2018/01/01 by Hiroaki Takeda, Yoshihiro Izumi, Masatomo Takahashi +8 · 28 citations
Biochemistry, Genetics and Molecular Biology · Chemistry · #Metabolomics and Mass Spectrometry Studies #Analytical Chemistry and Chromatography #Mass Spectrometry Techniques and Applications
paper · doi:10.1194/jlr.d083014
Lipidomics, the mass spectrometry-based comprehensive analysis of lipids, has attracted attention as an analytical approach to provide novel insight into lipid metabolism and to search for biomarkers. However, an ideal method for both comprehensive and quantitative analysis of lipids has not been fully developed. Here, we have proposed a practical methodology for widely targeted quantitative lipidome analysis using supercritical fluid chromatography fast-scanning triple-quadrupole mass spectrometry (SFC/QqQMS) and theoretically calculated a comprehensive lipid multiple reaction monitoring (MRM) library. Lipid classes can be separated by SFC with a normal-phase diethylamine-bonded silica column with high resolution, high throughput, and good repeatability. Structural isomers of phospholipids can be monitored by mass spectrometric separation with fatty acyl-based MRM transitions. SFC/QqQMS analysis with an internal standard-dilution method offers quantitative information for both lipid class and individual lipid molecular species in the same lipid class. Additionally, data acquired using this method has advantages, including reduction of misidentification and acceleration of data analysis. Using the SFC/QqQMS system, alteration of plasma lipid levels in myocardial infarction-prone rabbits to the supplementation of EPA was first observed. Our developed SFC/QqQMS method represents a potentially useful tool for in-depth studies focused on complex lipid metabolism and biomarker discovery.—Takeda, H., Y. Izumi, M. Takahashi, T. Paxton, S. Tamura, T. Koike, Y. Yu, N. Kato, K. Nagase, M. Shiomi, and T. Bamba. Lipidomics, the mass spectrometry-based comprehensive analysis of lipids, has attracted attention as an analytical approach to provide novel insight into lipid metabolism and to search for biomarkers. However, an ideal method for both comprehensive and quantitative analysis of lipids has not been fully developed. Here, we have proposed a practical methodology for widely targeted quantitative lipidome analysis using supercritical fluid chromatography fast-scanning triple-quadrupole mass spectrometry (SFC/QqQMS) and theoretically calculated a comprehensive lipid multiple reaction monitoring (MRM) library. Lipid classes can be separated by SFC with a normal-phase diethylamine-bonded silica column with high resolution, high throughput, and good repeatability. Structural isomers of phospholipids can be monitored by mass spectrometric separation with fatty acyl-based MRM transitions. SFC/QqQMS analysis with an internal standard-dilution method offers quantitative information for both lipid class and individual lipid molecular species in the same lipid class. Additionally, data acquired using this method has advantages, including reduction of misidentification and acceleration of data analysis. Using the SFC/QqQMS system, alteration of plasma lipid levels in myocardial infarction-prone rabbits to the supplementation of EPA was first observed. Our developed SFC/QqQMS method represents a potentially useful tool for in-depth studies focused on complex lipid metabolism and biomarker discovery.—Takeda, H., Y. Izumi, M. Takahashi, T. Paxton, S. Tamura, T. Koike, Y. Yu, N. Kato, K. Nagase, M. Shiomi, and T. Bamba. Imbalance of lipid metabolites in vivo is believed to affect the onset of serious diseases such as dyslipidemia and resultant atherosclerosis (1.Ekroos K. Jänis M. Tarasov K. Hurme R. Laaksonen R. Lipidomics: a tool for studies of atherosclerosis.Curr. Atheroscler. Rep. 2010; 12: 273-281Crossref PubMed Scopus (82) Google Scholar, 2.Takeda H. Koike T. Izumi Y. Yamada T. Yoshida M. Shiomi M. Fukusaki E. Bamba T. Lipidomic analysis of plasma lipoprotein fractions in myocardial infarction-prone rabbits.J. Biosci. Bioeng. 2015; 120: 476-482Crossref PubMed Scopus (27) Google Scholar). In recent years, cohort studies using metabolomics have also helped discover reliable biomarkers (3.Kettunen J. Tukiainen T. Sarin A.P. Ortega-Alonso A. Tikkanen E. Lyytikäinen L.P. Kangas A.J. Soininen P. Würtz P. Silander K. et al.Genome-wide association study identifies multiple loci influencing human serum metabolite levels.Nat. Genet. 