DOI: 10.5281/zenodo.18174936
title: Basophil signatures in the blood of mice with breast cancer

description: 1. Study overview  
This dataset contains processed proteomic quantification tables from a study investigating how basophils regulate regulatory T cells (Tregs) and CD8⁺ T cells during early lung metastasis in mouse cancer models. Basophils were shown to inhibit Tregs through histamine–HRH1 signaling and mast cell protease 8 (MCPT8)–mediated IL‑2Rα/CD25 cleavage, thereby enhancing CD8⁺ T‑cell cytotoxicity and limiting metastatic seeding of breast and lung cancer cells in the lung.

2. Biological system  
- Species: Mus musculus (mouse).  
- Disease/contexts:  
  - Breast cancer lung metastasis model using 4T1 mammary carcinoma cells (including 4T1‑CD9‑mCherry).  
  - KRAS-driven non–small cell lung cancer (Kras^G12D; Trp53^−/−, “KP”) model with GFP‑expressing tumor cells (KP‑GFP).  
- Immune populations studied: basophils, Tregs (CD4⁺CD25^hi FOXP3⁺), CD8⁺ T cells and other immune subsets in tumor, lung, spleen and blood.

3. Experimental design (proteomics)  
- Experimental focus: quantitative comparison of proteomes of sorted immune cell populations (primarily basophils and control populations) across different tumor and treatment conditions, to define basophil functional states (B0/B1) and their impact on tumor immunity.
- Experimental type: bottom‑up, label‑based quantitative proteomics using TMTpro isobaric tagging and LC–MS/MS in data‑dependent acquisition mode on an Orbitrap instrument.

4. Sample preparation  
- Cell isolation:  
  - Basophils and other immune subsets were isolated from mouse blood, spleen, lungs and tumors by fluorescence‑activated cell sorting (FACS) using standard lineage and activation markers as described in the associated manuscript.
- Lysis and digestion:  
  - Cell pellets were lysed in 8 M urea, 100 mM Tris‑HCl buffer.  
  - Proteins were reduced with 10 mM TCEP and alkylated with 40 mM chloroacetamide.  
  - Proteins were first digested with endoproteinase Lys‑C (∼4 h, 37 °C) and then, after dilution to 1 M urea, with trypsin overnight at 37 °C
- Peptide cleanup and labeling:  
  - Peptides were acidified with formic acid and desalted using SDB‑RPS StageTips.  
  - Dried peptides were resuspended in HEPES buffer (pH 8) and labeled with TMTpro reagents (90 min at room temperature); reactions were quenched with hydroxylamine.  
  - Labeled samples were mixed at a 1:1 ratio across TMT channels and fractionated by high‑pH reversed‑phase chromatography prior to LC–MS/MS.

5. LC–MS/MS acquisition  
- Instrumentation:  
  - NanoLC: Vanquish Neo nano‑UPLC.  
  - Mass spectrometer: Orbitrap Ascend Tribrid (Thermo Fisher Scientific).  
- Acquisition mode: data‑dependent acquisition (DDA, Top‑Speed, 3‑s cycles).  
- MS1:  
  - Resolution 120,000 (at m/z 200).  
  - Scan range m/z 400–1400.  
- MS2 (HCD):  
  - Resolution 45,000 (at m/z 200).  
  - Normalized collision energy (NCE) 35%.  
  - Isolation window 0.7 m/z.  
- Reporter ions from TMTpro tags were used for multiplexed quantification.

6. Database search and quantification  
- Software: Proteome Discoverer 3.0 (Thermo Fisher Scientific) using SEQUEST and MSFragger search engines.
- Database: UniProt Mus musculus reference proteome supplemented with common contaminants.  
- Enzyme: trypsin, full specificity, maximum two missed cleavages.  
- Fixed modifications:  
  - Carbamidomethyl (C).  
  - TMTpro modification on peptide N‑termini and lysine residues.  
- Variable modifications: methionine oxidation (M).  
- FDR control: peptide‑spectrum match (PSM) and protein FDR set to 1% using target–decoy strategies.
- Quantification:  
  - Reporter‑ion (TMTpro) intensities extracted in Proteome Discoverer.  
  - Sample loading normalization performed using total reporter‑ion signal.  
  - Additional between‑sample normalization and differential abundance analysis performed in R using Trimmed‑Mean of M values (TMM, edgeR) and limma; multiple testing corrected with Benjamini–Hochberg, typically using FDR < 0.05 as significance threshold.

7. Contents of this dataset  
- `QuantifiedProteins_4Nahal_Mansouri_woOutliers.txt`  
  - Protein‑level quantification table containing:  
    - Protein group identifiers, UniProt accessions and gene names.  
    - Descriptions and basic annotation fields.  
    - Peptide/PSM counts per protein.  
    - TMT reporter‑ion–based abundance values for all samples/channels.  
    - Log fold‑changes, p‑values, FDR values and flags (e.g. “FDR ≤ 0.01”) from downstream statistical analysis
- Optional additional tables (if included in the archive): per‑peptide or per‑PSM quantification, intermediate statistics tables, and analysis scripts.

8. Absence of raw MS data  
This deposit contains processed protein‑level quantitative data (no raw MS files)