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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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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 [26]. 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 [21]. 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 [16]. 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 [5] from spectra A and C and (2) calculated composition (open column) derived from individual PtdEtn molecular species listed in Table 1.
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pone-0001368-g005: 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 [26]. 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 [21]. 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 [16]. 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 [5] from spectra A and C and (2) calculated composition (open column) derived from individual PtdEtn molecular species listed in Table 1.

Mentions: To further validate the paired rule for pPtdEtn molecular species, we performed additional experiments to directly demonstrate the presence of very long aliphatic chains containing high degrees of unsaturation which are usually present in one of the paired pPtdEtn molecular species with low to very low abundance (Table 1). The bovine heart PtdEtn mixture was treated with 1 M LiOMe to cleave the FA ester bonds of PtdEtn as previously described [26]. The resultant solution was washed with 1∶4 (v/v) ethyl ether/hexane three times to remove the fatty acyl esters generated by this procedure. The resultant lysoPtdEtn containing an ether aliphatic chain at the sn-1 position of glycerol was recovered by using a modified Bligh and Dyer extraction procedure [21]. The ether lysoPtdEtn molecular species were analyzed by ESI/MS in the negative-ion mode and the presence of very long aliphatic chains containing high degrees of unsaturation in low abundance (e.g., the ion at m/z 510.2) was detected (Figure 5A). These low abundance ether lysoPtdEtn molecular species were more clearly visualized after Fmoc chloride treatment (Figure 5B) as previously described [16], particularly in the mass spectrum acquired by neutral loss of the Fmoc moiety (neutral loss of 222.2 u) (Figure 5C). Quantitative analyses of these ether lysoPtdEtn molecular species in comparison to an internal standard after 13C deisotoping [18], [27] corresponded well with the aliphatic chain composition of the ether PtdEtn molecular species listed in Table 1 (Figure 5D). These results support the hypothesis that paired pPtdEtn isomers are present in lipid extracts of biological samples.


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)

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 [26]. 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 [21]. 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 [16]. 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 [5] from spectra A and C and (2) calculated composition (open column) derived from individual PtdEtn molecular species listed in Table 1.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2147047&req=5

pone-0001368-g005: 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 [26]. 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 [21]. 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 [16]. 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 [5] from spectra A and C and (2) calculated composition (open column) derived from individual PtdEtn molecular species listed in Table 1.
Mentions: To further validate the paired rule for pPtdEtn molecular species, we performed additional experiments to directly demonstrate the presence of very long aliphatic chains containing high degrees of unsaturation which are usually present in one of the paired pPtdEtn molecular species with low to very low abundance (Table 1). The bovine heart PtdEtn mixture was treated with 1 M LiOMe to cleave the FA ester bonds of PtdEtn as previously described [26]. The resultant solution was washed with 1∶4 (v/v) ethyl ether/hexane three times to remove the fatty acyl esters generated by this procedure. The resultant lysoPtdEtn containing an ether aliphatic chain at the sn-1 position of glycerol was recovered by using a modified Bligh and Dyer extraction procedure [21]. The ether lysoPtdEtn molecular species were analyzed by ESI/MS in the negative-ion mode and the presence of very long aliphatic chains containing high degrees of unsaturation in low abundance (e.g., the ion at m/z 510.2) was detected (Figure 5A). These low abundance ether lysoPtdEtn molecular species were more clearly visualized after Fmoc chloride treatment (Figure 5B) as previously described [16], particularly in the mass spectrum acquired by neutral loss of the Fmoc moiety (neutral loss of 222.2 u) (Figure 5C). Quantitative analyses of these ether lysoPtdEtn molecular species in comparison to an internal standard after 13C deisotoping [18], [27] corresponded well with the aliphatic chain composition of the ether PtdEtn molecular species listed in Table 1 (Figure 5D). These results support the hypothesis that paired pPtdEtn isomers are present in lipid extracts of biological samples.

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