SOW updated.pdf

Statement of Work/Specification Sheet

Metabolomic Analysis of Swine Tissues

Background and Scope:

The USDA ARS ABBL requires the thorough analysis of metabolites present in plasma and tissue samples derived from the swine intestinal tract and feces, followed by quantification of these metabolites and statistical comparison of differences in metabolite concentrations between several treatment groups. This analysis is critical to understanding the adaptation of the digestive system during the transition of the young pig from a milk-based diet to solid foods at weaning. Identifying key metabolites that differ within the plasma or a specific region of the intestine or feces will provide critical information in determining what metabolites are positive or negative predicators of weaning adaptation and future experiments to enhance pig performance during weaning.

The global untargeted metabolite analysis will be required for 144 samples in total, comprised of samples from several tissues as mentioned above. The analysis requires quantification of amino acids and peptides as well as the products of amino acid metabolism, fatty acids and the products of their metabolism, phospholipids and the products of their metabolism, diglycerides, steroids, bile acids, carbohydrates and the products of their metabolism, nucleotides and the products of their metabolism, vitamins, and their cofactors.

The quantification, statistical analysis and data interpretation performed by the vendor is critical to the project and their final report is the basis for publication of the research, so it must be easily interpretable by scientists who do not routinely perform these methods or analyses.

Requirements/Specifications

1. The project is to be performed as a global untargeted profile.

2. Samples will be provided by ARS and received by the vendor where they will be coded and stored in ultracold freezers until sample preparation. These freezers should be constantly monitored.

3. Sample preparation and extraction for analysis is to be performed by the vendor.

4. Samples are to be analyzed by ultra-high performance liquid chromatography/tandem accurate mass spectrometry (UHPLC/MS/MS) methods with peak identification, deconvolution, and identification. Identification should be by comparison to the vendor’s curated chemical reference library (not an outside source library that was not generated on the equipment to be used in this analysis).

5. Lipid analysis should be able to quantify at least 12 lipid classes, including principle phospholipid, sphingolipid and neutral lipid classes.

6. Amino acid analysis should include metabolites resulting from amino acid metabolism.

7. Carbohydrate analysis should include quantification of metabolites of the TCA cycle, glycolysis, gluconeogenesis, and pentose metabolism.

8. Nucleotide analysis should include both purine and pyrimidine metabolism.

9. Data should be subjected to pathway analysis for data interpretation by trained PhD.

biochemists working for the vendor.

10. Data should be stored on servers that are backed up nightly (incremental) and fully backed up at least weekly by the vendor.

11. The final report should be prepared by trained biochemists and include a Word document that provides the general methodologies used to quantify the metabolites, how the data was analyzed, and interpretation of the data. The data interpretation should provide indication of the relationship of changes in metabolite concentrations that were measured with specific metabolic pathways and how these resultant changes in metabolic pathways are related to the biochemical scientific literature.

12. The final report should provide an Excel spreadsheet that is accessible by ARS scientists and is sorted by biochemical pathways and specific biochemicals identified. The data should also indicate the statistical values of significance of the comparison of biochemical between treatment groups. The excel spreadsheet should also contain the fold changes in each metabolite between treatment groups and the specific statistical value for each comparison for each metabolite.

13. The final report should contain a PowerPoint file with tables and charts of the significant data comparisons that can be used for publication of the data.