2012; 44: 269-276Crossref PubMed Scopus (431) Google Scholar, 4.Rhee E.P. Ho J.E. Chen M.H. Shen D. Cheng S. Larson M.G. Ghorbani A. Shi X. Helenius I.T. O'Donnell C.J. et al.A genome-wide association study of the human metabolism in a community-based cohort.Cell Metab. 2013; 18: 130-143Abstract Full Text Full Text PDF PubMed Scopus (208) Google Scholar). Accordingly, the development of a novel lipidomic analytical system that enables the acquisition of comprehensive and quantitative information of individual lipid molecular species is needed for identification and validation of potential biomarkers across multiple specimens and different equipment and/or facilities (5.Bowden J.A. Heckert A. Ulmer C.Z. Jones C.M. Koelmel J.P. Abdullah L. Ahonen L. Alnouti Y. Armando A. Asara J.M. et al.Harmonizing lipidomics: NIST interlaboratory comparison exercise for lipidomics using standard reference material 1950 metabolites in frozen human plasma.J. Lipid Res. 2017; 58: 2275-2288Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar). Recently, chromatographic and/or tandem mass spectrometric analytical approaches for lipidomics have been proposed (6.Cajka T. Fiehn O. Comprehensive analysis of lipids in biological systems by liquid chromatography-mass spectrometry.Trends Analyt. Chem. 2014; 61: 192-206Crossref PubMed Scopus (402) Google Scholar). Features of representative lipidomic methodologies are summarized in supplemental Table S1. The shotgun lipidomics approach, using direct infusion tandem mass spectrometry (DI/MS/MS), is often used to rapidly quantitate lipid molecular species in a short time (7.Han X. Gross R.W. Shotgun lipidomics: electrospray ionization mass spectrometric analysis and quantitation of cellular lipidomes directly from crude extracts of biological samples.Mass Spectrom. Rev. 2005; 24: 367-412Crossref PubMed Scopus (916) Google Scholar, 8.Ståhlman M. Ejsing C.S. Tarasov K. Perman J. Borén J. Ekroos K. High-throughput shotgun lipidomics by quadrupole time-of-flight mass spectrometry.J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 2009; 877: 2664-2672Crossref PubMed Scopus (173) Google Scholar, 9.Heiskanen L.A. Suoniemi M. Ta H.X. Tarasov K. Ekroos K. Long-term performance and stability of molecular shotgun lipidomic analysis of human plasma samples.Anal. Chem. 2013; 85: 8757-8763Crossref PubMed Scopus (61) Google Scholar). Each lipid level can be quantitated using mass spectrometry because the ion-suppression and/or ion-enhancement effects of the biological matrix can be normalized by adding the appropriate internal standards of each lipid class (e.g., chemically synthesized lipid standards not detected in vivo or stable isotope-labeled lipid standards). However, it is difficult to identify some isomeric compounds due to coelution, and low abundant lipid molecular species are poorly detected because of strong ionization suppression. In-source fragmentation caused by DI/MS/MS analysis can also confound lipid identification and quantification. LC/MS/MS techniques have been developed to identify and quantitate lipid molecular species. The advantages of LC/MS/MS methods over DI/MS/MS are as follows: i) improvement of detection sensitivity, since ESI mass spectrometry (ESI-MS) is a concentration-sensitive device (10.Hopfgartner G. Bean K. Henion J. Henry R. Ion spray mass spectrometric detection for liquid chromatography: a concentration- or a mass-flow-sensitive device?.J. Chromatogr. A. 1993; 647: 51-61Crossref Scopus (146) Google Scholar); ii) decrease in the effects of the biological matrix by liquid chromatography (LC) separation; and iii) consequently, an increase in the number of lipids detected. Reverse-phase LC (RPLC) is a widely used separation technique of lipid molecular species based on the hydrophobic interaction between nonpolar side chains of C18 particles and hydrophobic fatty acyl chains of lipids. RPLC can enhance the chromatographic resolution of a wide range of lipids including some isomers (11.Yamada T. Uchikata T. Sakamoto S. Yokoi Y. Fukusaki E. Bamba T. Development of a lipid profiling system using reverse-phase liquid chromatography coupled to high-resolution mass spectrometry with rapid polarity switching and an automated lipid identification software.J. Chromatogr. A. 2013; 1292: 211-218Crossref PubMed Scopus (93) Google Scholar, 12.Tsugawa H. Cajka T. Kind T. Ma Y. Higgins B. Ikeda K. Kanazawa M. VanderGheynst J. Fiehn O. Arita M. MS-DIAL: data-independent MS/MS deconvolution for comprehensive metabolome analysis.Nat. Methods. 