1. The CCE 5 th Annual Retreat Global Proteomics & Determination of Vitamin D Metabolites Update Jiri Adamec

2. Sample Collected Denaturation (DTT), alkylation (iodacetamide) and digestion (typsin) Sample clean up C18 spin columns LCMS evaluation on Agilent XCT Data Analysis Using Purdue Pipeline Lipid removal Methanol/chloroform extraction Peak List of Regulated Peaks Peptide/Protein ID of Peak List Using LC-MS/MS 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. Global Proteomics Update

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

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. New method was developed Analysis Vitamin D metabolites includes: –24,25(OH) 2 VD 3 and 24,25(OH) 2 VD 2 –1,25(OH) 2 VD 3 and 1,25(OH) 2 VD 2 –25OHVD 3 and 25OHVD 2 –VD 3 and VD 2 Quantitative capabilities (serum/plasma matix): –Lower limit of quantification: 10 pg/mL –Range: 10 pg/mL to 200 ng/mL –Alternative matrices possible Spiking sample with heavy standards HPLC-MRM-MS/MS Serum/plasma sample Standards of 2 H 6 VitD metabolites Absolute quantification Derivatization with Signal Enhancing Tag Vitamin D Update

6. Accuracy and Precision 24,25(OH) 2 VD 3 1,25(OH) 2 VD 3 25OHVD 3 VD 3 [Serum]meanPrecisionAccuracymeanPrecisionAccuracymeanPrecisionAccuracymeanPrecisionAccuracy pg/mL % CV%REpg/mL% CV%REpg/mL% CV%REpg/mL% CV%RE 10 3.4 102.2-1.8101.2-0.8101.8-0.1 20187.8-8.7213.53.2202.81.5203.0-0.6 100 1.1-0.33813.3-6.31015.71.11023.42.2 200 20.01014.40.71982.5-1.32004.10.0 400 4.60.02104.854087.61.93986.6-0.6 10009904.73755.1-6.310118.81.110055.60.5 2000210510.55.39994.5-0.121300.76.5191013.3-4.5 400040813.43.032855.6-4.440421.31.041112.42.8 840085103.71.376526.2-4.383366.0-0.877605.5-7.6 10000102933.62.984183.00.298974.9105773.75.8 20000195867.1-2.1101734.41.7207665.43.8215151.27.6 80000789006.1-1.4176502.63.8764883.6-4.47527310.3-5.9 170000179880125.8213562.66.81811677.96.6179800-5.8 20000020929010.64.6ND 1997205.1-0.12108204.75.4

7. 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 Vitamin D Analysis is Done through Intelimmune L.L.C. Inquiries and Purchase Orders: Intelimmune L.L.C. PO Box 372 North Webster, IN 46555 Phone: 800-588-9799 Phone: 574-834-4080 Fax: 574-834-7427 Email: info@intelimmune.cominfo@intelimmune.com Send samples to: Intelimmune, L.L.C. 3000 Kent Ave. Room B2-900 West Lafayette, IN 47906 phone: 765-412-5850