Analysis of diacylglycerols by ultra performance liquid chromatography-quadrupole time-of-flight mass spectrometry: Double bond location and isomers separation.

Diacylglycerols (DAGs) are important lipid intermediates and have been implicated in human diseases. Isomerism complicates their mass spectrometric analysis; in particular, it is difficult to identify fatty acid substituents and locate the double bond positions in unsaturated DAGs. We have developed an analytical strategy using ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC/Q-TOF MS) in conjunction with dimethyl disulfide (DMDS) derivatization and collision cross-section (CCS) measurement to characterize DAGs in biological samples. The method employs non-aqueous reversed-phase chromatographic separation and profile collision energy (CE) mode for MS(E) and MS/MS analyses. Three types of fragment ions were produced simultaneously. Hydrocarbon ions (m/z 50-200) obtained at high CE helped to distinguish unsaturated and saturated DAGs rapidly. Neutral loss ions and acylium ions (m/z 300-400) produced at low CE were used to identify fatty acid substituents. Informative methyl thioalkane fragment ions were used to locate the double bonds of unsaturated DAGs. Mono-methylthio derivatives were formed mainly by the reaction of DAGs with DMDS, where methyl thiol underwent addition to the first double bond farthest from the ester terminus of unsaturated fatty acid chains. The addition of CCS values maximized the separation of isomeric DAG species and improved the confidence of DAG identification. Fourteen DAGs were identified in mouse myotube cells based on accurate masses, characteristic fragment ions, DMDS derivatization, and CCS values.

[1]  P. Sjövall,et al.  TOF‐SIMS Analysis of Lipid Accumulation in the Skeletal Muscle of ob/ob Mice , 2008, Obesity.

[2]  H. Sovová,et al.  Comparison of various types of stationary phases in non-aqueous reversed-phase high-performance liquid chromatography-mass spectrometry of glycerolipids in blackcurrant oil and its enzymatic hydrolysis mixture. , 2009, Journal of chromatography. A.

[3]  Matej Oresic,et al.  Bioinformatics strategies for lipidomics analysis: characterization of obesity related hepatic steatosis , 2007, BMC Systems Biology.

[4]  A. Shibahara,et al.  cis-9,cis-15-octadecadienoic acid: a novel fatty acid found in higher plants. , 1993, Biochimica et biophysica acta.

[5]  E. Claude,et al.  Applications of ion-mobility mass spectrometry for lipid analysis , 2015, Analytical and Bioanalytical Chemistry.

[6]  Eoin Fahy,et al.  Lipidomics reveals a remarkable diversity of lipids in human plasma1[S] , 2010, Journal of Lipid Research.

[7]  R. Murphy,et al.  Analysis of Diacylglycerol Molecular Species in Cellular Lipid Extracts by Normal-Phase LC-Electrospray Mass Spectrometry. , 2011, International journal of mass spectrometry.

[8]  S. Carrasco,et al.  Diacylglycerol, when simplicity becomes complex. , 2007, Trends in biochemical sciences.

[9]  P. Zöllner.  Structure Determination of Diacylglycerols as Their Nicotinoyl Derivatives by Gas Chromatography-Mass Spectrometry , 1997 .

[10]  F. Hsu,et al.  Structural characterization of triacylglycerols as lithiated adducts by electrospray ionization mass spectrometry using low-energy collisionally activated dissociation on a triple stage quadrupole instrument , 1999, Journal of the American Society for Mass Spectrometry.

[11]  J. Lord,et al.  Diacylglycerol and Phosphatidate Generated by Phospholipases C and D, Respectively, Have Distinct Fatty Acid Compositions and Functions , 1997, The Journal of Biological Chemistry.

[12]  W. J. Dyer,et al.  A rapid method of total lipid extraction and purification. , 1959, Canadian journal of biochemistry and physiology.

[13]  J. Stone.  Regulation and Function of the RasGRP Family of Ras Activators in Blood Cells. , 2011, Genes & cancer.

[14]  T. Hankemeier,et al.  Localization of Fatty Acyl and Double Bond Positions in Phosphatidylcholines Using a Dual Stage CID Fragmentation Coupled with Ion Mobility Mass Spectrometry , 2011, Journal of the American Society for Mass Spectrometry.

[15]  F. Hsu,et al.  Multiple-stage linear ion-trap with high resolution mass spectrometry towards complete structural characterization of phosphatidylethanolamines containing cyclopropane fatty acyl chain in Leishmania infantum. , 2014, Journal of mass spectrometry : JMS.

[16]  Zhiqing Chen,et al.  Isolation and identification of C-19 fatty acids with anti-tumor activity from the spores of Ganoderma lucidum (reishi mushroom). , 2012, Fitoterapia.

