The global protein expression profile was determined using the iTRAQ quantitative proteomic approach. Protein preparation, iTRAQ labeling, strong cation exchange (SCX), nanoLC-MS/MS analysis, and protein identification and quantitation were performed following the procedures detailed in a previous study60 (link). Briefly, the tumor protein lysates from 6 randomly selected SE or EE mice were pooled, and then labeled with the iTRAQ labeling reagents 114 and 116 (iTRAQ Reagent 4-Plex Kit, Applied Biosystems), respectively. The iTRAQ-labeled samples were fractionated by SCX chromatography, and subsequently analyzed using the NanoLC system (NanoLC-2D Ultra, Eksigent, Dublin, CA, USA). Protein identifications and quantitations were performed with the ProteinPilot software (version 4.1; AB SCIEX, Foster City, CA, USA) and the mouse UniProtKB/Swiss-Prot proteome database. The FDR for the peptide identifications was estimated using a decoy database search strategy. We used a strict unused protein score cutoff of > 1.3 (confidence > 95%) for the protein identifications. Only those proteins identified from at least two peptides were included for the quantitative assay. The proteins were considered to be differentially expressed if their iTRAQ ratios were > 1.5 or < 0.67 in the comparison of EE versus SE.
Itraq reagent 4 plex kit
Manufactured by Thermo Fisher Scientific
The iTRAQ Reagent 4-Plex kit is a tool for quantitative protein analysis. It is designed to enable the simultaneous identification and quantification of proteins from up to four different samples in a single experiment.
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4 protocols using itraq reagent 4 plex kit
1
Quantitative Proteomic Profiling of Tumor Samples
2
Quantitative Proteomic Analysis of Cell Lines
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Cells were cultured to 80% confluence and collected. All protein extraction procedures were performed on ice. Cell pellets were dissolved in a cell lysis buffer (Pierce) containing protease and phosphatase inhibitors and incubated on ice for 45 min. The lysate was centrifuged at 12,000× g for 15 min at 4 °C, and the supernatant was collected. iTRAQ labeling was conducted using an iTRAQ Reagent 4-Plex kit (Applied Biosystems) based on the manufacturer’s protocol44 (link). One hundred micrograms of hFOB 1.19 or U2OS cell lysates was labeled with iTRAQ labeling reagents 114 or 115 (Applied Biosystems). After strong cation exchange and NanoLC−MS/MS analysis, protein identification and iTRAQ quantitation were performed using ProteinPilot4.1 software (AB SCIEX). Two physical replicates were performed. Figure 1A shows the flow chart of the proteomic analysis. Differentially expressed proteins underwent gene ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis.
3
Quantitative Proteomic Analysis of Lung Cancer Cells
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The nuclear proteins of NCI-H1299 and NCI-H358 cells were extracted and quantified by BCA protein quantitative method. iTRAQ labeling was conducted using an iTRAQ Reagent 4-Plex kit (Applied Biosystems, Foster City, CA) according to the manufacturer’s protocol. Briefly, 100 μg lysate of each sample was reduced with tris- (2-carboxyethyl) phosphine and alkylated with methyl methanethiosulfonate and then digested overnight at 37 °C with trypsin (mass spectrometry grade, Promega, Madison, WI). The ratio of trypsin to protein was 1:20. The iTRAQ labeled samples were then combined according to the specified set and transferred into a new EP tube, desalted with Oasis HLB cartridges (Waters, Milford, MA) and dried in a vacuum centrifuge (Concentrator Plus, Eppendorf, Germany) [7 (link)].
4
Quantitative Proteomic Analysis Using iTRAQ
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We performed iTRAQ labeling with an iTRAQ Reagent 4-Plex kit from Applied Biosystems (Foster City, CA) using the manufacturer's protocol. Experimental samples from two, four and six weeks of stimulation were labeled with iTRAQ labeling reagents 114, 118 and 121, respectively. Control samples from two, four and six weeks were labeled with reagents 113, 117 and 119, respectively.
One hundred microliters of pooled sample (n=3) for each group was reduced with tris-(2-carboxyethyl) phosphine and alkylated with methyl methanethiosulfonate (MMTS). Trypsin (mass spectrometry grade, Promega, Madison, WI) was used to digest samples overnight at 37°C at a 1:20 ratio (W/W) of Trypsin to protein.
The different labeling reactions were combined to perform the appropriate comparisons. Oasis HLB cartridges (Waters, Milford, MA) were used to desalt the mixtures, which were then dried in a vacuum centrifuge (Concentrator Plus, Eppendorf, Germany) (12).
One hundred microliters of pooled sample (n=3) for each group was reduced with tris-(2-carboxyethyl) phosphine and alkylated with methyl methanethiosulfonate (MMTS). Trypsin (mass spectrometry grade, Promega, Madison, WI) was used to digest samples overnight at 37°C at a 1:20 ratio (W/W) of Trypsin to protein.
The different labeling reactions were combined to perform the appropriate comparisons. Oasis HLB cartridges (Waters, Milford, MA) were used to desalt the mixtures, which were then dried in a vacuum centrifuge (Concentrator Plus, Eppendorf, Germany) (12).
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