HPKs from 3-5 healthy individual donors were pre-stimulated with TGFb (10ng/ml) plus IL-4 (20ng/ml) to upregulated the IL-9R (Das et al., 2019). Next day, HPKs were stimulated with IL-9 (50ng/ml) or in combination with IFNg (10ng/ml) for 24 hours at 37 degree C with 5% CO2. Sample preparation for LC-MS/MS was performed as described previously with few modifications. HPKs were lysed in TRIzol reagent (Invitrogen, Carlsbad, CA) and proteins were extracted as per the guidelines by the manufacturer. The instruments were set to MS1 resolution of 70000 and MS2 resolution of 17500. The MS spectra were analysed using the Thermo-scientific mass informatics platform Proteome discoverer version 2.2. Workflows for discovery proteomics were used with both Mascot and SequestHT as search engines. For label-free quantification, the standard LFQ workflows by Thermo were used. The samples were normalized with respect to the peak-area based total peptide amount. The permitted retention time shift (RT) was set to 2 min. Fragment mass tolerance was set to 0.02 Da and the precursor mass tolerance to 2ppm. The false discovery rate (FDR) was set to 0.01 (Strict). Importantly, only unique peptides were considered for peak-area based quantification. For statistical analysis of identified proteins, null hypothesis testing was performed using ANOVA (background based).
[doi:10.25345/C5PN55]
[dataset license: CC0 1.0 Universal (CC0 1.0)]
Keywords: IL-9, Keratinocyte, interferon-gamma, Label-free
Principal Investigators: (in alphabetical order) |
Rahul Purwar, IIT Bombay, India |
Submitting User: | soumitram |
Marathe S, Dhamija B, Kumar S, Jain N, Ghosh S, Prakash J, Srinivasan S, Das S, Sawant A, Desai S, Khan F, Syiemlieh A, Munde M, Nayak C, Gandhi M, Kumar A, Srivastava S, Venkatesh KV, Barthel SR, Purwar R.
Multi-omics analysis and systems biology integration identifies the roles of IL-9 in keratinocyte metabolic reprogramming.
J Invest Dermatol. Epub 2021 Mar 2.
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