Api4000 q trap hybrid triple quadrupole linear ion trap mass spectrometer

Cellular lipids were extracted by a modified Bligh and Dyer procedure (29 (link)) with the use of 0.1N HCl for phase separation as described in (30 (link)). C17-S1P (40 pmol) was employed as internal standard, and was added during the initial step of lipid extraction. The extracted lipids were dissolved in methanol/chloroform (4∶1, v/v), and aliquots were taken to determine the total phospholipid content (31 (link)). Analyses of sphingoid base-1-phosphates were performed by electrospray ionization tandem mass spectrometry (ESI-LC/MS/MS). Briefly, API4000 Q-trap hybrid triple quadrupole linear ion-trap mass spectrometer (Applied Biosystems, Foster City, CA) equipped with a turboionspray ionization source interfaced with an automated Agilent 1100 series liquid chromatograph and autosampler (Agilent Technologies, Wilmington, DE), as performed in (31 (link)).

All experiments were IRB approved at the University of Illinois Chicago and Indiana University. Lung Tissue Research Consortium provided deidentified clinical data linked to lung specimens collected by their Core Laboratory from lung biopsies, lobectomies, transplant and lung volume reduction surgeries. Lung specimens were >5 cm away from any excised tumours.
Following extraction and lipid phosphorus (Pi) measurements,^{1 (link)} sphingolipid analyses were performed via combined liquid chromatography–tandem mass spectrometry, using API4000 Q-trap hybrid triple quadrupole linear ion-trap mass spectrometer (Applied Biosystems-Sciex) with turbo ion spray ionisation source and Agilent 1100 series liquid chromatography (Agilent Technologies).^{6 (link)}Paraffin-embedded lung tissue sections were immunostained with active caspase-3 (Cell Signaling) or ceramide (Alexis) biotinylated antibodies (Animal Research Kit, Dako). Randomised deidentified images of distal lung parenchyma were acquired and quantified (Metamorph) with a macro from Dr Rubin Tuder (University of Colorado).
Statistical analysis included ANOVA (normality tested with Bartlett’s Test) and Pearson linear regression (Prism 6, GraphPad). Multiple regression models were run in R.