALA)). Remarkably, the isomeric ratio of GLA vs ALA esterified in neutral lipids was 3-fold higher than the ratio of their nonesterified moieties. Through this developed method, previously underestimated or unidentified alterations in fatty acid structural isomers can be determined facilitating the identification of novel biomarkers and maladaptive alterations in lipid metabolism during disease

Shotgun Lipidomics Approach to Stabilize the Regiospecificity of Monoglycerides Using a Facile Low-Temperature Derivatization Enabling Their Definitive Identification and Quantitation

Monoglycerides play a central role in lipid metabolism and are important signaling metabolites. Quantitative analysis of monoglyceride molecular species has remained challenging due to rapid isomerization via α-hydroxy acyl migration. Herein, we describe a shotgun lipidomics approach that utilizes a single-phase methyl <i>tert</i>-butyl ether extraction to minimize acyl migration, a facile low temperature diacetyl derivatization to stabilize regiospecificity, and tandem mass spectrometric analysis to identify and quantify regioisomers of monoglycerides in biological samples. The rapid and robust diacetyl derivatization at low temperatures (e.g., −20 °C, 30 min) prevents postextraction acyl migration and preserves regiospecificity of monoglyceride structural isomers. Furthermore, ionization of ammonium adducts of diacetyl monoglyceride derivatives in positive-ion mode markedly increases analytic sensitivity (low fmol/μL). Critically, diacetyl derivatization enables the differentiation of discrete monoglyceride regioisomers without chromatography through their distinct signature fragmentation patterns during collision induced dissociation. The application of this approach in the analysis of monoglycerides in multiple biologic tissues demonstrated diverse profiles of molecular species. Remarkably, the regiospecificity of individual monoglyceride molecular species is also diverse from tissue to tissue. Collectively, this developed approach enables the profiling, identification and quantitation of monoglyceride regioisomers directly from tissue extracts

Comparison of representative aliphatic or acyl chain profiles in different lipid domains of bovine heart.

<p>The profiles of both aliphatic chains (open column in Panel A) and fatty acyl chains (closed column in Panel A) of bovine heart ether-linked ethanolamine glycerophospholipids (PtdEtn) were derived from individual molecular species listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001368#pone-0001368-t001" target="_blank">Table 1</a>. The fatty acyl chain composition of bovine heart diacyl PtdEtn (Panel B) was also calculated from the identified individual molecular species as listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001368#pone-0001368-t001" target="_blank">Table 1</a>. The profile of acyl-CoA in bovine heart (Panel C) was re-plotted from previously published data <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001368#pone.0001368-DeMar1" target="_blank">[28]</a>.</p

Product ion analyses of synthetic 18∶0-20∶4 plasmenylethanolamine molecular species in the negative-ion mode.

<p>Product ion ESI/MS analysis of deprotonated 18∶0-20∶4 plasmenylethanolamine at <i>m/z</i> 750.54 was performed on an LTQ-Orbitrap mass spectrometer with a C-trap using an ion selective window of 1 Th by LTQ. Collision activation in C-trap was carried out with normalized collision energy of 55% and gas pressure of 1 mT. The resultant fragment ions were analyzed in the Orbitrap. The arrow indicates the absence of the 18:0 FA carboxylate in the spectrum after amplifying the position greater than1,000 fold.</p

Representative negative-ion ESI/MS analyses of bovine heart ethanolamine glycerophospholipid molecular species.

<p>Bovine heart lipids were extracted by a modified Bligh and Dyer procedure <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001368#pone.0001368-Bligh1" target="_blank">[21]</a> and the PtdEtn fraction was separated by using HPLC with a cation-exchange column as previously described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001368#pone.0001368-Gross3" target="_blank">[23]</a>. Analyses of PtdEtn molecular species were performed in the negative-ion mode by using an LTQ-Orbitrap mass spectrometer equipped with a Nanomate device as described under “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001368#s2" target="_blank">MATERIALS AND METHODS</a>”.</p

Identification and analyses of individual molecular species present in purified bovine heart ethanolamine glycerophospholipid^a.

