MassIVE MSV000084416

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Longitudinal Serum Proteomics In a Mouse Model of Lethal Endotoxemia

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Description

A total of thirty-two samples mouse serum samples were analyzed in duplicate (64 total injections) using an LC-MS system comprised of a nanoAcquity LC instrument (Waters nanoAcquity UPLC system, Milford, MA) connected to an Orbitrap Fusion Lumos tribrid mass spectrometer (Thermo Fisher Scientific, San Jose, CA). Peptides were trapped on a nanoAcquity trap column (180 um x 20 mm), washed with 99.5% mobile phase A (0.1% formic acid in water) and separated on a nanoAcquity C18 column (1.7 um BEH130Angstrom, 100 um x 100 mm) at 40 C. Peptides were eluted with a linear gradient of 3% to 40% mobile phase B (0.1 % FA in acetonitrile) over 90 min. Gradient changes were followed at 91min to 85% B and then increased to 95% B at 95 min. The gradient was changed back to 3% of B to equilibrate for 20 minutes prior to the next injection. Each injection consisted of 1.5 ug of purified peptides and injection order was randomized to minimize bias. Eluted peptides were ionized in positive ion polarity at a 2.1 kV of spraying voltage. MS1 full scans were recorded in the range of m/z 380 to 1580 with a resolution of 120,000 at 200 m/z using the Orbitrap mass analyzer. Automatic gain control was set at 8 x 105 with 50 ms of maximum injection time. Top speed (3sec) data dependent acquisition mode was used to maximize the number of MS2 spectra from each cycle. Higher-energy C-trap dissociation (HCD) was used to fragment selected precursor ions with a normalized collision energy of 30%. Tandem MS scans were performed in the ion trap mass analyzer with 35 ms maximum injection time, an ion target value set at 5 x 103, and dynamic exclusion set to 30s. [doi:10.25345/C5Z388] [dataset license: CC0 1.0 Universal (CC0 1.0)]

Keywords: TLR4, LPS, endotoxemia, mouse, serum, LC-MS

Contact

Principal Investigators:
(in alphabetical order) 		Robert K. Ernst, University of Maryland, Baltimore, United States of America
Submitting User: eharberts
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