2015; 12: 523-526Crossref PubMed Scopus (1283) Google Scholar). However, biological matrix effects cannot be normalized in all detected peaks, because it is not to appropriate internal standards to all the detected In normal-phase LC MS/MS can be used to each lipid class to the P. G. liquid chromatography class separation and species of phospholipids electrospray mass mass Spectrom. Scopus Google Scholar, S. T. of species in human using normal-phase liquid chromatography coupled with electrospray ionization tandem mass spectrometry.J. Chromatogr. B Biomed. Sci. PubMed Scopus Google Scholar, L. and of classes in human using normal-phase liquid chromatography coupled with electrospray mass spectrometry and the in Chromatogr. B Analyt. Technol. Biomed. Life Sci. PubMed Scopus Google Scholar). In the approach can to reliable identification and of each lipid class and some of individual lipid species by the of appropriate internal However, the of (e.g., and a reduction in ionization the of lipid molecular species is interaction chromatography MS/MS lipidome analysis has been to this M. J. D. of classes by interaction chromatography detected by electrospray ionization mass spectrometry.J. Chromatogr. A. 2010; PubMed Scopus Google Scholar, Y. J.M. of phospholipids by interaction liquid chromatography coupled to tandem mass the of compounds in Chromatogr. A. PubMed Scopus Google Scholar, E. M. M. M. A. P. quantitation of lipid classes using interaction liquid ionization mass spectrometry with internal standard and Chem. 2012; PubMed Scopus Google Scholar, A. G. M. J. G. T. interaction liquid chromatography separation of classes based on a Chromatogr. A. 2012; PubMed Scopus Google Scholar, Y. Y. K. lipidomics based on interaction chromatography coupled to time-of-flight mass 2013; PubMed Scopus Google Scholar, K. S. T. lipid analysis by interaction liquid chromatography coupled with electrospray ionization tandem mass spectrometry.J. Sci. 2015; PubMed Scopus Google Scholar). of for lipidomics is to that of The of is that of because (e.g., and as the However, since methods have and separation for nonpolar lipids and E. M. M. M. A. P. quantitation of lipid classes using interaction liquid ionization mass spectrometry with internal standard and Chem. 2012; PubMed Scopus Google methods are to lipids such as and M. J. D. of classes by interaction chromatography detected by electrospray ionization mass spectrometry.J. Chromatogr. A. 2010; PubMed Scopus Google Scholar, Y. J.M. of phospholipids by interaction liquid chromatography coupled to tandem mass the of compounds in Chromatogr. A. PubMed Scopus Google Scholar, A. G. M. J. G. T. interaction liquid chromatography separation of classes based on a Chromatogr. A. 2012; PubMed Scopus Google Scholar, Y. Y. K. lipidomics based on interaction chromatography coupled to time-of-flight mass 2013; PubMed Scopus Google Scholar, K. S. T. lipid analysis by interaction liquid chromatography coupled with electrospray ionization tandem mass spectrometry.J. Sci. 2015; PubMed Scopus Google Scholar). In to lipidome analysis with high of and are also to a on the of the Y. J.M. of phospholipids by interaction liquid chromatography coupled to tandem mass the of compounds in Chromatogr. A. PubMed Scopus Google Scholar, E. M. M. M. A. P. quantitation of lipid classes using interaction liquid ionization mass spectrometry with internal standard and Chem. 2012; PubMed Scopus Google Scholar, A. G. M. J. G. T. interaction liquid chromatography separation of classes based on a Chromatogr. A. 2012; PubMed Scopus Google Scholar, Y. Y. K. lipidomics based on interaction chromatography coupled to time-of-flight mass 2013; PubMed Scopus Google Scholar, K. S. T. lipid analysis by interaction liquid chromatography coupled with electrospray ionization tandem mass spectrometry.J. Sci. 2015; PubMed Scopus Google Scholar). supercritical fluid and is as a and chromatography is a chromatographic separation technique that an as a is the used for because can be to supercritical and such as chemically to and has low and the polarity of the in SFC can be by adding a such as as a In we have that with a reverse-phase column can be used for the analysis of lipids T. N. A. K. Y. A. Fukusaki E. and analysis of lipids by using supercritical fluid chromatography-mass spectrometry for Biosci. Bioeng. PubMed Scopus Google Scholar, T. A. S. Yoshida M. Fukusaki E. Bamba T. Development of profiling method using supercritical fluid spectrometry.J. Chromatogr. A. 2012; PubMed Scopus Google Scholar, T. Uchikata T. Sakamoto S. Yokoi Y. S. Yoshida M. Fukusaki E. Bamba T. fluid mass spectrometry based lipidomics coupled with automated lipid identification