1. Laser capture microdissectionand proteomics : Possibilities and limitation 분 자 유 전 학 실 험 실 석 사 1차 최 용 욱

2. Tissue heterogeneity & enrichment strategies • Tissue heterogeneity : complexity of many tissue samples 1) The biologically relevant cell type is often in the minority 2) Such samples aren't ideally suited to direct analysis • Enrichment strategies for selected cell types 1) Growth of cell line from tissue sample 2) Amplifying a specific cell population 3) primary and established cell lines

3. Fresh and primary cultures by using 2-D PAGE : alteration in the level & number of protein but significant similarity Fresh TCC Primary cultured TCC

4. Laser capture microdissection(LCM) • The limitation of tissue in size and quantity from patient

6. Laser Capture Microdissection(LCM) Steps prepare place capture microdissect extract incubate analyze

7. 2-D PAGE & LCM • 2-D PAGE is the central separation tool in proteomic analysis and its compatibility with LCM <2-D gels of LCM procured cercal epithelium & total cervical tissue>

8. SELDI • Surface-enhanced laser desorption/ionization(SELDI)

9. Protein Purification

10. Epitope mapping

11. Differential Protein Expression ProfilingProtein Purification

12. Assay development & protein-protein interaction Peptide Mapping

13. SELDI analysis of colon carcinogenesis

14. SELDI & LCM • SELDI is sensitive down to the femtomole(10-15)/ attomole(10-18) level ☞ much smaller amounts of material than 2-D PAGE • Profiles have been generated from as few as 25 to 50 cells ☞ Making SELDI ideally suited to the analysis of LCM procured material • Most appropriate for low molecular weight proteins (<20kDa)

15. Antibody technologies and LCM • selection of Ab specific for disease : LCM procured materialforepitope source ☞ Getting the disease specific Ab by comparing normal tissue • Immunoassays : increasingly used to analyze specific proteins in extracts from LCM captured material for biomarker • LCM based approach making use of protein lysate arrays provides a high throughput alternative for marker validation.

16. Concluding remarks • LCM - an important part to play in proteomics research from initial marker discovery to marker validation • Necessary to consider the need for the enrichment LCM – Even though it is too early to assess • Comparative analyses by reducing the level of false positives resulting from differential heterogeneity of cell type between normal and diseased samples • Profiling of protein modification using LCM procured material

17. <별첨1> ProteinChip Reader