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

Yang K, Zhao Z, Gross RW, Han X - PLoS ONE (2007)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Division of Bioorganic Chemistry and Molecular Pharmacology, Department of Internal Medicine, Washington University School of Medicine, St. Louis, Missouri, United States of America.

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.

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Related in: MedlinePlus

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 [21] and the PtdEtn fraction was separated by using HPLC with a cation-exchange column as previously described [23]. 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”.
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pone-0001368-g002: 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 [21] and the PtdEtn fraction was separated by using HPLC with a cation-exchange column as previously described [23]. 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”.

Mentions: PtdEtn molecular species were readily analyzed as deprotonated molecular ions by ESI/MS in the negative-ion mode (Figure 2) as previously described [18], [24]. Abundant pPtdEtn molecular species were present in isolated bovine heart PtdEtn as previously described [25]. However, isobaric ions to these pPtdEtn molecular species (corresponding to phosphatidylethanolamine (dPtdEtn) molecular species) were also present and were readily resolved using the high resolution present in an Orbitrap analyzer (Inset B of Figure 2). Tandem MS analysis of each individual isobaric ion peak corresponding to a representative deprotonated pPtdEtn molecular species always demonstrated the presence of multiple pPtdEtn isomers along with isomeric plasmanylethanolamine (aPtdEtn) and isobaric dPtdEtn molecular species (Figures 2 and 3 and Table 1). For example, product-ion MS analysis of an isobaric ion at m/z 772.5 (0.26% relative abundance to the base peak at m/z 766.54, Figure 2, inset A of Figure 2, and Table 1) from bovine heart PtdEtn in the negative-ion mode was performed with a mass selection window of 1 Th using an LTQ-Orbitrap mass spectrometer after collision-induced activation (CID). The CID analysis of this isobaric ion demonstrated multiple abundant fragment ions in the carboxylate region in an order of abundance at m/z 281.25 (i.e., 18∶1 FA), m/z 329.25 (i.e., 22∶5 FA), m/z 279.23 (i.e., 18∶2 FA), m/z 327.23 (i.e., 22∶6 FA), m/z 311.29 (i.e., 20∶0 FA), m/z 309.28 (i.e., 20∶1 FA), m/z 283.26 (i.e., 18∶0 FA), and m/z 303.23 (i.e., 20∶4 FA) (inset of Figure 3A). It is noteworthy that the ions at m/z 283.24 and 285.26 (inset of Figure 3A) correspond to aliphatic anions resulting from the loss of a CO2 molecule from 22∶6 FA (m/z 327.23) and 22∶5 FA (m/z 329.25), respectively. Loss of a CO2 molecule from polyunsaturated fatty acyl carboxylates (e.g., 20∶5, 22∶5, and 22∶6 FA) is common as previously demonstrated [5], [17]. Loss of a CO2 molecule from arachidonate (20∶4 FA) can also be detected, but is relatively less rapid in comparison to other aforementioned polyunsaturated fatty acids. The most common fragmentation pathway likely results from the resonance stabilized anion conjugated to olefinic π clouds. Since the content of the selected molecular ion at m/z 772.5 was markedly decreased in the lipid mixture following treatment with acid vapor as described previously [5], this ion largely represented a deprotonated pPtdEtn or isomeric pPtdEtn mixtures. Based on the identified FA carboxylates through accurate mass analyses, the accurate mass of the precursor ion (i.e., m/z 772.53), and the known information about the ion (i.e., predominantly deprotonated pPtdEtn), gave rise to the conclusion that the ion at m/z 772.53 is comprised of at least four pPtdEtn isomers, including 18∶2-22∶5, 18∶1-22∶6, 22∶6-18∶1, and 22∶5-18∶2 pPtdEtn molecular species in the order of relative abundance. The molar ratio of these isomers was approximately 6∶5∶5∶4 from the intensities of FA carboxylates after considering the differential fragmentation kinetics of different molecular species as previously described [24]. The relative ratios of each individual molecular species are listed in parentheses underneath the corresponding molecular species in Table 1.


