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A novel informatics concept for high-throughput shotgun lipidomics based on the molecular fragmentation query language.

Herzog R, Schwudke D, Schuhmann K, Sampaio JL, Bornstein SR, Schroeder M, Shevchenko A - Genome Biol. (2011)

Bottom Line: Shotgun lipidome profiling relies on direct mass spectrometric analysis of total lipid extracts from cells, tissues or organisms and is a powerful tool to elucidate the molecular composition of lipidomes.We present a novel informatics concept of the molecular fragmentation query language implemented within the LipidXplorer open source software kit that supports accurate quantification of individual species of any ionizable lipid class in shotgun spectra acquired on any mass spectrometry platform.

View Article: PubMed Central - HTML - PubMed

Affiliation: Max Planck Institute of Molecular Cell Biology and Genetics, Pfotenhauerstrasse 108, 01307 Dresden, Germany.

ABSTRACT
Shotgun lipidome profiling relies on direct mass spectrometric analysis of total lipid extracts from cells, tissues or organisms and is a powerful tool to elucidate the molecular composition of lipidomes. We present a novel informatics concept of the molecular fragmentation query language implemented within the LipidXplorer open source software kit that supports accurate quantification of individual species of any ionizable lipid class in shotgun spectra acquired on any mass spectrometry platform.

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Related in: MedlinePlus

Identification of PC and PC-O species by MFQL queries relying on complementary signature ions. (a) MS/MS spectrum of the precursor ion of the acetate adduct of PC 36:1 (m/z 846.6224), in which four signature ions are recognized: molecular ion (MS); fragment of neutral loss of acetate and methyl group (Δm/z = 74.0 (NL)); acyl anions of the two fatty acid moieties (FA 281.3 and FA 283.2; both boxed in the chemical structure at the top). (b) MS/MS spectrum of the acetate adduct of PC-O 34:3 (m/z 800.5808). Signature ions are the same as in (a), except m/z 464.4 representing the fragment produced by neutral loss of the sn-2 fatty acid moiety. (c) Quantitative profiles of PC and PC-O species reported from abundances of different signature ions. MS, precursor ions in MS spectra; NL 74, neutral loss Δm/z 74 in MS/MS spectra; FA/FAO, acyl anions of fatty acid moieties and (for PC-O) neutral loss of sn-2 fatty acid moiety. The relative abundance of species was normalized to the total abundance of species within each (PC or PC-O) class.
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Figure 11: Identification of PC and PC-O species by MFQL queries relying on complementary signature ions. (a) MS/MS spectrum of the precursor ion of the acetate adduct of PC 36:1 (m/z 846.6224), in which four signature ions are recognized: molecular ion (MS); fragment of neutral loss of acetate and methyl group (Δm/z = 74.0 (NL)); acyl anions of the two fatty acid moieties (FA 281.3 and FA 283.2; both boxed in the chemical structure at the top). (b) MS/MS spectrum of the acetate adduct of PC-O 34:3 (m/z 800.5808). Signature ions are the same as in (a), except m/z 464.4 representing the fragment produced by neutral loss of the sn-2 fatty acid moiety. (c) Quantitative profiles of PC and PC-O species reported from abundances of different signature ions. MS, precursor ions in MS spectra; NL 74, neutral loss Δm/z 74 in MS/MS spectra; FA/FAO, acyl anions of fatty acid moieties and (for PC-O) neutral loss of sn-2 fatty acid moiety. The relative abundance of species was normalized to the total abundance of species within each (PC or PC-O) class.

Mentions: Otherwise, species identification should rely on signature ions in MS/MS spectra, such as acyl anions of fatty acid moieties, products of neutral losses of fatty acid moieties, head group fragments, and so on. As an example, we demonstrate here how using multiple signature ions helped in identifying molecular species of structurally related PC and PC-O lipids (Figure 5). The analysis was performed in negative ion mode in which both PC and PC-O were detected as molecular adducts with acetate anions (Figure 11). Species of both classes have the phosphorylcholine head group attached to the glycerol backbone at the sn-3 position and the fatty acid moiety at the sn-2 position (Figure 5). However, at the sn-1 position ester (PC) species have another fatty acid moiety, whereas ether (PC-O) species have a fatty alcohol moiety. To identify PC species, four signature ions could be considered (Table 6, Figure 11): intact precursor ion; fragment ion produced by neutral loss of 74 Da that is specific for the head group fragment; and two acyl anions of fatty acid moieties (Figure 11a). PC-O species can be identified (and distinguished from PC species) also by four signature ions (Table 6). Compared to PC, accurate masses of the intact precursor ion and of the fragment of 74 Da neutral loss should meet different sc-constraints. The third signature ion is the fragment of neutral loss of the fatty acid moiety and the fourth is the acyl anion of the fatty acid moiety itself (Figure 11b). At the same time, masses of the fatty acid and fatty alcohol moieties should complement the intact precursor mass. The high mass resolution of the Orbitrap mass analyzer allowed us to distinguish peaks of intact isobaric PC and PC-O, as well as of SM (also present in the total extract) and first isotopic peaks of PC. In MS/MS spectra we could clearly distinguish peaks of neutral loss products from co-selected PC, PC-O and SM precursors.


