Quantification of Diacylglycerol Species from Cellular Extracts by Electrospray Ionization Mass Spectrometry Using a Linear Regression Algorithm

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• 作者：Hannah L. Callender ; Jeffrey S. Forrester ; Pavlina Ivanova ; Anita Preininger ; Stephen Milne ; H. Alex Brown
• 刊名：Analytical Chemistry
• 出版年：2007
• 出版时间：January 1, 2007
• 年：2007
• 卷：79
• 期：1
• 页码：263 - 272
• 全文大小：421K
• 年卷期：v.79,no.1(January 1, 2007)
• ISSN：1520-6882

文摘

Diacylglycerols (DAGs) play significant roles in bothintermediate metabolism and signal transduction. Theselipid species are second messengers involved in modulating a plethora of cellular processes. Evaluation of DAGspecies concentrations has been hampered by the lack ofa reliable method for molecular species analysis within acomplex mixture of cellular lipids. We describe a newmethod for quantitative analysis of DAG species fromcomplex biological extracts based on positive mode electrospray ionization mass spectrometry without prior derivatization. Quantification is achieved using internalstandards and calibration curves constructed by spikingcell extracts with different concentrations of DAG speciescontaining various acyl chain lengths and degrees ofunsaturation. The new mass spectral data processingalgorithm incorporates a multiple linear regression modelincluding a factor accountable for possible interactionsbetween experimental preparations and the slope of thecurve for the standards, allowing the examinations of theeffects of sample origin conditions (such as cell types,phenotypes, etc.) and instrument variability on this slope.Internal standards provide a basis for quantification of 28DAG molecular species detected in RAW 264.7 cells afterstimulation of a G-protein coupled receptor with plateletactivating factor. This method displays excellent reproducibility over the established range of concentrationswith variations of ges/entities/le.gif">10% and is highly sensitive with adetection limit of 0.1-0.4 pmol/ges/entities/mgr.gif">L depending upon acylchain composition. We have shown differential effects onvarious DAGs in response to a ligand which illustratesthe importance of examining lipids at the molecularspecies level rather than as a single homogeneous entity.