GENERAL INFORMATION ........................... 1. Dataset title: Dataset_Orbitrap_Raw-Data_Stress_Hypothalamus(1h-24h) 2. Authors: Infantes-López, María Inmaculada; Nieto-Quero, Andrea; Pedraza, Carmen; Perez-Martin, Margarita 3. Author contact information: Carmen Pedraza, mdpedraza@uma.es METHODOLOGICAL INFORMATION ................................. 1. Description of the methods for collection/generation of data: - Sample preparation. The samples used for this study were taken from the hypothalamus of each right hemisphere of the animals. The tissue was homogenised, the amount of protein in the extracts was determined by the BCA (bicinchoninic acid) method and standardised to a concentration of 1 µg/µl. - Tryptic digestion in solution and tagging by Tandem Mass Tag (TMT). Prior to digestion, the proteins were treated for the reduction of disulphide bonds between proteins and alkylation for the blocking of thiol groups. Subsequently, they were subjected to a purification process by acetone precipitation. Finally, they were digested with trypsin. The digested proteins were labelled with TMT 6plex isobaric labelling reagents. - Nanometre-scale high-performance liquid chromatography (nHPLC) and mass spectrometry using Orbitrap. Peptides were first separated using a pre-column, and then passed through an analytical column with the elution time varied according to the percentage of solvent. The peptides were positively ionised using a nanospray ion source. Finally, the peptides were analysed using the Quadrupole-Orbitrap hybrid mass spectrometer, Q-Exactive HF-X (Thermo Fisher Scientific). - Analysis of MS/MS or MS2 spectra. Spectra were searched (resulting in 25,097 peptide sequences) using the SwissProt database for Mus musculus. The acquired raw data were analysed on the 'Proteome Discoverer 2.4' platform (Thermo Fisher Scientific) with the 'Sequest HT' search engine using mass tolerances of 10 ppm and 0.02 Da for precursor ions and fragment ions, respectively. 2. Data processing methods After data generation by Q-Exactive HF-X Hybrid Quadrupole-Orbitrap Mass Spectrometer system, a functional enrichment study was performed. The significantly overexpressed and underexpressed proteins of the right hypothalamus of experimental groups with their respective controls (stress/control) were filtered (p <0.05; FDR <0.05; FC >1.2 for proteins overexpressed under stress; and FC <0.833 for proteins underexpressed under stress) [1]. Thus, log2(FC) and -log(p-value) were calculated. Considering FC as 'Abundance Ratio: (Experimental) / (Control)' and p-value as 'Abundance Ratio P-Value: (Experimental) / (Control)'. Through STRING, number of interactors was increased to 100 for both over- and underexpressed proteins. Then, MCODE was used as a clustering tool for the protein‒protein interaction network obtained from STRING. After that, a functional enrichment analysis was performed using DAVID [2,3] by introducing each protein gene name grouped into the clusters from the previous step. Biological processes in each of the different signalling pathways (protein clustering) were determined, which determined in turn what functions were affected. Finally, a network analysis of the GO terms obtained from DAVID was performed so that it was easier to interpret and integrate the biological implications of the results. [1] Dozio, V, Sanchez, JC. (2018). Profiling the proteomic inflammatory state of human astrocytes using DIA mass spectrometry. Journal of Neuroinflammation, 15(1), 331. https://doi.org/10.1186/s12974-018-1371-6 [2] Huang, DW, Sherman, BT, Lempicki, RA. (2009). Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nature Protocols, 4(1), 44-57. https://doi.org/10.1038/nprot.2008.211 [3] Huang, DW, Sherman, BT, Lempicki, RA. (2009). Bioinformatics enrichment tools: paths toward the comprehensive functional analysis of large gene lists. Nucleic Acids Research, 37(1), 1-13. https://doi.org/10.1093/nar/gkn923 3. Software or instruments needed to interpret the data: - SwissProt database for Mus musculus version 2017.10.25 - 'Proteome Discoverer 2.4' platform (Thermo Fisher Scientific). https://www.thermofisher.com/es/es/home/industrial/mass-spectrometry/liquid-chromatography-mass-spectrometry-lc-ms/lc-ms-software/multi-omics-data-analysis/proteome-discoverer-software.html 4. Environmental or experimental conditions: - 1h post-acute WIRS - 24h post-acute WIRS FILE OVERVIEW ---------------------- The raw dataset is distributed in one single file in .xlsx format. This provides information on the proteins detected in hypothalamus extracted after 1 and 24 hours post-acute stress. DATA-SPECIFIC INFORMATION: ------------------------------------------- 1. Name File: Orbitrap_Raw-Data_Stress_TMT-Hypothalamus(4BR-1h-24h).xlsx