Quantitative Proteomics of LA Bacterium — Research project

1. Quantitative Proteomics of LA Bacterium—Research project Zhang Xiaoli 2011.06.12

2. 1.Research approaches 2.Research project 3.Experiment routes

3. 1. Research approaches 1.1Stable isotope labeling In vivo (metabolic labeling) Definition: stable isotopes are incorporated into proteins during the growth of a model organism. (1) SILAC(stable isotope labeling with amino acid in cell culture) coupling with 2-LC-MS/MS analysis (2)15N labelled coupling with 2-LC-MS/MS analysis

4. (1) SILAC (stable isotope labeling with amino acid in cell culture)

5. (2)15N labeling coupling with 2-LC-MS/MS analysis

6. Advantages and Disadvantages of metabolic labeling • Advantages: • little measureable deleterious effect on the growth and • development of an organism; • (2) useful for experiments involving smaller model microbes • such as lactic acid bacterium, which requires a defined • medium and a set of controlled conditions to grow; • (3) Completely labeling ——high labeling efficiency • (4) Reduce sample handling errors; • (5) simplifying quantitation; • (6) inexpensive. • Disadvantages: • Only utilize to living cells or tissues

7. 1.2Stable isotope labeling in vitro (chemical labeling) Definition: stable isotopic labels are incorporated into the peptides during peptide preparation — (1)ICAT (isotope coded affinity tags) — (2)iTRAQ(isotope tagging for relative and absolute protein quantitation) — (3)18O-enriched water All of them coupled with 2-D LC/MS-MS analysis

8. (1) ICAT (isotope coded affinity tags)

9. (2) iTRAQ

10. (3) 18O-enriched water

11. Advantages and Disadvantages of chemical labeling • Advantages: • (1) Suitable for all types of samples • (2) Accurate quantification • Disadvantages: • Undesirable by-products • (2) Incomplete labeling due to existence of chemical contaminants • (3) Increases peptide complexity • (4) Pretreatment and digestion processes may lead to differences • in parallel samples • (5) Expensive

12. 1.3 Our employed approaches iTRAQ SILAC ——expensive ——inexpensive

13. 1.4 Utilization Conditions of SILAC ①heavy amino acids have to be essential ones; ②the living cells should be auxotrophic for the labeling amino acids; ③the mass difference between the heavy and the normal amino acids should be at least 4 Da; ④the selection of amino acids should match the enzyme used for protein digestion.

14. 2. Research project 1. Investigating the metabolites (major amino acids ) of LA bacterium. 2. SILAC-coupled 2-D LC-MS/MS analysis of proteomics of LA bacterium with oxidative stress 3. SILAC-coupled 2-D LC-MS/MS analysis of proteomics of LA bacterium with Lactic acid or nisin stress

15. 3. Short-term experiment schedule 1. 2011.06.13-2011.06.26: Determining the minimal chemical defined medium of LA bacterium. 2. 2011.06.27-2011.07.26: Investigating the metabolites (major amino acids ) of LA bacterium. 3. 2011.07.27-2011.08.26: Analyzing proteomics of LA bacterium with lactic acid or nisin stress.

16. Thanks for your attention!