[17]  John A. Bowden,et al.  Analysis of cholesteryl esters and diacylglycerols using lithiated adducts and electrospray ionization-tandem mass spectrometry. , 2011, Analytical biochemistry.

[18]  Bernhard O. Palsson,et al.  Ion Mobility-Derived Collision Cross Section As an Additional Measure for Lipid Fingerprinting and Identification , 2014, Analytical chemistry.

[19]  S. H. Tan,et al.  Double-bond location in monounsaturated fatty acids by dimethyl disulfide derivatization and mass spectrometry: Application to analysis of fatty acids in pheromone glands of four lepidoptera , 1985, Journal of Chemical Ecology.

[20]  A. Hichami,et al.  Diacylglycerols Containing Omega 3 and Omega 6 Fatty Acids Bind to RasGRP and Modulate MAP Kinase Activation* , 2004, Journal of Biological Chemistry.

[21]  Bernhard O. Palsson,et al.  Ion Mobility Derived Collision Cross Sections to Support Metabolomics Applications , 2014, Analytical chemistry.

[22]  J. Lord,et al.  Generation of diacylglycerol molecular species through the cell cycle: a role for 1-stearoyl, 2-arachidonyl glycerol in the activation of nuclear protein kinase C-betaII at G2/M. , 2002, Journal of cell science.

[23]  F. Hsu,et al.  Structural elucidation of diglycosyl diacylglycerol and monoglycosyl diacylglycerol from Streptococcus pneumoniae by multiple-stage linear ion-trap mass spectrometry with electrospray ionization. , 2012, Journal of mass spectrometry : JMS.

[24]  G. Francis.  Alkylthiolation for the determination of double-bond position in unsaturated fatty acid esters , 1981 .

[25]  R. Harris,et al.  Long-chain alkenes of the haptophytes Isochrysis galbana and Emiliania huxleyi , 1998, Lipids.

[26]  G. Shulman,et al.  Diacylglycerol activation of protein kinase Cε and hepatic insulin resistance. , 2012, Cell metabolism.

[27]  A. Shibahara,et al.  Determination of double-bond positions in methylene-interrupted dienoic fatty acids by GC-MS as their dimethyl disulfide adducts , 1991 .

[28]  A. Hichami,et al.  Implication of acyl chain of diacylglycerols in activation of different isoforms of protein kinase C , 2001, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[29]  A. Attygalle,et al.  Determination of double-bond position in some unsaturated terpenes and other branched compounds by alkylthiolation. , 1993, Analytical chemistry.

[30]  H. Callender,et al.  Quantification of diacylglycerol species from cellular extracts by electrospray ionization mass spectrometry using a linear regression algorithm. , 2007, Analytical chemistry.

[31]  S. Miyatani,et al.  Determining double-bond positions in monoenoic 2-hydroxy fatty acids of glucosylceramides by gas chromatography-mass spectrometry , 2000, Lipids.

[32]  R. Epand,et al.  Substrate specificity of diacylglycerol kinase‐epsilon and the phosphatidylinositol cycle , 2011, FEBS letters.

[33]  B. Perret,et al.  Identification and quantification of diacylglycerols in HDL and accessibility to lipase. , 1996, Journal of lipid research.

[34]  M. Wakelam,et al.  Diacylglycerols and phosphatidates: which molecular species are intracellular messengers? , 1998, Trends in biochemical sciences.

[35]  Su-yeon Lee,et al.  Measurements of Diacylglycerols in Skeletal Muscle by Atmospheric Pressure Chemical Ionization Mass Spectrometry , 2013, Lipids.

[36]  T. Croley,et al.  Mass Accuracy and Isotopic Abundance Measurements for HR-MS Instrumentation: Capabilities for Non-Targeted Analyses , 2014, Journal of The American Society for Mass Spectrometry.

[37]  G. Shulman,et al.  Role of diacylglycerol activation of PKCθ in lipid-induced muscle insulin resistance in humans , 2014, Proceedings of the National Academy of Sciences.

[38]  D. Oh,et al.  Simple determination of double-bond positions in long-chain olefins by cross-metathesis. , 2011, Angewandte Chemie.

[39]  F. Hsu,et al.  Electrospray ionization multiple-stage linear ion-trap mass spectrometry for structural elucidation of triacylglycerols: Assignment of fatty acyl groups on the glycerol backbone and location of double bonds , 2010, Journal of the American Society for Mass Spectrometry.

[40]  R. Murphy,et al.  Detection of the abundance of diacylglycerol and triacylglycerol molecular species in cells using neutral loss mass spectrometry. , 2007, Analytical biochemistry.

[41]  Juan Antonio Vizcaíno,et al.  Shorthand notation for lipid structures derived from mass spectrometry , 2013, Journal of Lipid Research.

[42]  André Nadler,et al.  The fatty acid composition of diacylglycerols determines local signaling patterns. , 2013, Angewandte Chemie.