PRIDE Assigned Tags:Biomedical Dataset
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Using quantitative proteomics-isobaric tags for relative and absolute quantification (iTRAQ), we characterize the mechanism of TIIA regulation in gastric cancer cell line AGS.
Sample Processing Protocol
AGS cells were treated with 5.3 μM (IC50) TIIA or 0.1% DMSO control medium for 48 hr after 24 hr of seeding (8 x 104 cells/well in 6-well plates). Cells were harvested with trypsin/EDTA and then total proteins were extracted by using lysis buffer (1% SDS (Bioman), 50 mM Tris-HCl (pH 6.8), 10% glycerol), and 1X protease inhibitor (Bioman) and sonication. Then each protein sample underwent reduction, alkylation, and gel-assisted trypsin digestion overnight to yield peptides, which were later extracted from the gels. Equal amounts of peptides from control and TIIA-treated samples were labeled by different iTRAQ reagents (AB SCIEX; control samples labeled by 114 or 115; TIIA-treated samples labeled by 116 or 117) and incubated at room temperature for 1 hr. Peptides were combined together and dried with a centrifugal evaporator (CVE-2000; EYELA). After iTRAQ-labeling, samples were desalted and analyzed by using a LC-ESI-Q-TOF mass spectrometer (Waters SYNAPT® G2 HDMS; Waters Corp.). Samples were injected into a 180 mm × 2 cm capillary trap column and separated by a 75 mm × 25 cm nanoACQUITY UPLC™ 1.7 mm Ethylene Bridged Hybrid C18 column using a nanoACQUITY Ultra Performance LC™ System (Waters Corp.). The mass spectrometer (MS) was operated in electrospray ionization sensitivity mode, and calibrated with a synthetic human [Glu1]-Fibrinopeptide B solution (1 pmol/ml; Sigma-Aldrich) delivered through a NanoLockSprayTM source, which was used for accurate mass measurements. Data was acquired in the data directed analysis (DDA) mode, which included one full MS scan (m/z 350-1700, 1 s) and three sequential MS/MS scans (m/z 100-1990. 1.5 s for each scan) on the three most intense ions present in the full scan mass spectrum.
Data Processing Protocol
The peak list resulting from MS/MS spectra was exported to mgf format by Mascot Distiller v2.3.2 (Matrix Science, London, United Kingdom) with charge state set to 2+, 3+, 4+, 5+, and other default parameters. Data files were merged and searched against the combined sequence database (containing 36,774 sequence entries) of the Swiss-Prot human database.
Lin LL, Hsia CR, Hsu CL, Huang HC, Juan HF. Integrating transcriptomics and proteomics to show that tanshinone IIA suppresses cell growth by blocking glucose metabolism in gastric cancer cells. BMC Genomics. 2015 Feb 5;16:41 PubMed: 25652794
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