Hydrophilic interaction liquid chromatography-electrospray ionization-tandem mass spectrometry of a complex mixture of native and oxidized phospholipids.
A mixture of native and oxidized phospholipids (PLs), generated by the soybean lipoxygenase type V-catalyzed partial oxidation of a lipid extract obtained from human platelets, was analyzed by Hydrophilic Interaction Liquid Chromatography-ElectroSpray Ionization-Tandem Mass Spectrometry (HILIC-ESI-MS/MS). The complexity of the resulting mixture was remarkable, considering that the starting lipid extract, containing (as demonstrated in a previous study) about 130 native PLs, was enriched with enzymatically generated hydroperoxylated derivatives and chemically generated hydroxylated forms of PLs bearing polyunsaturated side chains. Nonetheless, the described analytical approach proved to be very powerful; indeed, focusing on phosphatidylcolines (PCs), the most abundant PL class in human platelets, about fifty different native/oxidized species could be identified in a single HILIC-ESI-MS/MS run. Low-energy collision induced dissociation tandem MS (CID-MS/MS) experiments on chromatographically separated species showed single neutral losses of H2O2 and H2O to be typical fragmentation pathways of hydroperoxylated PCs, whereas a single H2O loss was observed for hydroxylated ones. Moreover, diagnostic losses of n-hexanal or n-pentanol were exploited to recognize PCs hydroperoxylated on the last but five carbon atom of a ɷ-6 polyunsaturated side chain. Despite the low resolution of the 3D ion trap mass analyzer used, the described HILIC-ESI-MS/MS approach appears very promising for the identification of oxidized lipids in oxidatively stressed complex biological systems.
Dipartimento di Chimica, Università degli Studi di Bari "Aldo Moro", Via E. Orabona 4, 70126 Bari, Italy; Centro Interdipartimentale SMART, Università degli Studi di Bari "Aldo Moro", Via E. Orabona 4, 70126 Bari, Italy. Electronic address: ilario.losito@uniba.it.
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Losito
Dipartimento di Chimica, Università degli Studi di Bari "Aldo Moro", Via E. Orabona 4, 70126 Bari, Italy.
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Facchini
Dipartimento di Chimica, Università degli Studi di Bari "Aldo Moro", Via E. Orabona 4, 70126 Bari, Italy.
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Diomede
Dipartimento di Bioscienze, Biotecnologie e Biofarmaceutica, Università degli Studi di Bari "Aldo Moro", Via E. Orabona 4, 70126 Bari, Italy.
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Conte
Dipartimento di Bioscienze, Biotecnologie e Biofarmaceutica, Università degli Studi di Bari "Aldo Moro", Via E. Orabona 4, 70126 Bari, Italy.
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Megli
Dipartimento di Chimica, Università degli Studi di Bari "Aldo Moro", Via E. Orabona 4, 70126 Bari, Italy; Centro Interdipartimentale SMART, Università degli Studi di Bari "Aldo Moro", Via E. Orabona 4, 70126 Bari, Italy.
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Cataldi
Dipartimento di Chimica, Università degli Studi di Bari "Aldo Moro", Via E. Orabona 4, 70126 Bari, Italy; Centro Interdipartimentale SMART, Università degli Studi di Bari "Aldo Moro", Via E. Orabona 4, 70126 Bari, Italy.
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Palmisano
D000447:Aldehydes / Q000737:chemistry; D001792:Blood Platelets / Q000737:chemistry; D002853:Chromatography, Liquid / Q000379:methods; D006801:Humans; D006861:Hydrogen Peroxide / Q000737:chemistry; D057927:Hydrophobic and Hydrophilic Interactions; D008084:Lipoxygenase / Q000737:chemistry; D010084:Oxidation-Reduction; D000439:Pentanols / Q000737:chemistry; D010713:Phosphatidylcholines / Q000097:blood / Q000737:chemistry; D010743:Phospholipids / Q000097:blood / Q000737:chemistry*; D013025:Glycine max / Q000737:chemistry; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods; D053719:Tandem Mass Spectrometry / Q000379:methods
D000447:Aldehydes; D000439:Pentanols; D010713:Phosphatidylcholines; D010743:Phospholipids; C010463:n-hexanal; D006861:Hydrogen Peroxide; D008084:Lipoxygenase; C024999:n-pentanol
Electrospray ionization tandem mass spectrometry; Human platelets; Hydrophilic interaction liquid chromatography; Oxidative stress; Oxidized phospholipids; Phosphatidylcholines