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| Status |
Public on May 07, 2019 |
| Title |
uuo-day3-2 |
| Sample type |
protein |
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| Source name |
Unilateral ureter obstruction (UUO) model
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| Organism |
Mus musculus |
| Characteristics |
strain: BALC/c
Sex: male
treatment: 3 days after unilateral ureter obstruction
tissue: kidney
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| Treatment protocol |
Unilateral ureter obstruction (UUO) model: Before surgery, mice were anaesthetized with an intraperitoneal injection of sodium pentobarbital (50 mg/kg of body weight), the left kidney was exposed via a flank incision and 3.0 silk suture thread was used to tie off the ureter at the lower pole. Before the obstruction and 3, 7, and 14 days later, mice were sacrificed, kidneys were removed and immediately snap frozen in liquid nitrogen.
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| Growth protocol |
Male BALC/c mice were obtained from Charles River Laboratories (USA). All experimental protocols concerning the use of laboratory animals were performed according to the NIH guidelines for the care and use of laboratory animals, and approved by the Institutional Animal Care and Use Committees (IACUC) of Harvard Medical School. Mice were housed in groups of three on a 12-h light/dark cycle with access to food and water ad libitum. At the age of 8-10 weeks they entered the experiment.
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| Extracted molecule |
protein |
| Extraction protocol |
Kidney samples were mechanically homogenized in lysis buffer (8 M urea, 1% SDS, Roche complete protease inhibitors and phosphatase inhibitors, 50 mM Tris pH 8.5). Approximately one third of a kidney was used for sample preparation. Protein concentration was determined using the BCA assay (Pierce, Rockford, IL). The homogenate was reduced with 5 mM DTT and alkylated with 15 mM iodoacetamide (Sigma, St. Louis, MO). 0.15 mg of protein was precipitated using chloroform:methanol. Pellets were washed twice with cold methanol and re-solubilized in 8M urea with 20 mM EPPS, pH 8.5. After diluting the samples to 4M urea using 20 mM EPPS, they were digested with Lys-C (Wako Chemicals, Richmond, VA) overnight at room temperature. On the next day, samples were further diluted to 1.5 M urea using 20 mM EPPS and digested for 6h at 37°C using Trypsin (Promega, Madison, WI). 60 µg of each sample were then brought to 10% (v/v) acetonitrile and labeled with 2:1 (TMT:Peptide) by mass of TMT-10 reagent (Pierce). The reaction was quenched with hydroxylamine (0.5% final volume). Afterwards, samples were acidified by adding formic acid to 2% final volume, combined, and desalted using a C18 Sep-Pak (Waters, Milford, MA). The now combined sample was fractionated using basic pH reversed phase chromatography using a 1200 HPLC (Agilent; Santa Clara, CA) equipped with a UV-DAD detector and fraction collection system. Then, the resulting 12 fractions were desalted using the C18 StageTip procedure. Each fraction was loaded onto a 100 µm id, 35 cm long column packed with 1.8 µm beads (Sepax, Newark DE) and separated using a 3h gradient from 8-27% buffer B (99% acetonitrile and 1% formic acid) and buffer A (96% water, 3% acetonitrile and 1% formic acid) using an Easy 1000 nano-LC (Thermo-Fisher Scientific, San Jose, CA). All MS analyses were performed on an Orbitrap Fusion Lumos mass spectrometer (Thermo-Fisher Scientific, San Jose, CA) applying a multi-notch MS3 method.
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| Label |
10-plex TMT proteomics
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| Label protocol |
n/a
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| Hybridization protocol |
n/a
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| Scan protocol |
n/a
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| Data processing |
Raw data was converted to mzXML
searched via Sequest against Uniprot version 28
Variable modifications of oxidized methionine and over-labelling of TMT on serine, threonine and tyrosine were considered
linear discriminant analysis was used to distinguish forward and reverse hits and reverse hits were filtered to an FDR of 1% at the protein level
using rules of parsimony shared peptides were collapsed into the minimally sufficient number of proteins
Quantitation filters of > 200 sum reporter ion S:N and > 0.7 isolation specificity were incorporated
version 28 Uniprot (UniProt Consortium 2018) database downloaded 02/04/2014
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| Submission date |
Feb 06, 2019 |
| Last update date |
May 07, 2019 |
| Contact name |
Lorena Pantano |
| E-mail(s) |
lpantano@iscb.org
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| Organization name |
Harvard Chan School of Public Health
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| Department |
Biostatistics
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| Lab |
Bioinformatic Core
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| Street address |
655 Huntington Ave
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| City |
Boston |
| State/province |
MA |
| ZIP/Postal code |
02115 |
| Country |
USA |
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| Platform ID |
GPL26156 |
| Series (2) |
| GSE118341 |
Multi Omics analysis of fibrotic kidneys in two mouse models |
| GSE126182 |
Multi Omics analysis of fibrotic kidneys in two mouse models (Unilateral ureter obstruction (UUO) model MS data set) |
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| Supplementary file |
Size |
Download |
File type/resource |
| GSM3592702_day3_2_uuo.csv.gz |
101.4 Kb |
(ftp)(http) |
CSV |
| Processed data provided as supplementary file |
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