2 Dimensional Gel Electrophoresis

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2
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  ???, ??????????? ????, ?????????? ??? ???????,
  ?????? ?????? ??????????.

3
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• ??????..

4
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• ?????, ????? ??????

5
????? ???????????

• 2 Dimensional Gel Electrophoresis
• ????? ????????
• Mass Spectrometry (MS)
• ?????????? ?????
• ?????????? ??????

6
2 Dimensional Gel Electrophoresis

• ???? ?????? ???? ?? ?? ???????.
• ????? ???????? ????? ??????
• ????? ????? ????? ??? ?-pI
• ????? ????? ??? ??? ???? ????????
• ?????
• ????? ?????? ??????

7
(No Transcript)
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A Sampling Of The Yeast Proteome

• B. Futcher, G. I. Latter, P. Monardo, C. S.
  McLaughlin, J. I. Garrels (1999)
• ????? ??????? ???? S.cerevisiae ?"? 2DE ??????
  ???? ????? ?? ??????? ?????? ???????? ??'?.
• ????? ?????
• ????? ??? ?????? ????? ???? ?? ?????? ?????? ??'?
  ?????? ?????.
• ????? ??? ??? ?????? ????????, ??????
• mRNA ???? ?- codon bias

9
?????? ???????

• ???? 1400 ?????? ?????? ?? ????? 2D-Gels
• ????? ???????
• ????? ????? ????????
• ????? ??? ??? ?????? ???????? ??????? mRNA
• ????? ??? ??? ??? codon bias ??????? ????????
• ??????? ??????? ??????? ??????? ????? ??????
• ??????????? ?? ???????

• ????? ?-3 ?????? ?'???, ??? ??? ??? ???????? ????
  ????? ????? ?????? ?? ????? ????.
• ????? ??????? ?????? ??????
• ????? ?-1400 ?????? ????? ??????? ??-1200 ???????

????!
????? ?????? ?????? ????? "???????" ??????
?????, ??????? ?????? ??????? ???? ????????
??????.
10
?????? ???????

• ???? 1400 ?????? ?????? ?? ????? 2D-Gels
• ????? ???????
• ????? ????????
• ????? ??? ??? ?????? ???????? ??????? mRNA
• ????? ??? ??? ??? codon bias ??????? ????????
• ??????? ??????? ??????? ??????? ????? ??????
• ??????????? ?? ???????

• ???? 169 ?????? ???????? 148 ??????? ?????, ?????
  ?"? MS
• ????? ???????? ???? ????? ????? ??????????S) 35)

11
?????? ???????

• ???? 1400 ?????? ?????? ?? ????? 2D-Gels
• ????? ???????
• ????? ????? ????????
• ????? ??? ??? ?????? ???????? ??????? mRNA
• ????? ??? ??? ??? codon bias ??????? ????????
• ??????? ??????? ??????? ??????? ????? ??????
• ??????????? ?? ???????

• ???? mRNA
• SAGE
• ??????????? ?? cRNA ?- chip technology
• ????? ??? ??? ?????? (??????? ?? ?? ??? ???)
• ????? ??????? ???? ?????? ??? ??????? ??? ??????
  ?- mRNA ???? ?????? ??????
• ???? ?????? mRNA ?????, ?????? ???????? ???????
  ??? ????? x,10x ????? ?????? ?????? ???? ?????.

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rp0.76 ( plt0.00001 ) rs0.74 ( plt0.0001 )
13
?????? ???????

• Codon bias- ????? ??????? ???????? ?? ???
• ??????? ????? ???????? ????? ?"? ?-mRNA
• ???? ?? ???????? ?????? ?? C.b ????.
• ????? ?????? ??? ??? ?-C.b
• ???? ???? ?????? ?? ????
• ??????? ???? ??????.
• ???? ??? C.b ????? CAI
• ????? ????? ???? ????????
• ????? ?????

• ???? 1400 ?????? ?????? ?? ????? 2D-Gels
• ????? ???????
• ????? ????? ????????
• ????? ??? ??? ?????? ???????? ??????? mRNA
• ????? ??? ??? ??? codon bias ??????? ????????
• ??????? ??????? ??????? ??????? ????? ??????
• ??????????? ?? ???????

rs0.80 ( plt0.0001 )
14
?????? ???????

• ???? 1400 ?????? ?????? ?? ????? 2D-Gels
• ????? ???????
• ????? ????? ????????
• ????? ??? ??? ?????? ???????? ??????? mRNA
• ????? ??? ??? ??? codon bias ??????? ????????
• ??????? ??????? ??????? ??????? ????? ??????
• ??????????? ?? ???????

