In a case-control study (n=41 per group) nested in the Cooper Center Longitudinal Study we tested the ability of HDL to protect endothelium by stimulating endothelial nitric oxide synthase activity and suppressing NFκB-mediated inflammatory response in endothelial cells and analyzed HDL protein composition using targeted SRM-LCMS proteomics.
HDL was denatured, reduced/alkylated and digested with trypsin. Thirty six proteins were quantified by targeted proteomics with 15N-apoA-I spiked in as internal standard.
Tryptic digests of HDL were chromatographed using a nanoAquity UPLC (Waters, MA) with a C18 trapping column (XBridge BEH C18 100 Å, 5 µm, 0.1 x 40 mm, Waters, MA) (trapping flow rate 4 μL/min), a capillary XBridge BEH C18 analytical column (XBridge BEH C18 100Å, 3.5µm, 100x0.075 mm, Waters, MA), and a 30 min linear gradient of acetonitrile, 0.1% formic acid (7-35%) in 0.1% formic acid in water at a flow rate of 0.6 µL/min. The NanoAquity UPLC was connected to a Thermo TSQ Vantage triple-quadrupole mass spectrometer with electrospray ionization. The instrument was operated in SRM mode with 10 ms dwell time and the peptides were monitored collision energies optimized to maximize the signal.
HDL was isolated by ultracentrifugation from plasma of individual with T2 diabetes and healthy controls. Samples were randomized prior to the isolation by UC and split into two sets. Four samples failed the LCMS analysis and were excluded from the Skyline documents. Key to the samples is available upon request.