LC–MS dataset for quantification of antipyrine (ANP) in mouse brain after SeeThrough protocol

LC–MS raw and processed data supporting the quantification of antipyrine penetration into mouse brain after SeeThrough protocol.## 1 | Repository Layout<br>```MS_data/├─ README.md ← this file├─ metadata.tsv ← sample metadata (SampleID, group, conc_uM, bio_rep, tech_rep)├─ raw/ ← unprocessed Thermo *.RAW files│  ├─ calibration/ ← calibration samples (bio rep 1‑3)│  └─ quantification/│     ├─ control/ ← CTRL group│     └─ seethrough/ ← SeeThrough (ST) group├─ processed/ ← processed data│  ├─ calibration_peakArea.csv│  ├─ quant_peakArea_control.csv│  ├─ quant_peakArea_seethrough.csv│  ├─ quant_summary_bio.csv ← bio‑rep mean peak area + concentration│  └─ summary_ST_conc.csv ← regression parameters &amp; grand‑mean concentration└─ analysis/ ← re‑analysis scripts   └─ peak_integration.py```<br>## 2 | File‑naming Scheme| Component | Meaning | Example ||-----------|---------|---------|| `CAL` / `CTRL` / `ST` | sample type | `CAL_1uM_bio1_tech1.RAW` || `1uM`, `2uM`, … | nominal conc. (calibration only) | – || `bioX` | biological replicate (1–3) | `bio2` || `techY` | technical replicate (1–2) | `tech2` |<br>## 3 | Sample PreparationFiltered‑brain supernatant (100 µL) was mixed with acetonitrile (300 µL) to precipitate proteins. After centrifugation, 200 µL supernatant was dried, re‑dissolved in 0.1 % TFA, and 1 µL was injected into the nano‑LC system.<br>## 4 | LC Conditions- **Trap column**: 0.075 × 20 mm, 3 µm C18 (Acclaim PepMap 100, Thermo) - **Analytical column**: 0.075 × 150 mm, 3 µm C18 (NTCC‑360/75‑3‑153, Nikkyo) - **Mobile phases**: A = 0.1 % FA, B = ACN + 0.1 % FA - **Gradient @ 300 nL min⁻¹**: 1 % B 5 min → 1–40 % B 35 min → 40–90 % B 1 min → 90 % B 4 min<br>## 5 | MS ConditionsOrbitrap, positive ESI (2.2 kV); scan 70–300 m/z; 70 000 FWHM (m/z 200).<br>## 6 | Data Processing1. Xcalibur Qual Browser v4.6 → extract XIC at *m/z* 189.102 2. Export peak areas to CSV 3. Store files listed under `processed/` 4. Run `analysis/peak_integration.py` to: - build the calibration line (slope = 3.991 × 10⁹, intercept = 4.588 × 10⁹, R² = 0.9997) - compute bio‑rep mean peak areas &amp; concentrations (`quant_summary_bio.csv`) - calculate grand‑mean ST concentration = 0.688 µM &lt; 1 µM (`summary_ST_conc.csv`)