Glucose
Ethanol
15
?????? ???????

• ???? 1400 ?????? ?????? ?? ????? 2D-Gels
• ????? ???????
• ????? ????? ????????
• ????? ??? ??? ?????? ???????? ??????? mRNA
• ????? ??? ??? ??? codon bias ??????? ????????
• ??????? ??????? ??????? ??????? ????? ??????
• ??????????? ?? ???????

• ????? ??????? ????? ??????????? ?"? ?????
  ?????????? (P32), ???? ?- 2D-Gel ??????
• ?????? ?- 300 ?????? ???????? 150-200 ???????
• ????? ??? ???????? ???? ?"? ?????? ??'??? ??
  ????? ?????????? S35

16
????? ?????

• ??? ??????...
• ????? 1200 ??????? ??? ???? ?? ??? ????????? ???
  ????
• ????? ???? 148 ???????
• ???? ???? ?? ???? ???????? ?-2 ?????? ??? 100
  ????' ???
• ?- 4000 ????' ????? ???? ?? ????' mRNA
• ??? ??? ?????? ??? ???? ?????? ???? ??? ?- C.b ??
  ???? ??????? ???? ??????.
• ??? ??? ???? ?????? ???? ???? ?- mRNA

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?????????? ?????? Mass Spectrometer

• Mass Spectrometer - ????? ?????? ??? ????????
  ?????? ?? ?? ???? ??? ????? ????.
• ????? ???????? ????? ?????? ????? (???????)
  ????????
• ????? ???????? ??????
• ????? ???????? ??????? ???? ????
• "?????" ?? ?????? ???? ???? ???????
• ????? ???? ?????? ?????? ???????? ????? ?????????
  ????? ????? ??????

18
????? ????????

• Matrix Assisted Laser Desorption Ionisation
  (MALDI)
• Electrospray Ionisation (ESI)

19
????? ????? ????????

• Time Of Flight (Tof)
• Quadropole Mass Filter
• Ion Trap
• MS/MS

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ESI Tof
MALDI Tof
21
??? ??????? ?? Mass Spectrometer
22
????? ???????? ????? ???? ??????
????? ??????? ?- Mascot DB
????? ??????? ?????
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(No Transcript)
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• ??????? ?????? ??? ?????? ??????, ???? ??????
  ????? ??????? ?????? ????? ??? ???? ???????.
• ????? ?????? ????????? ?? ??????? ???? ????? ????
  ?????? ???????? ??????? ????????

25
Functional Organization Of The Yeast Proteome By
Systematic Analysis Of Protein Complexes

• Anne-Claude Gavin et al (2002)
• ?????
• ????? ????????? ???????? ???? S.cerevisiae ?????
  TAP ?-MS ????? ??? ????? ??? ??????????? ??????
  ???? ??????? ????? ?????? ???? ?????? ??????
  ?????.

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???? ??????
27
??????, ???????? ??????? ?????

• ???? 589 ???????? ???? ???, 78 ???? ???????????
  ?? ??????? ?????.
• ?? ???? 22 ???????? ??????? ?
• ?? ???????? ??????????
• ?????? ??????????? ?? ??????, ?????? ?? ??? ?????
  ??????????? ???????
• ??? ????? ???????????? ???'...
• ????? ??? ??????? ?????
• ?????? ??? ???????? ???????? ????? ????.
• ??????? ??????? ?-20 ?? ???? ????????? ?????
  ?????? ??????? ( ??????? ?????? )

28
?????? ???????? ??????????

• 589 ???????? ???????? ????? ??????????
• 245 ????? ?-98 ????????? ?????? ?-YPD
• 242 ????? ?-134 ????????? ?????
• 102 ?? ????? ??? ???????
• ???? ?????????? ?? ?-2 ??? 83 ???????
• ????????? ????? ?-9 ???????? ????? ?? ???????
  ?????
• ???? 304 ??????? ??? ??? ???? ?????? ?????
  ??????? ?-231, ??-113 ?????? ???? ???? ??? ???

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????? ??? ??????????? ?? ????????
30
????? ????? ?? ????????? ????? ?????

• ??????? ????? ?????????? ????? ?????? ??????????
  ??????? ???????? ?????????? ??????
• "??????? ?????????"
• ????? ???? ?? ???? ????????? ?????? ???????????
  ???? ????? ???? ?? ???? ?????? ????????? ??? ???
  ????? ????? ???? ????

31
????? ?????

• ????? 1739 ???? ????? 1143 ?????????? ????
• ?????? 232 ????????? ?????
• ????? ??????? ????? ????? ???? 344 ???????
• ????? ????? ?????? ?????? ????????? ??????? ?????
  ?????, ?????? ??????? ???????? ????? ???????
  ????????? ?????? ????

