University of Lleida Biochemistry of Oxidative Stress - I151F characterization

Quantitation of frataxin-related proteins in I151F mice
Data License: CC BY 4.0
  • Organism: Mus musculus
  • Instrument: 6420 Triple Quadrupole LC/MS
  • SpikeIn: Yes
  • Keywords: Friedreich ataxia, frataxin, iron-sulfur
  • Lab head: Jordi Tamarit Submitter: Jordi Tamarit
Abstract
By targeted proteomics we analyzed the content of several frataxin-related proteins in tissues collected from WT and FXNI151F mice. The proteins analyzed were two components of the tricarboxylic acids cycle (CS and Aco2), several components of the OXPHOS system (SDHA, SDHB, CY1, QCR2, COX2, ATPA, and ATPB), two mitochondrial chaperones (HSP60 and GRP75), three components of the Pyruvate dehydrogenase complex (PDHA1, DLAT and DLDH), and two superoxide dismutases (SOD1 and SOD2). Three glycolytic enzymes where also included in the analysis (GAPDH, PKM, and Enolases A, B, G). Two of the proteins analyzed contain iron-sulfur clusters (ACO2 and SDHB), while two of them (CYC and CY1) contain heme groups. The relative content of these proteins was analyzed in Brain, Cerebellum and Heart from WT and MUT mice, sacrificed at 21 or 39 weeks of age. Significant differences in the content of several proteins were observed between WT and MUT mice. The proteins more affected were ACO2 and the two components of OXPHOS complex II (SDHA and SDHB). These proteins showed a marked decrease in cerebellum and brain from MUT mice, both at 21 and 39 weeks. In heart, we could only observe loss of complex II in 21 weeks-old mice, but not in 39-week old mice, while ACO2 content was not altered in heart at any age.
Experiment Description
Tissue homogenates (50 ug of protein) were precipitated with cold acetone (9 volumes) and resuspended in 1% sodium deoxycholate, 50 mM ammonium bicarbonate. Then, proteins were subjected to reduction by 12 mM DTT and alquilation by 40 mM IAM. Mass spectrometry grade trypsin (SOLu-Trypsin, Sigma) was added to a final enzyme:substrate ratio of 1:50. After overnight digestion at 37ºC, formic acid was added to precipitate sodium deoxycholate. The resulting peptide mix was purified and enriched using 100 ul Pierce C18 ZipTips. Eluted fraction from the C18 ZipTip was evaporated using a Concentrator Plus (Eppendorf) and peptides were resuspended in 3% acetonitrile plus 0,1% formic acid containing a heavy peptide standards mixture. Heavy peptides were obtained from JPT (SpikeTidesTM_L). All peptide samples were analyzed on a triple quadrupole spectrometer (Agilent 6420) equipped with an electrospray ion source. Chromatographic separations of peptides were performed on an Agilent 1200 LC system using a Supelco Bioshell A160 Peptide C18 column (1 mm x 15 cm). Peptides (up to 15 micrograms of protein digest) were separated with a linear gradient of acetonitrile/water, containing 0.1% formic acid, at a flow rate of 75 ul/min. A gradient from 3 to 60% acetonitrile in 45 minutes was used. The mass spectrometer was operated in multiple reaction monitoring mode. Transitions were obtained from SRM atlas or Prosit and imported into Skyline software , which was also used to analyze results. Once validated and optimized, the SRM assays were used to quantify all the analyzed peptides using scheduled SRM mode in a single run (retention time window, 120 s; cycle time, 1 sec)
Sample Description
WT and FXNI151F mice were sacrified by cervical dislocation at 21 or 39 weeks of age. Tissues (brain, cerebellum and heart) were dissected immediately and snap‐frozen in liquid nitrogen and stored at −80°C untill used. A piece of tissue sample (20–100 mg) was cut into 2–3 mm2 pieces. All the pieces were placed in 1.5ml screw cap polypropylene tubes in the presence of lysis buffer consisting in 50 mM tris(hydroxymethyl)aminomethane (Tris) HCl pH 7.5 containing a protease inhibitor cocktail (Roche). Glass beads (0.5–1.0 mm) were added to the mixture which was homogenized in a BioSpec Mini-Beadbeater. Following homogenization, SDS was added to the mixture at 4% final concentration. This homogenate was vortexed for 1 minute, heated at 98ºC for 5 minutes, sonicated and subsequently centrifuged at 12000g for 10 min.
Created on 4/13/21, 3:28 PM
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MRM Brain_2021-02-24_11-23-27.sky.zip2021-04-13 15:28:2822459026841
MRM Heart_2021-02-26_13-08-25.sky.zip2021-04-13 15:28:2822438625638
MRM Cerebelum_2021-02-24_13-23-28.sky.zip2021-04-13 15:28:2822438625640