MassIVE MSV000085952

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GNPS - Beneficial effects of low glucose on neuronal cell survival in an in vitro ischemic penumbral model

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Description

HT22 cells were cultured under hypoxia for 17.5 h with 0.69 mM low glucose (H+LG), hypoxia without glucose, or normoxia with normal 22 mM glucose (N+G). Proteomic analysis was performed by the Duke Proteomics and Metabolomics Shared Resource (address correspondence to mwfoster(at)duke.edu. Cells (n=3 replicates per condition) were lysed by sonication in 0.5% ALS-1, reduced with DTT and alkylated with iodoacetamide followed by digestion with trypsin digestion overnight. A QC pool was made by mixing equal amounts of all samples. Samples were analyzed using a nanoACQUITY UPLC system (Waters) coupled to a Fusion Lumos high resolution accurate mass tandem mass spectrometer (Thermo) via a nanoelectrospray ionization source. 750 ng of each sample was analyzed using block randomization with interspersed analyses of the QC pool. Briefly, the sample was first trapped on a Symmetry C18 180 micron by 20 mm trapping column (5 microl/min at 99.9/0.1 v/v H2O/MeCN) followed by an analytical separation using a 1.7 micron AQCUITY HSS T3 C18 75 micron x 250 mm column (Waters) with a 90 min gradient of 5 to 30% MeCN with 0.1% formic acid at a flow rate of 400 nl/min and column temperature of 55 degC. Data collection on the Fusion Lumos MS was performed in data-dependent acquisition (DDA) mode with a 240,000 resolution (at m/z 200) full MS scan from m/z 375 to 1600 with a target AGC value of 2e5 ions and 50 ms maximum injection time (IT) with internal calibration enabled. Peptides were selected for MS/MS using with advanced peak determination enabled, peptide monosotopic peak determination, and including charge states 2-5. MS/MS used HCD fragmentation and detection in the ion trap. Briefly, a 2 s method used an isolation width of 0.7 m/z, a normalized collision energy of 30 plus/minus 5%, a rapid ion trap scan rate, max AGC of 5e3 ions, max IT of 300 ms and use of all available parallelization time. A 20 s dynamic exclusion was enabled. Data was analyzed using Rosetta Elucidator v.4 (see Supplemental Data). Database searching with Mascot Server v 2.5 used a Swissprot Mus Musculus Database (downloaded 09/05/2017) appended with common contaminants and reverse decoys. Search parameters included precursor mass tolerance of 5 ppm, product ion mass tolerance of 0.6 Da, trypsin specificity with up to two missed cleavages, fixed modification on Cys (carbamidomethyl) and variable deamidation (N/Q)and acetylation (protein N-terminus). Data was annotated at a 1% peptide FDR using the PeptideTeller algorithm in Elucidator. Peptides were quantified by area under the curve and normalized to robust mean, and protein intensities were calculated as the sum of peptide intensities. [doi:10.25345/C5H175] [dataset license: CC0 1.0 Universal (CC0 1.0)]

Keywords: neuronal cells ; ischemic penumbra ; hypoxia ; low glucose

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Principal Investigators:
(in alphabetical order) 		JiaHua Xie, NCCU, USA
Submitting User: mwfoster
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