Cancer Proteomics by 4IVnaOnS

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									    Cancer Proteomics
: Hepatocellular Carcinoma


         생명과학부
          임승외
          2001.11.21
               Content

• Introduction(Cancer proteomics)
• Hepatocellular carcinomas(HCC)
• 2D-gel analysis
   – 2D-gel electrophoresis
   – Image analysis
   – MALDI-TOF
   – Identification
• Results
               Introduction
• Cancer proteomics
 : Protein-expression analysis
 -> Tumor marker or Drug target !




      Genetic events in colorectal cancer
Proteomics Research Flow Diagram


   1. Sample                Normal Vs. Tumor
     Preparation

                            2D gels, HPLC
   2.Separation

                            Mass spectrometry
   3. Analysis

   4.Identification         Bioinformatics
   (of disease associated
        proteins)
Hepatocellular Carcinoma(HCC)
• HCC 환자 중 90% 이상이 HBV, HCV에 의한
  (각각 70%, 20%) 만성 간질환을 동반
• Human의 경우, 대개 liver cirrhosis를 거쳐
  HCC로 발전함.




• 정상에서 HCC로 발전하기까지 약 20-40년이
  소요.
• Hepatocyte genome의 종양성 돌연변이
     1.    Sample Preparation
• Human cell line
: HepG2, Huh7, Chang

• Human tissue
: Normal tissue Vs. Tumor tissue

-> Total protein
      2. Separation -1st IEF
• IPG(immobilized pH gradients) strip
 -> Sample loading
    2. Separation - 2nd SDS-PAGE
• IEF가 끝난후 strip을 equiliburation 후 2nd SDS-
  PAGE로 Mw대로 분리.
           pH3                                            pH10

            3.7   4.1   5.3             7.1   8.1   8.8

                              2nd SDS-PAGE


                                                    8.8
                  4.1




      MW                                      8.1

                                        7.1



                        5.3

            3.7
        Gel Staining & Image Scan


•   CBB stain
•   Silver stain
•   SYPRO dye stain
•   RI labeling




                      Human liver (SYPRO orange stain)
     3. Analysis- Image Analysis
Imagemaster 2D Elite 3.1
            3. Analysis- MALDI-TOF

•   Matrix Assisted Laser
    Desorption Ionization -
    Time of Flight




     MALDI-TOF 동영상
4. Identification
5. Result




            2001. Electrophoresis

								
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