《a highly efficient, high-throughput lipidomics platform for the quantitative detection of eicosanoids in human whole blood：一个高效的高通量的脂质组学平台对全血中的类花生酸类物质的定量检测》由会员分享，可在线阅读，更多相关《a highly efficient, high-throughput lipidomics platform for the quantitative detection of eicosanoids in human whole blood：一个高效的高通量的脂质组学平台对全血中的类花生酸类物质的定量检测（8页珍藏版）》请在金锄头文库上搜索。
1、A highly efficient, high-throughput lipidomics platform for the quantitativedetection of eicosanoids in human whole bloodJiao Songa,1, Xuejun Liub,1, Jiejun Wuc, Michael J. Meehand, Jonathan M. Blevitta, Pieter C. Dorresteind,Marcos E. Millaa,aImmunology Discovery, Janssen Research and Development,
2、La Jolla, CA 92121, USAbInformatics, Janssen Research and Development, La Jolla, CA 92121, USAcChemistryWest Coast, Janssen Research and Development, La Jolla, CA 92121, USAdSchool of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, La Jolla, CA 92093, USAa r t i c l ei n f
3、 oArticle history:Received 23 August 2012Received in revised form 10 October 2012Accepted 15 October 2012Available online 26 October 2012Keywords:UPLCMRM/MSLipidomicsEicosanoidsHuman whole bloodHigh-throughput platformAutomated softwarea b s t r a c tWe have developed an ultra-performance liquid chr
4、omatographymultiple reaction monitoring/massspectrometry (UPLCMRM/MS)-based, high-content, high-throughput platform that enables simulta-neous profiling of multiple lipids produced ex vivo in human whole blood (HWB) on treatment withcalcium ionophore and its modulation with pharmacological agents. H
5、WB samples were processed in a96-well plate format compatible with high-throughput sample processing instrumentation. Weemployed a scheduled MRM (sMRM) method, with a triplequadrupole mass spectrometer coupled toa UPLC system, to measure absolute amounts of 122 distinct eicosanoids using deuterated
6、internalstandards. In a 6.5-min run, we resolved and detected with high sensitivity (lower limit of quantificationin the range of 0.4460 pg) all targeted analytes from a very small HWB sample (2.5ll). Approximately90% of the analytes exhibited a dynamic range exceeding 1000. We also developed a tail
7、ored softwarepackage that dramatically sped up the overall data quantification and analysis process with superiorconsistency and accuracy. Matrix effects from HWB and precision of the calibration curve were evaluatedusing this newly developed automation tool. This platform was successfully applied t
8、o the globalquantification of changes on all 122 eicosanoids in HWB samples from healthy donors in response tocalcium ionophore stimulation.? 2012 Elsevier Inc. All rights reserved.Arachidonic acid (AA)2produced from phospholipids by cytosolicphospholipase A2(cPLA2) is a gateway metabolic intermedia
9、te forthe production of multiple bioactive lipids 1. Those lipids, namedeicosanoids, have been the subject of intense research over recentyears due to their signaling functions in virtually every organ systemand physiological processes 2. In particular, leukotrienes and pros-taglandins, produced via
10、 the 5-lipoxygenase (5-LO) and cyclooxy-genase (COX) pathways, respectively, play major roles in theinitiation and maintenance of inflammatory responses leading tomultiple diseases 3, including asthma and atopic reactions as wellas cardiovascular disease and cancer 4,5. Multiple therapeuticagents ra
11、nging from preclinical research to clinical assets specificallytarget gene products required for leukotriene and prostaglandin bio-synthesis and signaling, demonstrating the critical importance of theeicosanome in the modulation of proinflammatory and autoimmuneresponses 6.The first committed step i
12、n leukotriene biosynthesis consists inthe sequential conversion of AA to 5-(S)-hydroperoxy-6,8,11,14-eicosatetraenoic acid (5-HpETE) and then leukotriene A4(LTA4)710 by the interaction of 5-LO with the 5-LO-activating protein(FLAP). LTA4is then hydrolyzed to LTB4by the LTA4hydrolase, orconjugated wi
13、th reduced glutathione via the concerted action ofLTC4synthase and FLAP, to generate the cysteinyl leukotriene (Cys-LT) LTC4, which is then exported from producing cells and con-verted by the action of extracellular peptidases to LTD4and theterminal product LTE411. Both LTB4and CysLTs act as potent
14、che-moattractants and leukocyte-activating mediators and are well-established biomarkers for the monitoring of the pharmacological0003-2697/$ - see front matter ? 2012 Elsevier Inc. All rights reserved.http:/dx.doi.org/10.1016/j.ab.2012.10.022Corresponding author. Fax: +1 858 450 2040.E-mail address
15、: (M.E. Milla).1These authors contributed equally.2Abbreviations used: AA, arachidonic acid; cPLA2, cytosolic phospholipase A2; 5-LO,5-lipoxygenase; COX, cyclooxygenase; 5-HpETE, 5-(S)-hydroperoxy-6,8,11,14-eicosa-tetraenoic acid; LTA4, leukotriene A4; FLAP, 5-LO-activating protein; CysLT, cysteinyl
16、leukotriene; LC, liquid chromatography; MS/MS, tandem mass spectrometry; MS,mass spectrometry; HWB, human whole blood; IS, internal standard; SPE, solid phaseextraction; UPLC, ultra-performance liquid chromatography; sMRM, scheduledmultiple reaction monitoring; DP, declustering potential; CE, collision energy; RE,relative error; LLOQ, lower limit of quantification; ULOQ, upper limit of quantifica-tion; HPLC, high-performance liquid chromatography; TIC, total ion chromatogram;XIC, extracted ion c
