Document: Mice. All studies were approved by the Animal Care and Use Committee of the University of California, Los Angeles. Mice were housed (1-3/cage) in a pathogen-free barrier facility (22°C) with a 12 h light/dark cycle with free access to food and water. All the strains were a fed low-fat diet (Wayne Rodent BLOX 8604; Harlan Teklad Laboratory). 60-80 day old mice fasted for 16 h at 7pm and sacrificed at 9am the following morning. Mice were bled from the retro-orbital sinus into tubes containing EDTA (final concentration 1 mM), after isofluorane inhalation. Plasma was collected and stored at −80 o C until analysis. Plasma HDL-C measurements. Plasma cholesterol levels (Invitrogen) were determined biochemically following the manufacturer's guidelines. (Shao et al., 2005) . Efflux of [ 3 H]cholesterol was measured after a 4 h incubation in medium with APOB depleted serum HDL (2.8% v/v). ABCA1-specific cholesterol efflux capacity was calculated as the percentage of total [ 3 H]cholesterol (medium plus cell) released into the medium of BHK cells stimulated with mifepristone after the value obtained with cells stimulated with medium alone was subtracted. HDL isolation. Serum HDL was prepared by adding calcium (2 mM final concentration) to plasma and using PEG (polyethylene glycol, 8kDa, Sigma) to precipitate lipoproteins containing APOB (VLDL, IDL, LDL). After centrifugation at 10,000 g for 30 min at 4 o C, serum HDL was harvested from the supernatant. HDL was isolated from serum or EDTA-anticoagulated plasma, using sequential ultracentrifugation (d=1.063-1.21 mg/mL) (Vaisar et al., 2007) . HDL was stored on ice in the dark and used within 1 week of preparation. For each isolation batch, control samples from the same pooled mouse plasma were included and further processed by tryptic digest and MS to control for experimental variability. Liquid chromatography-electrospray ionization tandem mass spectrometric (LC-ESI-MS/MS) analysis. HDL (10 μg protein) isolated by ultracentrifugation and 0.5 ug of yeast carboxypeptidase were solubilized with 0.1% RapiGest (Waters) in 200 mM ammonium bicarbonate, reduced with dithiothreitol, alkylated with iodoacetamide, and digested with trypsin (1:20, w/w HDL protein; Promega) for 3 h at 37°C. After a second aliquot of trypsin (1:20, w/w HDL protein) was added, samples were incubated overnight at 37°C. After RapiGest was removed by acid hydrolysis, samples were dried and stored at −20°C until analysis. Prior to analysis, samples were reconstituted in 5% acetonitrile, 0.1% formic acid (Pamir et al., 2016) . Tryptic digests of mouse HDL (1 μg protein) were injected onto a C18 trap column (Paradigm Platinum Peptide Nanotrap, 0.15 x 50 mm; Michrom Bioresources Inc.), desalted (50 μL/min) for 5 min with 1% acetonitrile/0.1% formic acid, eluted onto an analytical reverse-phase column (0.15 x 150 mm, Magic C18AQ, 5 μm, 200 A; Michrom Bioresources Inc.), and separated on a Paradigm M4B HPLC (Michrom Bioresources Inc.) at a flow rate of 1 μL/min over 180 min, using a linear gradient of 5% to 35% buffer B (90% acetonitrile, 0.1% formic acid) in buffer A (5% acetonitrile, 0.1% formic acid). Electrospray ionization (ESI) was performed using a Captive . CC-BY-NC-ND 4.0 International license is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It . https://doi.org/10.1101/405811 doi: bioRxiv preprint Spray source (Michrom BioResources, Inc, Auburn, CA) at 10 mL/min
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