Serum proteome of male and female Wistar rats after chronic Asparagus racemosus (Shatavari) root extract: PEAKS Studio project exports
Description
This record holds the complete data and analysis chain behind the article, from the mass-spectrometry search results to the scripts that produce the published tables and figures. 1. PEAKS Studio project exports (HTML and Excel). Three projects: Rat-male_11 with LFQ Rat-male_12 (male comparisons), Rat-female_11 with LFQ Rat-female_12 (female comparisons), and Rat M and F_20 with LFQ Rat M and F_21 (the baseline male-versus-female comparison). The HTML exports carry the peptide-spectrum matches and the annotated spectra behind every identification reported in the paper; the Excel exports carry the protein and peptide tables. Spectra were searched against the Rattus norvegicus RefSeq proteome (GCF_015227675.2, mRatBN7.2 annotation; 74,754 sequences) together with the contaminant database supplied with the software. 2. PHYPLU-D-26-00917_data_and_scripts.zip. The protein abundance matrices of the three PEAKS Q projects (208, 185 and 169 protein groups over 9, 9 and 18 injections); the sample-to-run mapping; a self-contained reimplementation of the descriptive screening described in the paper, with its output for all five comparisons; the Gene Ontology annotation tables for the four treatment and recovery comparisons, with the script that builds them and the script that draws Figure 3; and a script that recomputes the technical-replicate reproducibility figures quoted in the paper. A README describes every file, the normalization that PEAKS applied, the median-centering of the baseline comparison and the handling of contaminant entries. 3. MOET results. The complete output of the RGD Multi-Ontology Enrichment runs for the four treatment and recovery comparisons, reported in the paper for completeness and not used to support any conclusion in it. These four files are also present inside the analysis bundle above, at go_analysis/, and are provided here separately for convenience; the two copies are identical. Study design. Pooled sera from six groups of 12 Wistar rats each — male and female, each sex comprising a distilled-water control, a group given Asparagus racemosus root extract at 1000 mg/kg/day for 180 days, and a satellite group given the same regimen and then followed for 28 days without treatment — were analysed by nanoLC-TIMS-MS/MS in three technical injections per pool. The three injections measure analytical reproducibility and carry no information about variation between animals. Instrument raw files. The Bruker .d files were not retained by the analytical facility and are no longer available, so a submission to the ProteomeXchange Consortium through the PRIDE partner repository was not possible: a raw file is a mandatory component of every PRIDE submission, complete or partial. Every identification in the paper can nevertheless be traced to its peptide-spectrum matches and annotated spectra in the PEAKS exports deposited here, and every analysis downstream of the database search can be reproduced from the abundance matrices.
Files
Steps to reproduce
1. Animals and serum. Male and female Wistar rats received Asparagus racemosus root extract at 1000 mg/kg/day, or distilled water, by gavage for 180 days; a satellite group had the same regimen then 28 days without treatment. At necropsy, equal volumes of serum from the 12 animals of a group were pooled, giving six pools (MC, MT, MS, FC, FT, FS). 2. Sample preparation. Pool protein was measured by Bradford assay and 40 micrograms were mixed with Laemmli buffer and 1X DTT, heated at 95 C for 5 min and separated briefly by SDS-PAGE (12.5% gel, 200 V, about 10 min) with Coomassie R-250 staining. Gel pieces were destained in 50% acetonitrile / 0.1 M NH4HCO3, reduced with 10 mM DTT (60 C, 45 min), alkylated with 100 mM iodoacetamide (room temperature, 30 min, dark) and digested with trypsin at 1:50 enzyme:protein, 37 C overnight. Peptides were extracted with 5% formic acid in 50% acetonitrile, dried, desalted on C18 ZipTips and stored at -20 C. 3. LC-MS/MS. Peptides were redissolved in 0.1% formic acid and quantified colorimetrically. 200 ng were loaded on a 25 cm x 75 um, 1.9 um C18 column (PepSep) at 50 C and 300 nL/min over a 55 min gradient (2-40% B in 45 min, then 85% B for 10 min; A = 0.1% formic acid in water, B = 0.1% formic acid in acetonitrile) on a nanoflow LC coupled to a Bruker timsTOF Pro in ddaPASEF mode: one TIMS-MS survey and ten PASEF MS/MS scans per cycle, 100 ms ramp, 1/K0 1.6 to 0.6 Vs cm-2, singly charged ions excluded by polygon filter, 40 s active exclusion above 20,000 counts, collision energy ramped with mobility. The 18 sample injections (six pools x three injections) were acquired in one sequence of 28 injections that also held six injections of a pooled QC sample (equal volumes of all six pools) and four 0.1% formic acid blanks. The three injections of a pool were consecutive and the pools ran in the order MC, MT, MS, FC, FT, FS. 4. Identification and quantification. Spectra were searched in PEAKS Studio 10.6 against the Rattus norvegicus RefSeq proteome (GCF_015227675.2, mRatBN7.2 annotation, 74,754 sequences) together with the contaminant database supplied with the software: parent tolerance 20 ppm, fragment tolerance 0.05 Da, fixed carbamidomethylation of cysteine, variable oxidation of methionine and N-terminal acetylation, FDR 1% or less at the peptide-spectrum-match level. Label-free quantification used PEAKS Q, protein abundance being the summed intensity of the three most intense unique peptides. Three projects were run separately: male, female, and combined for the baseline comparison. 5. Downstream analysis, from the analysis bundle in this record. python3 descriptive_screening.py . screening/ regenerates the fold changes and listings; python3 replicate_cv.py . the reproducibility figures; python3 go_analysis/build_go_tables.py rgd.gaf.gz go-basic.obo out/ goa_human.gaf.gz the Gene Ontology tables; python3 go_analysis/make_figure3.py . Figure3.png Figure 3. README.txt describes each file.
Institutions
- Kasetsart UniversityBangkok, Bangkok
- Chulabhorn Research InstituteBangkok, Bangkok
- Ministry of Public HealthNonthaburi, Nonthaburi