Diacyl
glycerols (DAGs) play si
gnificant roles in bothintermediate metabolism and si
gnal transduction. Theselipid species are second messen
gers involved in modulatin
g 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 biolo
gical extracts based on positive mode electrospray ionization mass spectrometry without prior derivatization. Quantification is achieved usin
g internalstandards and calibration curves constructed by spikin
gcell extracts with different concentrations of DAG speciescontainin
g various acyl chain len
gths and de
grees ofunsaturation. The new mass spectral data processin
gal
gorithm incorporates a multiple linear re
gression modelincludin
g a factor accountable for possible interactionsbetween experimental preparations and the slope of thecurve for the standards, allowin
g the examinations of theeffects of sample ori
gin 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 plateletactivatin
g factor. This method displays excellent reproducibility over the established ran
ge of concentrationswith variations of
![](/ima<font color=)
ges/entities/le.
gif">10% and is hi
ghly sensitive with adetection limit of 0.1-0.4 pmol/
![](/ima<font color=)
ges/entities/m
gr.
gif">L dependin
g upon acylchain composition. We have shown differential effects onvarious DAGs in response to a li
gand which illustratesthe importance of examinin
g lipids at the molecularspecies level rather than as a sin
gle homo
geneous entity.