Relative Quantification of Phospholipids Based on Isotope-Labeled Methylation by Nanoflow Ultrahigh Performance Liquid Chromatography-Tandem Mass Spectrometry: Enhancement in Cardiolipin Profiling

Jong Cheol Lee, Seul Kee Byeon, Myeong Hee Moon

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Abstract

In this study, lipid analysis based on isotope-labeled methlylation (ILM) was performed by nanoflow ultrahigh performance liquid chromatography-eletrospray ionization-tandem mass spectrometry (nUPLC-ESI-MS/MS) for enhanced detection and quantification of targeted phospholipids. ILM depends on methylation of phosphate groups by (trimethylsilyl)diazomethane, and the ILM based quantitation with reversed phase nUPLC-ESI-MS/MS provides advantages in PL profiling such as enhanced detectability of methylated PLs owing to increased hydrophobicity and substantial increase in resolution due to the increase of retention. Efficacy of ILM in nUPLC-ESI-MS/MS analysis was evaluated in the selected reaction monitoring (SRM) method by varying the mixing ratio of H-/D-methylated PL standards, which resulted in the successful quantification of 24 species, including phosphatidic acid (PA), phosphatidylserine (PS), phosphatidylglycerol (PG), ceramide-1-phosphate (Cer1P), phosphoinositides, and cardiolipin (CL), with ∼6.6% variation in the calculated ratio of H-/D-methylated PLs. The method was applied to the lipid extracts from a DU145 cell line after D-allose treatment, resulting in the quantification of 83 PLs of which results were not statistically different from those obtained by conventional quantification methods. Morever, detection and quantification of CLs and PAs were evidenced to be highly effective when used with the ILM method as 43 CLs and 20 PAs from cellular lipid extracts were analyzed while only 18 CLs and 12 PAs were identified when conventional methods were carried out. This proves the ILM combined with LC-MS to be a promising method for analysis of the aforementioned classes of lipids. Overall, the study highlighted the applicability of targeted quantification by the ILM method in lipidomic analysis and demonstrated an improvement in the detection of less abundant anionic PLs.

Original languageEnglish
Pages (from-to)4969-4977
Number of pages9
JournalAnalytical Chemistry
Volume89
Issue number9
DOIs
Publication statusPublished - 2017 May 2

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Cardiolipins
Methylation
Liquid chromatography
Isotopes
Mass spectrometry
Phospholipids
Lipids
Diazomethane
Phosphatidylglycerols
Phosphatidic Acids
Phosphatidylserines
Hydrophobicity
Phosphatidylinositols
Ionization
Phosphates
Cells
Monitoring

All Science Journal Classification (ASJC) codes

  • Analytical Chemistry

Cite this

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title = "Relative Quantification of Phospholipids Based on Isotope-Labeled Methylation by Nanoflow Ultrahigh Performance Liquid Chromatography-Tandem Mass Spectrometry: Enhancement in Cardiolipin Profiling",
abstract = "In this study, lipid analysis based on isotope-labeled methlylation (ILM) was performed by nanoflow ultrahigh performance liquid chromatography-eletrospray ionization-tandem mass spectrometry (nUPLC-ESI-MS/MS) for enhanced detection and quantification of targeted phospholipids. ILM depends on methylation of phosphate groups by (trimethylsilyl)diazomethane, and the ILM based quantitation with reversed phase nUPLC-ESI-MS/MS provides advantages in PL profiling such as enhanced detectability of methylated PLs owing to increased hydrophobicity and substantial increase in resolution due to the increase of retention. Efficacy of ILM in nUPLC-ESI-MS/MS analysis was evaluated in the selected reaction monitoring (SRM) method by varying the mixing ratio of H-/D-methylated PL standards, which resulted in the successful quantification of 24 species, including phosphatidic acid (PA), phosphatidylserine (PS), phosphatidylglycerol (PG), ceramide-1-phosphate (Cer1P), phosphoinositides, and cardiolipin (CL), with ∼6.6{\%} variation in the calculated ratio of H-/D-methylated PLs. The method was applied to the lipid extracts from a DU145 cell line after D-allose treatment, resulting in the quantification of 83 PLs of which results were not statistically different from those obtained by conventional quantification methods. Morever, detection and quantification of CLs and PAs were evidenced to be highly effective when used with the ILM method as 43 CLs and 20 PAs from cellular lipid extracts were analyzed while only 18 CLs and 12 PAs were identified when conventional methods were carried out. This proves the ILM combined with LC-MS to be a promising method for analysis of the aforementioned classes of lipids. Overall, the study highlighted the applicability of targeted quantification by the ILM method in lipidomic analysis and demonstrated an improvement in the detection of less abundant anionic PLs.",
author = "Lee, {Jong Cheol} and Byeon, {Seul Kee} and Moon, {Myeong Hee}",
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T1 - Relative Quantification of Phospholipids Based on Isotope-Labeled Methylation by Nanoflow Ultrahigh Performance Liquid Chromatography-Tandem Mass Spectrometry

