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Identification of carbonylated lipids from different phospholipid classes by shotgun and LC-MS lipidomics

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Abstract

Oxidized lipids play a significant role in the pathogenesis of numerous oxidative stress-related human disorders, such as atherosclerosis, obesity, inflammation, and autoimmune diseases. Lipid peroxidation, induced by reactive oxygen and nitrogen species, yields a high variety of modified lipids. Among them, carbonylated lipid peroxidation products (oxoLPP), formed by oxidation of the fatty acid moiety yielding aldehydes or ketones (carbonyl groups), are electrophilic compounds that are able to modify nucleophilic substrates like proteins, nucleic acid, and aminophospholipids. Some carbonylated phosphatidylcholines possess even pro-inflammatory activities. However, little is known about oxoLPP derived from other phospholipid (PL) classes. Here, we present a new analytical strategy based on the mass spectrometry (MS) of PL-oxoLPP derivatized with 7-(diethylamino)coumarin-3-carbohydrazide (CHH). Shotgun MS revealed many oxoLPP derived from in vitro oxidized glycerophosphatidylglycerols (PG, 31), glycerophosphatidylcholine (PC, 23), glycerophosphatidylethanolamine (PE, 34), glycerophosphatidylserines (PS, 7), glycerophosphatidic acids (PA, 17), and phosphatidylinositiolphosphates (PIP, 6) vesicles. This data were used to optimize LipidXplorer-assisted identification, and a python-based post-processing script was developed to increase both throughput and accuracy. When applied to full lipid extracts from rat primary cardiomyocytes treated with peroxynitrite donor SIN-1, ten PL-bound oxoLPP were unambiguously identified by LC-MS, including two PC-, two PE-, one PG-, two PS-, and three PA-derived species. Some of the well-known carbonylated PC were detected, while most PL-oxoLPP were shown for the first time.

Overview of analytical and bioinformatics approach for detection and identification of carbonylated phospholipids.

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Abbreviations

AA:

Arachidonic acid

CHH:

7-(Diethylamino)coumarin-3-carbohydrazide

CID:

Collision-induced dissociation

DAG:

Diacylglycerols

DBE:

Double bond equivalents

DDA:

Data-dependent acquisition

DHA:

Docosahexaenoic acid

ESI:

Electrospray

FA:

Fatty acid

GPF:

Gas phase fractionation

HG:

Head group

HOOA-PC:

1-Palmitoyl-2-(5-hydroxy-8-oxo-oct-6-enoyl)-glycerophosphatidylcholine

LA:

Linoleic acid

LC-MS:

Liquid chromatography coupled to mass spectrometry

LPP:

Lipid peroxidation products

MFQL:

Molecular fragmentation query language

MS:

Mass spectrometry

MS/MS:

Tandem mass spectrometry

MTBE:

Methyl-tert-butyl ether

nCE:

Normalized collision energy

OA:

Oleic acid

OAP:

Oxygen addition products

OCP:

Oxygen cleavage products

oxoLPP:

Carbonylated lipid peroxidation products

oxPC:

Oxidized phosphatidylcholine

PA:

Glycerophosphatidic acids

PAPC:

1-Palmitoyl-2-arachidonyl-glycerophosphatidylcholine

PAPE:

1-Palmitoyl-2-arachidonoyl-glycerophosphatidylethanolamine

PAPG:

1-Palmitoyl-2-arachidonoyl-glycerophosphatidylglycerol

PC:

Glycerophosphatidylcholine

PDPC:

1-Palmitoyl-2-docosahexaenoyl-glycerophosphatidylcholine

PDPE:

1-Palmitoyl-2-docosahexaenoyl-glycerophosphatidylethanolamine

PE:

Glycerophosphatidylethanolamine

PI:

Glycerophosphatidylinositols

PIP:

Phosphatidylinositiolphosphates

PL:

Glycerophospholipids

PLPA:

1-Palmitoyl-2-linoleoyl-glycerophosphatidic acid

PLPC:

1-Palmitoyl-2-linoleoyl-glycerophosphatidylcholine

PLPE:

1-Palmitoyl-2-linoleoyl-glycerophosphatidylethanolamine

PLPG:

1-Palmitoyl-2-linoleoyl-glycerophosphatidylglycerol

PLPS:

1-Palmitoyl-2-linoleoyl-glycerophosphatidylserine

POPA:

1-Palmitoyl-2-oleoyl-glycerophosphatidic acid

POPC:

1-Palmitoyl-2-oleoyl-glycerophosphatidylcholine

POPE:

1-Palmitoyl-2-oleoyl-glycerophosphatidylethanolamine

POPG:

1-Palmitoyl-2-oleoyl-glycerophosphatidylglycerol

POPS:

1-Palmitoyl-2-oleoyl-glycerophosphatidylserine

POVPC:

1-Palmitoyl-2-(5-oxo-valeryl)-glycerophosphatidylcholine

PS:

Glycerophosphatidylserines

PUFA:

Polyunsaturated fatty acid

RNS:

Reactive nitrogen species

ROS:

Reactive oxygen species

RPC:

Reversed phase chromatography

SAPC:

1-Stearoyl-2-arachidonoylglycerophosphatidylcholine

SAPIP:

1-Stearoyl-2-arachidonoyl-gylcerophosphatidylinositolphosphate

SIN-1:

3-Morpholinosydnonimine

SLPIP:

1-Stearoyl-2-linoleoyl-glycerophosphatidylinositolphosphate

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Acknowledgments

The authors are thankful to Prof. Ralf Hoffmann (Institute of Bioanalytical Chemistry, University of Leipzig) for providing access to his laboratory and mass spectrometers. Financial support from European Regional Development Fund (ERDF, European Union and Free State Saxony; 100146238 and 100121468 to M.F) and a stipend to I.M. provided by Universität Leipzig are gratefully acknowledged.

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Authors and Affiliations

  1. Institute of Bioanalytical Chemistry, Faculty of Chemistry and Mineralogy, Universität Leipzig, Johannisallee 29, 04103, Leipzig, Germany

    Zhixu Ni, Ivana Milic & Maria Fedorova

  2. Center for Biotechnology and Biomedicine, Universität Leipzig, Deutscher Platz 5, 04103, Leipzig, Germany

    Zhixu Ni, Ivana Milic & Maria Fedorova

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  1. Zhixu Ni
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  2. Ivana Milic
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Correspondence to Maria Fedorova.

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Published in the topical collection Lipidomics with guest editor Michal Holčapek.

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Ni, Z., Milic, I. & Fedorova, M. Identification of carbonylated lipids from different phospholipid classes by shotgun and LC-MS lipidomics. Anal Bioanal Chem 407, 5161–5173 (2015). https://doi.org/10.1007/s00216-015-8536-2

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