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

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

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

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