for lipid Chromatogr. A. 2013; PubMed Scopus Google Scholar). and that SFC coupled with a quadrupole mass system with a normal-phase column has been to lipidome analysis M. M. High-throughput and comprehensive lipidomic analysis using supercritical fluid chromatography-mass Chem. 2015; PubMed Scopus Google Scholar). and also that lipid class separation using SFC be to lipid class separation methods and in of short analysis time and chromatographic resolution for a wide of lipids. However, SFC separation of individual lipid chromatographic and mass spectrometric separation of individual lipid including or and quantitative have not been fully is to chromatographic and mass spectrometric separation for of a wide range of lipids. reaction monitoring (MRM) using triple-quadrupole mass spectrometry has and quantitative for reliable monitoring of lipids. mass spectrometric separation of isomers and can be using fatty acyl-based MRM transitions. In lipidome fragmentation was based on the lipid class and side rapid development of fast-scanning has also to MRM MRM for analysis of lipids widely targeted H. Arita M. Kanazawa M. A. Bamba T. Fukusaki E. a data and metabolite identification tool for multiple reaction monitoring based widely targeted Chem. 2013; 85: PubMed Scopus Google Scholar, J. Y. for targeted metabolomics by liquid chromatography-mass PubMed Google Scholar). In the we to an methodology for widely targeted quantitative analysis of individual lipid molecular species of lipid including and in complex biological using SFC coupled with fast-scanning (SFC/QqQMS) and theoretically calculated a comprehensive lipid MRM on this analytical we the of of EPA on the plasma lipid using myocardial infarction-prone rabbits M. T. The and of a to the 2009; Full Text Full Text PDF PubMed Scopus Google Scholar, M. T. Yamada S. S. J. Development of an for myocardial PubMed Scopus Google Scholar). was from and from was from lipid standards from for Yoshida was used as the SFC Table the used to an lipid MRM library. by the and and in with the for of on and of in to the and of and of and for the of and in the of the of and rabbits The rabbits in in a with and a EPA was using a and of EPA was to rabbits of rabbits was a the rabbits standard and was from the of the a of of Lipid from plasma was using and method with rapid method of lipid and J. PubMed Scopus Google Scholar). lipid a or internal standard was to for of the lipid molecular species T. Tarasov K. D. Ekroos K. quantitative molecular PubMed Scopus Google Scholar). The of internal standard into the plasma as follows: and and and and and and In of plasma was with of internal standard and of and the for The plasma was by for for The was to adding of and of separation of and was by for of the to The plasma lipid for was to a of with a of for SFC/QqQMS analysis. was by of plasma SFC/QqQMS system was of an system and a triple-quadrupole mass with an ESI In to enhance ionization was used as a The SFC and systems and data acquisition by of each lipid in MRM for each lipid class using The SFC as follows: and with of of B of column of analytical analytical as follows: MRM including the and of each with the of the using standard The SFC as follows: and with of of of column analytical and The analysis as follows: and The MRM time as follows: on number of MRM and and polarity the of of each standard the and each with the internal standard of and and and and and and standard was in analytical by adding the same of lipid standard as the in the representative plasma lipid for the and of lipid molecular species using The quantitative calculated using the of the chromatographic of each to that of the internal standard of representative lipid class. level was using a the between and of EPA was using and The of lipid was by the between and of the SFC/QqQMS system used in this study is in supplemental S1. Recently, a analytical SFC system, the has been developed. The SFC system from a novel and is based on LC including and quantitative performance using with particles have the L. E. D. analytical supercritical chromatography using with 2014; PubMed Scopus Google Scholar). fast-scanning the has also been for and and MRM and it is to MRM for analysis of In the of the and a of used as the and is into the a of in be that in an (e.g., In we that SFC/QqQMS the analytical fully for SFC/QqQMS Y. Y. Izumi Y. Bamba T. of chromatographic and mass spectrometric for supercritical fluid spectrometry.J. Chromatogr. A. 2017; PubMed Scopus Google Scholar). of the and of the fragmentation of each of the