a<p>Bovine heart lipids were extracted by a modified Bligh and Dyer procedure <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001368#pone.0001368-Bligh1" target="_blank">[21]</a> and the ethanolamine phospholipid (PtdEtn) fraction was separated by using HPLC as previously described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001368#pone.0001368-Gross3" target="_blank">[23]</a>. Analyses of PtdEtn molecular species were performed in the negative-ion mode by using an LTQ-Orbitrap mass spectrometer with an electrospray ion source. The determined monoisotopic masses (column 1) of PtdEtn molecular species were externally calibrated relative to the base peak. The molecular formulas listed in column 2 were derived from accurate mass analyses of monoisotopic mass and were grouped into each isobaric mass. The prefix “a”, “d”, and “p” stand for alkyl-acyl PtdEtn, diacyl PtdEtn, and plasmalogen PtdEtn, respectively. The relative abundance listed in column 3 was normalized to the isobaric base peak of the ion at <i>m/z</i> 766.5 after ¹³C de-isotoping and represents X±SD of at least four different analyses. The notation m∶n represents the fatty acyl (or ether aliphatic) chain containing m carbons and n double bonds. The numbers in the parentheses represent the relative composition of each individual molecular species of an isobaric ion. The symbols of “<” and “>” indicate that the data represent the best estimation from the analyses.</p>b<p>Identification of individual pPtdEtn molecular species was performed based on both accurate mass analyses and acidic vapor treatment. Identification of individual aPtdEtn molecular species was performed based on the accurate mass analyses, the paired rule, and the information of the identified pPtdEtn counterparts as discussed in the text. Identification of individual dPtdEtn molecular species was conducted solely based on accurate mass analyses. The abundance of each of the paired dPtdEtn molecular species cannot be accurately determined at the current stage of lipidomic technology.</p

Representative negative-ion ESI/MS analyses of individual ethanolamine glycerophospholipid molecular species in mouse cerebellar lipid extracts.

<p>Mouse cerebellar lipid extracts were prepared by a modified Bligh and Dyer procedure <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001368#pone.0001368-Bligh1" target="_blank">[21]</a>. Spectrum A was acquired in the negative-ion mode by using a QqQ mass spectrometer directly from a lipid extract that was diluted to less than 50 pmol of total lipids/µl after addition of approximately 25 pmol LiOH/µl to the lipid solution. Spectrum B was taken in the negative-ion mode after the diluted lipid solution used in spectrum A was treated with acid vapor and a small amount of LiOH (approximately 25 pmol LiOH/µl) was added to the infused solution. Spectrum C was acquired in the negative-ion mode as that of spectrum A but in the precursor-ion mode. The tandem mass spectrometry of precursor-ion scanning of 196 Th (i.e., phosphoethanolamine) was conducted through scanning the first quadrupole in the interested mass range and monitoring the third quadruple with the ion at <i>m/z</i> 196 while collision activation was performed in the second quadrupole at collision energy of 50 eV. Spectrum D was acquired in the negative-ion mode directly from a diluted mouse cerebellum lipid extract after addition of Fmoc chloride as previously described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001368#pone.0001368-Han5" target="_blank">[16]</a>. Spectrum E was acquired in the negative-ion mode as that of spectrum D but in the neutral loss mode. Tandem mass spectrometry of neutral loss scanning was conducted through coordinately scanning the first and third quadrupoles with a mass difference of 222.2 u (i.e., loss of a Fmoc) while collisional activation was performed in the second quadrupole at collision energy of 32 eV. “IS” denotes internal standard. All mass spectral traces are displayed after normalization to the base peak in each individual spectrum. All spectra are displayed after being normalized to the base peak in individual spectrum.</p

Pathways involved in the biosynthesis of plasmenylethanolamine.

<p>The enzymes that may be involved in non-selective utilization of acyl CoA pool are highlighted with broken-lined frames. ** CDP-ethanolamine: 1-<i>O</i>-alkyl-2-acyl-<i>sn</i>-glycerol ethanolamine phosphotransferase.</p

The structures of the paired isomers of plasmenylethanolamine molecular species.

<p>The structures of the paired isomers of plasmenylethanolamine molecular species.</p

Kuma, Kengo

Kuma è impegnato da anni in una seria critica a quello che definisce il ‘metodo del calcestruzzo’, nel desiderio di trovare un’alternativa all’uso di questo materiale che ‘governa’ il mondo perché ha un metodo di produzione universale. Il suo interesse è rivolto all’incontro con i materiali che egli chiama sostanze e al tema della sparizione, ben sintetizzato dal suo motto: «Voglio cancellare l’architettura!»