The sample preparation, data acquisition, preprocessing, statistical analysis, metabolite identification and database construction have been described previously. For more detailed description, please refer to the publication by Gou et al.18.MaterialsAdult Arctic lampreys (Lethenteron camtschaticum) in the spawning migration stage were collected from the Songhua River in Heilongjiang Province, China. Fourteen different tissues, heart (H), liver (L), kidney (K), brain (B), supraneural body (S), muscle (M), intestine (I), gill (G), eye (E), testis (T), ovary (O), buccal gland (Bu), blood (X), and notochord (N), were carefully dissected and rinsed in sterile phosphate-buffered saline (PBS: 10 mM phosphate buffer, 2.7 mM potassium chloride, 137 mM sodium chloride, pH 7.4). The secretion from the buccal gland was extracted using a syringe. All samples were snap-frozen in liquid nitrogen and stored at −80 °C until LC-MS analysis (Fig. 1a).Metabolite extractionTo extract the samples, 30 mg of each sample was placed into a 2 mL Eppendorf tube along with 20 μL of internal standard (2-chloro-L-phenylalanine, 0.3 mg/mL) and 400 μL of an extraction solution composed of 80% methanol in water (V/V) (Fig. 1a). Two small steel balls were then added to each tube. The tubes were pre-cooled at −20 °C for 2 minutes before grinding the samples at 60 Hz for 2 minutes using a Tissuelyser-48 grinding miller (Jingxing Limited Company, Shanghai, China). The mixture was briefly vortexed and then sonicated at room temperature (25–28 °C) for 10 minutes. Subsequently, the samples were centrifuged at 13,000 rpm and 4 °C for 10 minutes. Following centrifugation, 300 μL of the supernatant was transferred to a brown glass vial and dried using a freeze concentration centrifugal dryer. The dried residue was reconstituted in 300 μL of a methanol and water mixture (1/4, V/V), vortexed for 30 seconds, and then placed at −20 °C for 2 hours. The samples were centrifuged again at 13,000 rpm and 4 °C for 5 minutes. The supernatants (150 μL) were carefully extracted using crystal syringes, filtered through a 0.22 μm PTFE filter (Acrodisc® CR 13 mm; PALL), and transferred into LC vials for LC-MS analysis. Pooled QC samples were created by combining 20 μL aliquots from each extracted sample.LC-MS analysisA Dionex Ultimate 3000 UHPLC system coupled with a Q-Exactive quadrupole-Orbitrap mass spectrometer, equipped with a heated electrospray ionization (ESI) source (Thermo Fisher Scientific, Waltham, MA, USA), was utilized for spatial metabolomics analysis in both positive and negative ion modes (Fig. 1b). The separation was carried out on an ACQUITY UPLC HSS T3 column (1.8 μm, 2.1 × 100 mm). The binary gradient elution system consisted of (A) water with 0.1% formic acid (V/V) and (B) acetonitrile with 0.1% formic acid (V/V). The following gradient program was used: 5% B from 0–2 min, 5–25% B from 2–4 min, 25–50% B from 4–8 min, 50–80% B from 8–10 min, 80–100% B from 10–14 min, held at 100% B for 1 min, followed by 100% to 5% B from 15–15.1 min, and holding at 5% B from 15.1–18 min. The flow rate was maintained at 0.35 mL/min, with the column temperature set to 45 °C. All samples were kept at 4 °C during analysis, and a 2 μL injection volume was used. The mass spectrometer scanned a mass range from m/z 66.7 to 1000.5, with a resolution of 70,000 for full MS scans and 35,000 for HCD MS/MS scans. The collision energy was adjusted to 10, 20, and 40 eV. The mass spectrometer operated under the following conditions: spray voltage of 3800 V (+) and −3000 V (−); sheath gas flow rate of 35 arbitrary units; auxiliary gas flow rate of 8 arbitrary units; and a capillary temperature of 320 °C. The quality control (QC) samples were injected at regular intervals (every 10 samples) throughout the analytical run to ensure the repeatability of the data (Fig. 1b).Data preprocessing and metabolite annotationThree distinct software tools were employed for data preprocessing and metabolite identification: MS-DIAL (v.4.9)25,26, Progenesis QI (v.2.4, Waters), and Compound Discoverer (v.3.3.3, Thermo Scientific). Among them, Progenesis QI and MS-DIAL were primarily used for metabolite identification. For MS-DIAL, the raw data were converted into.abf files using the Reifycs ABF Converter (Figure S1). Then, the .abf files were imported to the MS-DIAL platform. For Progenesis QI and Compound Discoverer, the raw data were directly used for data analysis (Fig. 1c). The specific parameters used for lamprey metabolomics data processing using the three software are detailed in Supplementary Table S1.Lamprey spatial metabolomics database constructionThe LampreyDB database was constructed using MySQL (v.8.0) and Django (v.3.0.6) as the backend framework. The frontend was developed with HTML and JavaScript. Custom scripts, along with the Python library Beautiful Soup (https://pypi.org/project/beautifulsoup4/), were used to create the interactive anatomical heatmap. Additional visual elements, such as MS spectrum plots, were generated using the Plotly Python library (https://plotly.com/python/). The database is hosted on Microsoft Azure cloud service.