lipid classes and by SFC analysis in both and of each lipid in the or and/or in the or by the of to the and In this the MS/MS of the individual lipid for each molecular acquired by with the lipid was difficult to in using on the abundant and the for each lipid we the and Table the of the and MRM for each lipid class. The of in SFC on of targeted lipids was first by SFC analysis with a column as the reference column because of the of In the of using with of the lipids such as and the by the of to the is that of a of in as the to an improvement in for compounds (e.g., because of and of the J. C.J. the range of supercritical fluid chromatography by of Chem. 2013; PubMed Scopus Google Scholar). it is that improvement in was in the lipid species. Using this we the of the The with of a with with The first enables the of to the column to enhance In the the is between the and in a of In the in SFC with the the of and lipids such as are this In to enhance the ionization for we the of the a the of the was to study that system using a normal-phase column a of molecular species in M. M. High-throughput and comprehensive lipidomic analysis using supercritical fluid chromatography-mass Chem. 2015; PubMed Scopus Google Scholar). However, isomers and and isomers (e.g., and not in the the separation of each lipid class and some lipid including isomers and we used different normal-phase including and developed based on we the SFC the system in this SFC The was to in the first this the of lipids such as was in all SFC using each of the different lipid classes with In SFC using the the the was from to we the of column on lipid class separation and system the column the of each lipid class and system in all SFC using each of the different lipid class separation not In this the column was to we for the of each lipid class and the same each and we to it to the of we the In the of in the SFC using the was and the system was the of the the separation of lipid classes using different SFC the same SFC Table the of of each lipid class. In the of using the of and with a as a hydrophobic lipids, such as and a the Table In SFC separation with the column the interaction with lipids such as and for lipids was to a in SFC separation using the column using the column of the for this is that the has to the and separation of lipids. using the separation of isomers such as (e.g., and and lipids (e.g., and also are as and in the metabolism of and However, the in between and in vivo is not the Suoniemi M. H. Ekroos K. in human plasma and by Chem. 2015; PubMed Scopus Google Scholar). the in the of and or and have not been analytical method be useful in for of the and of on we to the column for widely targeted lipidome analysis. SFC with the column good separation of internal standard of lipid classes In SFC separation using the the for was In to data across a MRM including and and polarity and chromatographic separation of lipid classes not ion-enhancement and/or ion-suppression effects lipid also to identification and quantitative of isomers of using a and lipids and SFC separation of each lipid class using a SFC/QqQMS (MRM) of internal standard of lipid of lipids in the of isomers with different side chains (e.g., and was based on MS/MS the fragmentation of each of the lipid classes and the MRM for identification and of each lipid Table The MRM for phospholipids including and to the MS/MS from side chains in the on the MRM from side and and be monitored of individual molecular species not be by MRM from of side fatty acyl the and of the the isomers cannot be monitored and and and have the same MRM as individual levels cannot be by the lipid separation method of SFC with the In the we molecular species by lipid levels of isomers using the same Table In to molecular information of with side separation in LC/MS/MS or is T. N. Fukusaki E. Bamba T. of in by supercritical fluid mass spectrometry.J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 2014; PubMed Scopus Google Scholar). The SFC/QqQMS analytical system was by the of SFC column SFC separation and MRM In to the MS/MS of each lipid class and Table we widely targeted MRM Table In MRM using are for quantitative analysis of targeted including However, this approach has are to the targeted In methods are a in of comprehensive and lipid In the an lipid MRM is to comprehensive lipids using fast-scanning targeted lipid classes with a of side targeted lipid molecular species compounds Table on the representative MS/MS from each we MRM for the of lipids in biological used for Table Our of a