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

ABSTRACT: 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.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Mus Musculus (ncbitaxon:10090)

SUBMITTER: JiaHua Xie

PROVIDER: MSV000085952 | MassIVE | Sat Aug 15 06:00:00 BST 2020

REPOSITORIES: MassIVE

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Project 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.

Project description:Heart tissue was lysed by sonication in 5% SDS (w/v) in 50 mM TEAB (pH 8.5) 450 microg of each sample was reduced with 10 mM DTT at 80 degC for 10 min and alkylated with 25 mM iodoacetamide in the dark for 30 min at room temperature. SDS was removed, and samples were digested with 20 microg of Sequencing Grade Modified Trypsin (Promega) using an S-trap mini device (Protifi) at 47 degC for 2 h. Lyophilized peptides were reconstituted in 100 microl of 200 mM TEAB buffer and labeled with 41 TMT10 reagents (lot# UL292365). After 2 h, reactions were quenched with hydroxylamine and combined. TMT-labeled peptides were fractionated by high pH reversed-phase (HPRP) chromatography. Briefly, peptides were reconstituted in 400 microl of 20 mM ammonium formate, and 125 microl was fractionated using a 2.1 mm x 5 cm BEH C18 column (Waters) and Waters ACQUITY iClass UPLC. Separations utilized a flow rate of 0.4 ml/min and column temperature of 55 degC, and mobile phases consisted of 20 mM ammonium formate, pH 10 (MPA) and neat MeCN (MPB). Separations used a gradient as follows: 0 min, 3% B; 3 min, 7% B; 50 min, 50% B, 51 min, 90% B; 55 min, 90% B; 56 min, 3% B; 60 min, 3% B. Forty-eight equal fractions were collected over 52 minutes into a 96-well plate containing 10 microl of 20% TFA per well. This analysis was repeated 2 times total for fractionation of 2 mg peptides. The first 3 fractions of each injection were excluded, and the remaining samples were re-concatenated into 12 fractions. Approximately 5 microg of each fraction was separately aliquoted for analysis of the unenriched proteome, and samples were lyophilized. Phosphopeptide enrichment used 200 microg of each of the 12 HPRP fractions was reconstituted in 80% MeCN/1% TFA containing 1M glycolic acid (buffer A), phosphopeptides were enriched using GL Sciences p10 TiO tips. After loading, tips were washed twice with buffer A followed by 2 times with 80% MeCN/1% TFA before elution with 20% MeCN/5% aqueous ammonia. After addition of neat formic acid, samples were lyophilized. Finally, peptides were desalted using C18 Stage Tips, lyophilized and reconstituted in 12 microl of 10 mM citrate in 1% TFA/2% MeCN. The unenriched fractions were reconstituted in 10 microl of 1% TFA/2% MeCN. 1D-LC-MS/MS was performed on 4.5 microl of each of the phospho-enriched fractions or on 0.75 microg of unenriched fractions. Samples were analyzed using a nanoACQUITY UPLC system (Waters) coupled to a coupled to a Exploris 480 high resolution accurate mass tandem mass spectrometer (Thermo) via a nanoelectrospray ionization source and FAIMS Pro Interface. Samples were first trapped on a Symmetry C18 180 microm x 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 microm Acquity HSS T3 C18 75 microm 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 was performed in data-dependent acquisition (DDA) mode with 3 FAIMS compensation voltages (-40, -60 and -80). Each CV used a 60,000 resolution full MS scan from m/z 375 to 1600 with a normalized AGC target of 300%, peptide monoisotopic peak determination, an intensity threshold of 5E3 ions, precursor fit of 70% with 0.7 m/z fit window, charge state of 2-5 and 40 s dynamic exclusion. MS/MS used a top6 method with 30,000 resolution and TurboTMT enabled, an isolation width of 0.7 m/z, NCE of 36, AGC target of 300% and maximum IT of 120 ms. Advanced precursor determination was enabled. The analysis of unenriched samples used a similar method except that a 1 s total scan time was used for each CV and MS/MS used an auto IT.

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

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