Shotgun lipidomics identifies a paired rule for the presence of isomeric ether phospholipid molecular species

Abstract

Background: Ether phospholipids are abundant membrane constituents present in electrically active tissues (e.g., heart and the brain) that play important roles in cellular function. Alterations of ether phospholipid molecular species contents are associated with a number of genetic disorders and human diseases.

Methodology/principal findings: Herein, the power of shotgun lipidomics, in combination with high mass accuracy/high resolution mass spectrometry, was explored to identify a paired rule for the presence of isomeric ether phospholipid molecular species in cellular lipidomes. The rule predicts that if an ether phospholipid A'-B is present in a lipidome, its isomeric counterpart B'-A is also present (where the ' represents an ether linkage). The biochemical basis of this rule results from the fact that the enzymes which participate in either the sequential oxidation of aliphatic alcohols to fatty acids, or the reduction of long chain fatty acids to aliphatic alcohols (metabolic precursors of ether lipid synthesis), are not entirely selective with respect to acyl chain length or degree of unsaturation. Moreover, the enzymatic selectivity for the incorporation of different aliphatic chains into the obligatory precursor of ether lipids (i.e., 1-O-alkyl-glycero-3-phosphate) is also limited.

Conclusions/significance: This intrinsic amplification of the number of lipid molecular species present in biological membranes predicted by this rule and demonstrated in this study greatly expands the number of ether lipid molecular species present in cellular lipidomes. Application of this rule to mass spectrometric analyses provides predictive clues to the presence of specific molecular species and greatly expands the number of identifiable and quantifiable ether lipid species present in biological samples. Through appropriate alterations in the database, use of the paired rule increases the number of identifiable metabolites in metabolic networks, thereby facilitating identification of biomarkers presaging disease states.

Figure 1. The structures of the paired isomers of plasmenylethanolamine molecular species.

Figure 2. Representative negative-ion ESI/MS analyses of bovine heart ethanolamine glycerophospholipid molecular species.

Bovine heart lipids were extracted by a modified Bligh and Dyer procedure and the PtdEtn fraction was separated by using HPLC with a cation-exchange column as previously described . 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 “MATERIALS AND METHODS”.

Figure 3. Product ion analyses of individual molecular species present in isolated bovine heart ethanolamine glycerophospholipids.

Product ion ESI/MS analyses of PtdEtn molecular species in isolated bovine heart ethanolamine glycerophospholipids at m/z 772.5 (Panel A) and 774.5 (Panel B) were performed by using an LTQ-Orbitrap mass spectrometer. Analyses were conducted using a peak width setting of 1 Th by selection of the molecular ion in the linear ion trap (LTQ), collision activation in the C-trap with a normalized collision energy of 55% and gas pressure of 1 mTorr, and analysis of the resultant product ions in the Orbitrap.

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

Product ion ESI/MS analysis of deprotonated 18∶0-20∶4 plasmenylethanolamine at m/z 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.

Figure 5. Quantitative analyses of ether lysoPtdEtn produced by alkaline hydrolysis of fatty acyl esters in bovine heart PtdEtn.

Bovine heart PtdEtn solution was treated with LiOMe leading to the cleavage of the FA ester bonds in the PtdEtn pool as previously described . The resultant lysoPtdEtn mixture containing an ether aliphatic chain at the sn-1 position of glycerol was recovered using a modified procedure of Bligh and Dyer . The mass spectrum in panel A was acquired in the negative-ion mode by using a QqQ mass spectrometer directly from the lysoPtdEtn solution that was diluted to less than 50 pmol of total lipids/µl. The mass spectrum in panel B was acquired in the negative-ion mode directly from a diluted lysoPtdEtn solution after addition of Fmoc chloride as previously described . The mass spectrum in panel C was acquired in the negative-ion mode as that of spectrum B but in the neutral loss mode. The 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 the collisional activation was performed in the second quadrupole at collision energy of 32 eV. “IS” denotes internal standard. Panel D shows the comparison of ether aliphatic chain profiles in bovine heart PtdEtn determined by using different approaches: (1) direct quantitation of the resultant ether lysoPtdEtn molecular species from alkaline hydrolysis of PtdEtn (closed column) through a two-step quantitation procedure from spectra A and C and (2) calculated composition (open column) derived from individual PtdEtn molecular species listed in Table 1.

Figure 6. Representative negative-ion ESI/MS analyses of individual ethanolamine glycerophospholipid molecular species in mouse cerebellar lipid extracts.

Mouse cerebellar lipid extracts were prepared by a modified Bligh and Dyer procedure . 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 m/z 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 . 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.

Figure 7. Comparison of representative aliphatic or acyl chain profiles in different lipid domains of bovine heart.

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 Table 1. The fatty acyl chain composition of bovine heart diacyl PtdEtn (Panel B) was also calculated from the identified individual molecular species as listed in Table 1. The profile of acyl-CoA in bovine heart (Panel C) was re-plotted from previously published data .

Figure 8. Pathways involved in the biosynthesis of plasmenylethanolamine.

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