widely targeted quantitative lipidomics methodology was as follows: i) of a of from each the lipid internal standards ii) of lipids by of the using different MRM in the is to MRM the and iii) targeted quantitative analysis of lipids in all by a MRM method and supplemental the method using lipids standard The using the of the chromatographic of each lipid class to that of the internal The of the analytical validation is summarized in Table each lipid class good and in supplemental Table SFC/QqQMS method using the column also for on different The SFC/QqQMS method with over The of for the lipid classes in the range of SFC/QqQMS the of and standards to be and and and in the method Table lipidome the of each lipid class was that in the MRM The of and lipid MRM also for of lipid approach also comprehensive and analysis of validation of SFC/QqQMS for of for each used for validation of quantitative of of by the analysis of individual lipid molecular species in the all plasma in this plasma used to used for validation of quantitative of by the analysis of individual lipid molecular species in the all plasma in this plasma used to in a studies that the ionization of lipid molecular species was on and on side chains (7.Han X. Gross R.W. Shotgun lipidomics: electrospray ionization mass spectrometric analysis and quantitation of cellular lipidomes directly from crude extracts of biological samples.Mass Spectrom. Rev. 2005; 24: 367-412Crossref PubMed Scopus (916) Google Scholar, X. Gross R.W. ionization mass spectrometric analysis of human plasma Sci. PubMed Scopus Google Scholar). in the ionization of lipid molecular species in and number of T. Tarasov K. D. Ekroos K. quantitative molecular PubMed Scopus Google Scholar). the lipid separation the of individual lipid species as the internal standard with the species. In to the quantitative a standard was each lipid molecular species in plasma using the SFC/QqQMS chemically synthesized lipid standards of lipid molecular species in plasma for of the standard by adding the same of lipid standard as calculated for lipid levels in the plasma lipid The calculated from to the range for that this lipidomics method using SFC/QqQMS quantitative analysis with good and are to serious such as have the association between and of atherosclerosis from the of supplementation of fatty in and J. PubMed Google Scholar, A. D. K. Y. Y. S. S. Yamada H. T. T. et of fatty in by Full Text Full Text PDF PubMed Scopus Google Scholar). of such as EPA and and of in was that effects of A. D. K. Y. Y. S. S. Yamada H. T. T. et of fatty in by Full Text Full Text PDF PubMed Scopus Google Scholar). In the we the alteration of plasma lipid by of EPA using the analytical used rabbits and EPA Table the and plasma levels in both and EPA in was levels by of we lipid with the was from a of of the plasma of lipid molecular species in plasma extracts of rabbits Table on the of lipid we MRM and quantitative analysis in the plasma extracts by the with the MRM of the lipid molecular species in in SFC separation using the lipid species with the same different fully classes some using novel analytical system, the alteration of each lipid class as as individual lipid molecular including isomers of phospholipids by of be the plasma of each lipid class in both the and the EPA The quantitative levels of plasma and by of the individual lipid the quantitative lipid data of the EPA between and using a Lipid molecular species with an EPA side lipid molecular species with a side also by of EPA and of the acyl by as in fatty PubMed Scopus Google Scholar). the high levels of lipid molecular species with a side in plasma based on the supplementation of a decrease in some lipid classes and was due to a decrease in lipids with a side that of the and with EPA In the we have developed a practical methodology for widely targeted quantitative lipidome analysis using SFC/QqQMS and theoretically calculated a comprehensive lipid MRM library. Lipid classes can be separated by SFC using the column with high resolution, high throughput, and good repeatability. The lipid separation and MRM also the of individual lipid species as the internal standard with the species. Our analytical to the of individual lipid classes as as individual lipid molecular including and isomers in analysis of comprehensive lipids in biological (e.g., and comparison of on different using different and/or of of lipids also the biological of lipid molecular species. all of the into developed analytical system is a tool for in-depth studies on complex lipid and biomarker