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

Yang K, Zhao Z, Gross RW, Han X - PLoS ONE (2007)

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 [21] and the PtdEtn fraction was separated by using HPLC with a cation-exchange column as previously described [23]. 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”.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2147047&req=5

pone-0001368-g002: 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 [21] and the PtdEtn fraction was separated by using HPLC with a cation-exchange column as previously described [23]. 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”.
Mentions: PtdEtn molecular species were readily analyzed as deprotonated molecular ions by ESI/MS in the negative-ion mode (Figure 2) as previously described [18], [24]. Abundant pPtdEtn molecular species were present in isolated bovine heart PtdEtn as previously described [25]. However, isobaric ions to these pPtdEtn molecular species (corresponding to phosphatidylethanolamine (dPtdEtn) molecular species) were also present and were readily resolved using the high resolution present in an Orbitrap analyzer (Inset B of Figure 2). Tandem MS analysis of each individual isobaric ion peak corresponding to a representative deprotonated pPtdEtn molecular species always demonstrated the presence of multiple pPtdEtn isomers along with isomeric plasmanylethanolamine (aPtdEtn) and isobaric dPtdEtn molecular species (Figures 2 and 3 and Table 1). For example, product-ion MS analysis of an isobaric ion at m/z 772.5 (0.26% relative abundance to the base peak at m/z 766.54, Figure 2, inset A of Figure 2, and Table 1) from bovine heart PtdEtn in the negative-ion mode was performed with a mass selection window of 1 Th using an LTQ-Orbitrap mass spectrometer after collision-induced activation (CID). The CID analysis of this isobaric ion demonstrated multiple abundant fragment ions in the carboxylate region in an order of abundance at m/z 281.25 (i.e., 18∶1 FA), m/z 329.25 (i.e., 22∶5 FA), m/z 279.23 (i.e., 18∶2 FA), m/z 327.23 (i.e., 22∶6 FA), m/z 311.29 (i.e., 20∶0 FA), m/z 309.28 (i.e., 20∶1 FA), m/z 283.26 (i.e., 18∶0 FA), and m/z 303.23 (i.e., 20∶4 FA) (inset of Figure 3A). It is noteworthy that the ions at m/z 283.24 and 285.26 (inset of Figure 3A) correspond to aliphatic anions resulting from the loss of a CO2 molecule from 22∶6 FA (m/z 327.23) and 22∶5 FA (m/z 329.25), respectively. Loss of a CO2 molecule from polyunsaturated fatty acyl carboxylates (e.g., 20∶5, 22∶5, and 22∶6 FA) is common as previously demonstrated [5], [17]. Loss of a CO2 molecule from arachidonate (20∶4 FA) can also be detected, but is relatively less rapid in comparison to other aforementioned polyunsaturated fatty acids. The most common fragmentation pathway likely results from the resonance stabilized anion conjugated to olefinic π clouds. Since the content of the selected molecular ion at m/z 772.5 was markedly decreased in the lipid mixture following treatment with acid vapor as described previously [5], this ion largely represented a deprotonated pPtdEtn or isomeric pPtdEtn mixtures. Based on the identified FA carboxylates through accurate mass analyses, the accurate mass of the precursor ion (i.e., m/z 772.53), and the known information about the ion (i.e., predominantly deprotonated pPtdEtn), gave rise to the conclusion that the ion at m/z 772.53 is comprised of at least four pPtdEtn isomers, including 18∶2-22∶5, 18∶1-22∶6, 22∶6-18∶1, and 22∶5-18∶2 pPtdEtn molecular species in the order of relative abundance. The molar ratio of these isomers was approximately 6∶5∶5∶4 from the intensities of FA carboxylates after considering the differential fragmentation kinetics of different molecular species as previously described [24]. The relative ratios of each individual molecular species are listed in parentheses underneath the corresponding molecular species in Table 1.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Division of Bioorganic Chemistry and Molecular Pharmacology, Department of Internal Medicine, Washington University School of Medicine, St. Louis, Missouri, United States of America.

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.

Show MeSH
Related in: MedlinePlus