A novel informatics concept for high-throughput shotgun lipidomics based on the molecular fragmentation query language.

Herzog R, Schwudke D, Schuhmann K, Sampaio JL, Bornstein SR, Schroeder M, Shevchenko A - Genome Biol. (2011)

Identification of PC and PC-O species by MFQL queries relying on complementary signature ions. (a) MS/MS spectrum of the precursor ion of the acetate adduct of PC 36:1 (m/z 846.6224), in which four signature ions are recognized: molecular ion (MS); fragment of neutral loss of acetate and methyl group (Δm/z = 74.0 (NL)); acyl anions of the two fatty acid moieties (FA 281.3 and FA 283.2; both boxed in the chemical structure at the top). (b) MS/MS spectrum of the acetate adduct of PC-O 34:3 (m/z 800.5808). Signature ions are the same as in (a), except m/z 464.4 representing the fragment produced by neutral loss of the sn-2 fatty acid moiety. (c) Quantitative profiles of PC and PC-O species reported from abundances of different signature ions. MS, precursor ions in MS spectra; NL 74, neutral loss Δm/z 74 in MS/MS spectra; FA/FAO, acyl anions of fatty acid moieties and (for PC-O) neutral loss of sn-2 fatty acid moiety. The relative abundance of species was normalized to the total abundance of species within each (PC or PC-O) class.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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Figure 11: Identification of PC and PC-O species by MFQL queries relying on complementary signature ions. (a) MS/MS spectrum of the precursor ion of the acetate adduct of PC 36:1 (m/z 846.6224), in which four signature ions are recognized: molecular ion (MS); fragment of neutral loss of acetate and methyl group (Δm/z = 74.0 (NL)); acyl anions of the two fatty acid moieties (FA 281.3 and FA 283.2; both boxed in the chemical structure at the top). (b) MS/MS spectrum of the acetate adduct of PC-O 34:3 (m/z 800.5808). Signature ions are the same as in (a), except m/z 464.4 representing the fragment produced by neutral loss of the sn-2 fatty acid moiety. (c) Quantitative profiles of PC and PC-O species reported from abundances of different signature ions. MS, precursor ions in MS spectra; NL 74, neutral loss Δm/z 74 in MS/MS spectra; FA/FAO, acyl anions of fatty acid moieties and (for PC-O) neutral loss of sn-2 fatty acid moiety. The relative abundance of species was normalized to the total abundance of species within each (PC or PC-O) class.
Mentions: Otherwise, species identification should rely on signature ions in MS/MS spectra, such as acyl anions of fatty acid moieties, products of neutral losses of fatty acid moieties, head group fragments, and so on. As an example, we demonstrate here how using multiple signature ions helped in identifying molecular species of structurally related PC and PC-O lipids (Figure 5). The analysis was performed in negative ion mode in which both PC and PC-O were detected as molecular adducts with acetate anions (Figure 11). Species of both classes have the phosphorylcholine head group attached to the glycerol backbone at the sn-3 position and the fatty acid moiety at the sn-2 position (Figure 5). However, at the sn-1 position ester (PC) species have another fatty acid moiety, whereas ether (PC-O) species have a fatty alcohol moiety. To identify PC species, four signature ions could be considered (Table 6, Figure 11): intact precursor ion; fragment ion produced by neutral loss of 74 Da that is specific for the head group fragment; and two acyl anions of fatty acid moieties (Figure 11a). PC-O species can be identified (and distinguished from PC species) also by four signature ions (Table 6). Compared to PC, accurate masses of the intact precursor ion and of the fragment of 74 Da neutral loss should meet different sc-constraints. The third signature ion is the fragment of neutral loss of the fatty acid moiety and the fourth is the acyl anion of the fatty acid moiety itself (Figure 11b). At the same time, masses of the fatty acid and fatty alcohol moieties should complement the intact precursor mass. The high mass resolution of the Orbitrap mass analyzer allowed us to distinguish peaks of intact isobaric PC and PC-O, as well as of SM (also present in the total extract) and first isotopic peaks of PC. In MS/MS spectra we could clearly distinguish peaks of neutral loss products from co-selected PC, PC-O and SM precursors.

Bottom Line: Shotgun lipidome profiling relies on direct mass spectrometric analysis of total lipid extracts from cells, tissues or organisms and is a powerful tool to elucidate the molecular composition of lipidomes.We present a novel informatics concept of the molecular fragmentation query language implemented within the LipidXplorer open source software kit that supports accurate quantification of individual species of any ionizable lipid class in shotgun spectra acquired on any mass spectrometry platform.

View Article: PubMed Central - HTML - PubMed

Affiliation: Max Planck Institute of Molecular Cell Biology and Genetics, Pfotenhauerstrasse 108, 01307 Dresden, Germany.

ABSTRACT
Shotgun lipidome profiling relies on direct mass spectrometric analysis of total lipid extracts from cells, tissues or organisms and is a powerful tool to elucidate the molecular composition of lipidomes. We present a novel informatics concept of the molecular fragmentation query language implemented within the LipidXplorer open source software kit that supports accurate quantification of individual species of any ionizable lipid class in shotgun spectra acquired on any mass spectrometry platform.

Show MeSH
Related in: MedlinePlus