T2 - Enhancement in Cardiolipin Profiling

AU - Lee, Jong Cheol

AU - Byeon, Seul Kee

AU - Moon, Myeong Hee

PY - 2017/5/2

Y1 - 2017/5/2

N2 - In this study, lipid analysis based on isotope-labeled methlylation (ILM) was performed by nanoflow ultrahigh performance liquid chromatography-eletrospray ionization-tandem mass spectrometry (nUPLC-ESI-MS/MS) for enhanced detection and quantification of targeted phospholipids. ILM depends on methylation of phosphate groups by (trimethylsilyl)diazomethane, and the ILM based quantitation with reversed phase nUPLC-ESI-MS/MS provides advantages in PL profiling such as enhanced detectability of methylated PLs owing to increased hydrophobicity and substantial increase in resolution due to the increase of retention. Efficacy of ILM in nUPLC-ESI-MS/MS analysis was evaluated in the selected reaction monitoring (SRM) method by varying the mixing ratio of H-/D-methylated PL standards, which resulted in the successful quantification of 24 species, including phosphatidic acid (PA), phosphatidylserine (PS), phosphatidylglycerol (PG), ceramide-1-phosphate (Cer1P), phosphoinositides, and cardiolipin (CL), with ∼6.6% variation in the calculated ratio of H-/D-methylated PLs. The method was applied to the lipid extracts from a DU145 cell line after D-allose treatment, resulting in the quantification of 83 PLs of which results were not statistically different from those obtained by conventional quantification methods. Morever, detection and quantification of CLs and PAs were evidenced to be highly effective when used with the ILM method as 43 CLs and 20 PAs from cellular lipid extracts were analyzed while only 18 CLs and 12 PAs were identified when conventional methods were carried out. This proves the ILM combined with LC-MS to be a promising method for analysis of the aforementioned classes of lipids. Overall, the study highlighted the applicability of targeted quantification by the ILM method in lipidomic analysis and demonstrated an improvement in the detection of less abundant anionic PLs.

AB - In this study, lipid analysis based on isotope-labeled methlylation (ILM) was performed by nanoflow ultrahigh performance liquid chromatography-eletrospray ionization-tandem mass spectrometry (nUPLC-ESI-MS/MS) for enhanced detection and quantification of targeted phospholipids. ILM depends on methylation of phosphate groups by (trimethylsilyl)diazomethane, and the ILM based quantitation with reversed phase nUPLC-ESI-MS/MS provides advantages in PL profiling such as enhanced detectability of methylated PLs owing to increased hydrophobicity and substantial increase in resolution due to the increase of retention. Efficacy of ILM in nUPLC-ESI-MS/MS analysis was evaluated in the selected reaction monitoring (SRM) method by varying the mixing ratio of H-/D-methylated PL standards, which resulted in the successful quantification of 24 species, including phosphatidic acid (PA), phosphatidylserine (PS), phosphatidylglycerol (PG), ceramide-1-phosphate (Cer1P), phosphoinositides, and cardiolipin (CL), with ∼6.6% variation in the calculated ratio of H-/D-methylated PLs. The method was applied to the lipid extracts from a DU145 cell line after D-allose treatment, resulting in the quantification of 83 PLs of which results were not statistically different from those obtained by conventional quantification methods. Morever, detection and quantification of CLs and PAs were evidenced to be highly effective when used with the ILM method as 43 CLs and 20 PAs from cellular lipid extracts were analyzed while only 18 CLs and 12 PAs were identified when conventional methods were carried out. This proves the ILM combined with LC-MS to be a promising method for analysis of the aforementioned classes of lipids. Overall, the study highlighted the applicability of targeted quantification by the ILM method in lipidomic analysis and demonstrated an improvement in the detection of less abundant anionic PLs.

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