GENERAL INFORMATION ........................... 1. Dataset title: HIPPOCAMPAL PROTEOMICS IN A MICE MODEL OF EARLY LIFE ADVERSITY IN COMBINATION WITH A SECOND ACUTE STRESS IN ADULTHOOD. 2. Authors: Munoz-Martin, Jose; Perez-Martin, Margarita; Pedraza, Carmen 3. Author contact information: Carmen Pedraza, mdpedraza@uma.es // Margarita Perez-Martin, marper@uma.es METHODOLOGICAL INFORMATION ................................. 1. Experimental treatments and sample collection/processing: - Animals: C57BL6J male and female mice (Age P62-65). - Stress procedure: In short, female and male C57BL/6J mice were subjected to 3h daily maternal separation (MS) for 21 days since birth. Around day 60 (P62 and 64), mice underwent a single 2h movement restriction stress (RS), defining the following experimental groups: CTRL, RS, MS, MS+RS (N=6-7, males and females separately). 24h after RS, mice were perfused with PBS 1X and left hippocampus was collected and snap-frozen with dry-ice and stored -80ºC. Environmental or experimental conditions, time of collection of hippocampi as follows: - CTRL: control conditions, animal facility rearing. Collected P62 (9:00 am-14:00 pm) - RS: Restriction Stress (Adulthood, P62, single acute 2h). Collected P63 (9:00 am-14:00 pm). - MS: Maternal separation (Infancy, P1-21, 3h daily). Collected P64 (9:00 am-14:00 pm). - MS+RS: Maternal separation + Restriction Stress (P64). Collected P65 (9:00 am-14:00 pm). - Sample preparation. 5-6 frozen hippocampi per experimental group were quickly homogenized individually with 150 μl of lysis buffer RIPA (20 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 0.1% SDS, 1% Triton X-100, 1X protease inhibitors (cOmplete™ Mini Protease Inhibitor Cocktail Tablets (Roche). Samples were centrifuged at 16,000 × g for 10 min at 4°C. The supernatant was recovered and stored at 4°C overnight. Protein concentration was quantified the following day by the BCA (bicinchoninic acid) method (Pierce™ BCA Protein Assay Kit (Thermo Scientific). Protein extraction yield ranged from 5 µg/µl to 9 µg/µl. - Sample processing. Samples were standardized to a concentration of 1 µg/µl and processed by gel-assisted proteolysis. Briefly, protein extracts were immobilized within a polyacrylamide matrix, reduced with dithiothreitol, and alkylated with iodoacetamide to modify cysteine residues. Proteolytic digestion was performed using sequencing-grade trypsin (Promega, USA). Peptides were subsequently extracted from the gel matrix and prepared for mass spectrometry analysis. 2. Liquid Chromatography–High-Resolution Mass Spectrometry The Ultra-High-Performance Liquid Chromatography coupled to High-Resolution Mass Spectrometry (UHPLC-HRMS) analysis was performed by Proteomics Service of the Central Research Support Services (SCAI) at the University of Málaga (UMA). Briefly, peptide samples were injected into an Easy-nLC 1200 UHPLC system (Thermo Fisher Scientific, USA) coupled to a Q-Exactive™ HF-X Hybrid Quadrupole-Orbitrap mass spectrometer (Thermo Fisher Scientific, USA). Peptides were initially loaded onto a trap column and subsequently separated on a 50 cm analytical column using a 180 min linear gradient at a flow rate of 300 nLmin−1 (acetonitrile as eluent). Peptides were measured in positive electrospray ionization mode using a data-dependent acquisition (DDA) method to obtain the corresponding MS/MS spectra. A total of 3,357,048 MS/MS spectra were acquired during the run. 3. Data analysis and protein identification The acquired raw data were analyzed using the Proteome Discoverer TM 2.5 platform (Thermo Fisher Scientific, USA). MS/MS spectra were identified using the Sequest HT® search engine against the Mus musculus Swiss-Prot subset of the UniProt database (version 2025-02-05). Trypsin was specified as the proteolytic enzyme, allowing up to two missed cleavages. Carbamidomethylation of cysteine residues was set as a fixed modification, while methionine oxidation was included as a variable modification. Precursor ion mass tolerance was set to ±10 ppm, and fragment ion mass tolerance was set to ±0.02 Da. Protein identifications were validated using the Percolator® algorithm. This resulted in 840,155 PSMs or protein spectrum matches (peptide sequences that matches with the database); 28,294 Peptide groups and 3,669 Protein groups. A total number of 4,228 proteins were identified. Only 2,895 proteins fulfilled the adequation criteria: They were master proteins within their group, identified with at least two peptides detected and high confidence (FDR < 1%). Label-free quantification was performed using the Minora Feature Detector within Proteome Discoverer TM 2.5, with abundances determined from precursor ion intensities. Normalization across samples was conducted based on total peptide content. Protein abundance ratios obtained from 5-6 independent biological replicates per condition (males and females, separately) were evaluated using a one-way analysis of variance (ANOVA) applied to the whole protein dataset. Differentially expressed proteins were defined as those with p < 0.05. To classify proteins as over- or under-expressed following treatment, log2-transformed abundance ratios were used: values > 1 indicated upregulation, whereas values < −1 indicated downregulation. These results are compiled in the EXCEL file (hippocampal_proteomics_results_MSJUL22.xlsx). 4. Determination of protein expression and functional enrichment among experimental conditions The results, under the tab Protein RAW and compiled in the EXCEL file (hippocampal_proteomics_results_MSJUL22.xlsx) was processed as follows: The overexpressed and underexpressed proteins of experimental groups with their respective controls (CTRL), namely Abundance ratios, were filtered first to find those which were significant with a p-value<0.05 or -log10 (p-value) = 1.30. Secondly, only proteins with Fold-changes (FC) > 2.0 for overexpressed and FC<0.5 for underexpressed proteins compared to their CTRL were maintained with a subsequent filter. These FC are also represented as log2(FC) 1 or -1 for up or down regulated, respectively. Furthermore, comparisons for single stressor versus combined stressors (RS vs MS+RS, MS vs MS+RS) were performed. Males and females were analyzed separately. Volcano plots were represented with Proteome Discoverer 2.5 using log2 (FC) for X-axis and -log10(p-value) for Y-axis and can be found in the file volcano_plots_hippocampal_proteomics_MSJUL22.pdf Further downstream analysis regarding protein clusterization and functional enrichment via STRING, version 12.0 (https://string-db.org) can be consulted in the scientific publication: Munoz-Martin et al., 2026. SOFTWARE USED: ------------------------------ - UniProt database (version 2025-02-05). Mus musculus Swiss-Prot subset. - Proteome Discoverer 2.5 platform (Thermo Fisher Scientific, USA). 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 - STRING, version 12.0 (https://string-db.org) FILE OVERVIEW ---------------------- The raw dataset as well as the refined comparisons among experimental conditions are distributed in one EXCEL file (hippocampal_proteomics_results_MSJUL22.xlsx). Volcano plots for every comparison can also be found distributed in the pdf file (volcano_plots_hippocampal_proteomics_MSJUL22.pdf). DATA-SPECIFIC INFORMATION: ------------------------------------------- 1. Name File: hippocampal_proteomics_results_MSJUL22.xlsx 2. Name File: volcano_plots_hippocampal_proteomics_MSJUL22.pdf