The analysis of microbial proteomes : Strategies and data exploitation

1. The analysis of microbial proteomes: Strategies and data exploitation Electrophoresis – 2000 – 21-1178 생명과학부 분자유전학실험실 유영희

2. contents • Introduction • Overview of approaches and techniques available for the analysis of microbial proteomes • Analysis of specific subsets of the microbial proteome • Global analysis of microbial proteomes • Application of microbial proteomic information to other studies • Concluding remarks • References

3. 1. introduction • Microbes will serve as important test-beds for new functional genomic technologies • Microbes are ideally suited for these purposes for several reasons ◈ relatively simple and experimentally tractable ◈ certain microbes have been extensively characterized ( their biochemistry, genetics and physiology ) ◈ microbes are important in their own right and hence merit particular attention in functional genomic studies.

4. 1. introduction • This review … ◈ describes the advantages and disadvantages of the various strategies available for the analysis of microbial proteomes ◈ highlights the ways in which such information can be exploited by both microbiologists and other investigators

5. 2. Overview of approaches and techniques(available for the analysis of microbial proteomes )

6. 2. Overview of approaches and techniques(available for the analysis of microbial proteomes ) • Gel-based… 1. 1D or 2D system ◈ 장점 : high-resolution fractionation of most proteins ◈ 단점 : - cannot adequately resolve with extreme physicochemical properties ( very hydrophobic proteins or very high or low pI) - relatively labour-intensive - a low throughput

7. 2. Overview of approaches and techniques(available for the analysis of microbial proteomes ) • Gel-based… 2. Affinity probe procedure ◈ the use of a tagged protein containing a binding domain to pull out other associated proteins prior to their separation by electrophoresis.

8. 2. Overview of approaches and techniques(available for the analysis of microbial proteomes ) • Non-gel based… 1. two-hybrid method : to detect protein-protein interaction. ◈ 장점 - increase the versatility and throughput - allowing a more comprehensive mapping of protein- protein interaction

9. 2. Overview of approaches and techniques(available for the analysis of microbial proteomes ) • Non-gel based… 2. CIEF-ESI-MS (capillary isoelectric focusing electrophoresis/electrospray ionisation MS) : first dimension은 protein net charge에 의해서 liquid phases에서 separate. focusing후 electrospray ionisation mass spectrometer 에서 mass analysis ◈ 장점 : sensitivity

10. 2. Overview of approaches and techniques(available for the analysis of microbial proteomes ) • Non-gel based… 3. SELDI-MS (surface-enhanced laser desorption/ionisation mass spectrometry) : often referred to as protein chip most useful for identifying proteins that show large differences in expression 4. ICATs (isotope-coded affinity tags)

11. 3. Analysis of specific subsets of the microbial proteome 3.1 Stimulons and regulons - stimulons : the set of proteins that change in response to a stimulus. - stimulons ≥ regulons (most stimulons consist of multiple regulons) ◈ most common objectives in proteomics is.. - to define proteins that are up- or down-regulated when a cell is exposed to a particular stimulus or environment

12. 3. Analysis of specific subsets of the microbial proteome • Example : PhoPQ signal transduction system of Salmonella - constitutive mutant termed pho-24 ( external stimulus의 유무와 상관없이 항상 switched on ) - wild type과 pho-24의 expression pattern을 비교 33KD 위치의 spot이 induce peptide mass fingerprinting identified GAPDH(glyceradehyde 3-phosphate dehydrogenase) thereby suggesting that this glycolytic enzyme is positively regulated by the system

13. 3. Analysis of specific subsets of the microbial proteome 3.2 analysis of subcellular compartments and macromolecular complexes ◈ microbial cells은 복잡한 macromolecular architecture를 가진다. ◈ proteomics should therefore be an efficient route for the identification of new components associated with such structure.

14. 3. Analysis of specific subsets of the microbial proteome • Example - by using electrospray ionization to introduce E.coli ribosomes into a mass spectrometer and to dissociate them possible to investigate the dynamics and state of folding of specific protein and their interaction with other components. (PNAS-1998-95-7391)

15. 3. Analysis of specific subsets of the microbial proteome 3.3 analysis of other microbial proteomic subsets ◈ 이론적으로 … - interesting protein의 subsets은 proteomics에 의해서 characterise 될 수 있다. ◈ 실질적으로… - whole cell extracts에서 어떻게 purify 하며, 어느 정도 detection 할 수 있는지가 중요한 factor 이다.

16. 4. Global analysis of microbial proteomes • the ultimate goal is to produce a database that describes all of the proteins expressed in a cell under a specified set of conditions • Example - the gene-protein index of E.coli (Neidhardt and colleagues) the data available for the proteome of Heamophilus influenzae is being applied in studies on the identification of new targets for therapeutic drugs

17. 5. Application of microbial proteomic information to other studies • Proteomic information from microbes는 microbiologists뿐 아니라 다른 researcher에게도 유용한 자료. • Eukaryotic microbes는 animals/plants의 protein과 상대적으로 많은 homologues ( central metabolic process ) - 그러므로 microbial gene products의 연구는 human homologues proteins의 function의 연구에 도움 • Proteomic information from microbes can also be of more direct use in the biochemical characterization of certain enzymes

18. 5. Application of microbial proteomic information to other studies • Example : existing proteomic information can be used to define the recognition sites of novel protease

21. 6. Concluding remarks • As technology develops… : microbiologists will eventually have a complete description of all the key processes being carried out in a cell under a specific set of conditions • At the applied level, proteomic data will find widespread use : in the identification and characterization of targets for therapeutic drugs and the metabolic engineering of microbes