2. Identification of regulatory proteins from human cells using 2D-GE and LC-MS/MS Victor Paromov Christian Muenyi William L. Stone

3. Proteomics techniques used to identify proteins Obtain cell lysates Run two-dimensional gel electrophoresis (2D-GE) for each sample (in triplicates) Compare the gels and identify over-expressed and/or under-expressed proteins compared to protein spots from control treatment Identify those proteins (for each spot) by: –Cutting out the gel spot and trypsinizing the protein –Run LS/MS/MS on LTQ XL –Identify proteins by matching the tryptic peptides to theoretical fragments (Protein Database search)

4. S #1 S #2 Vehicle2.5 mM CEES Proteomics Study of CEES toxicity in human keratinocytes: EpiDerm tissues were exposed to vehicle or 2.5 mM CEES for 18 h. Cell lysates were separated by 2D gel electrophoresis, Coomassie blue-stained and photographed (three gels per sample). Average differences in protein expression were quantified with Dymension-2 Software. The proteins differentially expressed after CEES exposure are marked with red circles. Protein spots were excised from the gel, destained, trypsinized, and subjected to LC/MS/MS analysis. 2DGE Example comparing Vehicle- vs CEES-treated human keratinocytes

5. The protein identified belong to the 14-3-3 family of regulatory proteins. The 14-3-3 proteins (zeta/delta, theta, and sigma) have same MW = 28 kDa and pI = 4.5. Results for the Protein Database search for a protein spot #543. ← chromatogram of LC/MS data ← Three homologous proteins identified with highest probability.

6. Included in the report is the amino acid residue coverage for 14-3-3 protein Sigma. Top panel: protein sequence with identified peptides (shown in red). Bottom panel: The list of identified tryptic peptides.

7. The list of B and Y ions as they match against the experimental data (matched B ions – red, matched Y ions – blue). MS/MS fragment ion matches for a peptide fragment of 14-3-3 protein Sigma (YLAEVATGDDK) The mass errors of the measurement. MS/MS data of fragment ions that matched theoretical data of database

8. Conclusion: LC/MS/MS analysis and UniProt database search with “rigorous” filtering of the search results allow identification of three 14-3-3 regulatory proteins (zeta, theta, and sigma).