The CCE 5 th Annual Retreat Global Proteomics & Determination of Vitamin D Metabolites Update

1. The CCE 5th Annual RetreatGlobal Proteomics & Determination of Vitamin D MetabolitesUpdate Jiri Adamec

2. Global Proteomics Update Sample Collected • 110 plasma samples were received from IUPUI • 64 have been analyzed including 33 samples from healthy controls, 19 samples from patients with detected polyps, 10 samples from patients clinically diagnosed with colon cancer, 2 samples from patients diagnosed with rectal cancers, and 1 additional sample without diagnosis. • Raw data have been converted into mzXML format for evaluation by the bioinformatics team. • Remaining samples are being prepped for LCMS global proteomic analysis. Lipid removal Methanol/chloroform extraction Denaturation (DTT), alkylation (iodacetamide) and digestion (typsin) Sample clean up C18 spin columns Data Analysis Using Purdue Pipeline Peak List of Regulated Peaks Peptide/Protein ID of Peak List Using LC-MS/MS LCMS evaluation on Agilent XCT

3. Peptide Quantification – Step 1 Peak Extraction (Molecular Feature Extractor) Peak Alignment Data Normalization (normalized to median) Filtering (by relative frequency) Data Analysis (PCA, clustering, Volcano, etc.) Identification of regulated peaks Identification of regulated peaks using LC-MS/MS (Step 2)

4. Protein/Peptide ID – Step 2 • After data analysis and determination of statistical significance, a complete list of peaks of signature peptides will be generated • This list of peaks is subsequently subjected to targeted MS/MS acquisition mode. In our experience, 85% or more of tandem spectra from peaks of interest are obtained in this mode • We employ Spectrum Mill and X!Tandem as primary software packages for database searching

5. Standards of 2H6VitD metabolites Serum/plasma sample Spiking sample with heavy standards HPLC-MRM-MS/MS Absolute quantification Vitamin D Update • New method was developed • Analysis Vitamin D metabolites includes: • 24,25(OH)2VD3 and 24,25(OH)2VD2 • 1,25(OH)2VD3and 1,25(OH)2VD2 • 25OHVD3and 25OHVD2 • VD3 and VD2 • Quantitative capabilities (serum/plasma matix): • Lower limit of quantification: 10 pg/mL • Range: 10 pg/mL to 200 ng/mL • Alternative matrices possible Derivatization with Signal Enhancing Tag

6. Accuracy and Precision

7. Vitamin D Analysis is Done through Intelimmune L.L.C. • Inquiries and Purchase Orders: • IntelimmuneL.L.C. • PO Box 372 • North Webster, IN  46555 • Phone: 800-588-9799 • Phone: 574-834-4080 • Fax:  574-834-7427 • Email: info@intelimmune.com • Send samples to: • Intelimmune, L.L.C. • 3000 Kent Ave. • Room B2-900 • West Lafayette, IN  47906 • phone:  765-412-5850 • Instrument includes • Shimadzu UPLC system • ABI 4000 Qtrap mass spectrometer • Pricing: • Serum/plasma samples • $60.00 ($45.00 for CCE project) per sample for any two forms of vitamin D or its metabolite plus $20 ($15.00 for CCE project) per sample for determination of each additional metabolite. • Note: • Pricing for tissue, body fluids or matrices other than serum/plasma will be dependent upon sample preparation requirements and number of metabolite assays. • Minimum samples: 20