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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 individual ethanolamine glycerophospholipid molecular species in mouse cerebellar lipid extracts.Mouse cerebellar lipid extracts were prepared by a modified Bligh and Dyer procedure [21]. 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 [16]. 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.
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pone-0001368-g006: 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 [21]. 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 [16]. 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.

Mentions: Next, we examined whether this paired rule was applicable to the analysis of acid-labile, plasmalogen molecular species present in lipid extracts from multiple mammalian tissues or extracellular fluid samples including brain, heart, intestine, kidney, liver, and dorsal root ganglia of rat, rabbit, mouse, and/or human. A shotgun lipidomics approach was utilized to identify and quantify lipid molecular species following intrasource separation as described previously [18]–[20]. For example, PtdEtn molecular species from mouse cerebellum were analyzed by survey scanning in the negative-ion mode using an LTQ-Orbitrap mass spectrometer after direct infusion of diluted lipid extract solutions supplemented with a small amount LiOH (similar to Figure 6A). CID analyses with accurate mass were performed to identify the paired rule of pPtdEtn molecular species.


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 individual ethanolamine glycerophospholipid molecular species in mouse cerebellar lipid extracts.Mouse cerebellar lipid extracts were prepared by a modified Bligh and Dyer procedure [21]. 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 [16]. 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.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2147047&req=5

pone-0001368-g006: 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 [21]. 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 [16]. 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.
Mentions: Next, we examined whether this paired rule was applicable to the analysis of acid-labile, plasmalogen molecular species present in lipid extracts from multiple mammalian tissues or extracellular fluid samples including brain, heart, intestine, kidney, liver, and dorsal root ganglia of rat, rabbit, mouse, and/or human. A shotgun lipidomics approach was utilized to identify and quantify lipid molecular species following intrasource separation as described previously [18]–[20]. For example, PtdEtn molecular species from mouse cerebellum were analyzed by survey scanning in the negative-ion mode using an LTQ-Orbitrap mass spectrometer after direct infusion of diluted lipid extract solutions supplemented with a small amount LiOH (similar to Figure 6A). CID analyses with accurate mass were performed to identify the paired rule of pPtdEtn molecular species.

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