32
?????????? ??????

• ??? ???? ??????? ?? ???????
• ????? ????? ????? ???????
• ????? ?????? ??????? ???? ???? ?????
• ?????????? ???? ????????? ??????
• markers
• ????? ????? ?- MS ?????????? ???????

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Quantitative Proteomic Analysis Of Myc
Oncoprotein FunctionY.Shiio, S.Donohoe, E.C.Yi,
D.R.Goodlett, R.Aebersold, R.N.Eisenman (2002)

• myc - ?? ????? ????? ???? ??? ???????
• ?????? ????? ???????
• ??????
• ????? ???
• ????? ???
• ?????????
• ????????
• ????? ????????? ??????

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???? ?????

• ?????? ????? ??????? ??? ??? Myc(-) ???? Myc()
• ?????
• ????? ???? ?????
• ????? ????????? ??? ???
• ????? ???????
• ??????????? ?"? cation exchanger
• ????? ?"? LC-MS/MS

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????? ???????? ??????? ??? Myc(-) ?- Myc()

• ??? Myc(-) ?????????? ?????, ?????? ?????
• ??? Myc() ?????????? ?????, ?????? ?????
• ????? ?? 528 ??????? ????? 177 ???? ???? ????? ??
  ???? ??? 2
• ??????? ?????? ????? ????? ?????? ?????? ????????

36
(No Transcript)
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(No Transcript)
38
????? ????

• ????? ????? ???????? ??????? ?????? ???????
• ????? ????? ???????? ??????? ??????? ?????
• ????? ????? ?????????

• ??? ????? ??????? ???? ?? ?????
• ????? ???? ????? ?????? ????, ???????????, ?"?
  ?-ATP

39
?????? ??? ???
Actin stress fibers
Focal adhesions

• ???? Myc() ???? ???????? ???????
  ?????? ??? ???? ?????? ?????? ?????
  ????
• ???? ????? ?????? ?????
  
• ?????????? ????? ?"?????"

Myc(-)
Myc()
40
?????? ?????? ????? mRNA

• ?????? ?????? ????? ??? ??????? ????? ???????
  ????? mRNA ???? Myc()
• ????? ??????? ??????? ??????? ???????? ??? ?????
  ??????? ??"?
• ???? ??????? ??? ??????? ??? ????? ??? ?? Myc
  ???? ??????? ???? ????

41
????? ?????

• ????? ??????? ?????? ??????? ??? ??? Myc(-)
  ????Myc() ???? ??????? ??? ????? ?? ??
• ??????? ??? ?????? ??? ???? ???????? ???????????
  ????? ???????
• ?????? ??? ???????? ???? ??? ?????? ????? ??????
  ?????? ?? ??????? ???? Myc

42
?? ?? ??? ????

• 2 Dimensional Gel Electrophoresis
• ????? ????????
• Mass Spectrometry (MS)
• ????????? ?????
• ?????????? ??????

43
??? ???? ????...

• ????? ????? ???? ?????? ???? ???? (MS)
• ????? ????? ?????? ?????? ??? '????? ??????'
  ??'?
• ????? ?????? ???-??????????? ???? ?? ?????
• ??????? ????????? ??? ????????
• ?????????? ???????? ?????? ??????? ??????
  ??????????? ???? ?????
• ????? ???? ??????
• ???? ???? ???? ????? ??????
• ???????????? ???? ??????? (????? ???)
• ???? ????? ????? ???? ?? "????? ???"

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Going from sequence to consequence is of
course what proteomics is all about. Greg
Petsko C E News, November 26 (2001)
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References

• B. FUTCHER, G. I. LATTER, P. MONARDO, C. S.
  MCLAUGHLIN, AND J. I. GARRELS. A Sampling Of The
  Yeast Proteome. Molecular And Cellular Biology,
  1999, 19, 7357-7368.
• A. C. GAVIN et al. Functional Organization Of The
  Yeast Proteome By Systematic Analysis Of
  Complexes. Nature, 2002, 415, 141-147
• Y. SHIIO, S. DONOHOE, E. C. YI, D. R. GOODLETT,
  R. AEBERSOLD, AND R. N. EISENMAN. Quantitative
  Proteomic Analysis Of Myc Oncoprotein Function,
  EMBO J., 2002, 21, 5088-5096
• M. TYRES, M. MANN. From Genomics To Proteomics,
  Nature, 2003, 422, 193-197
• R. AEBERSOLD, M. MANN. Mass Spectrometry-Based
  Proteomics, Nature, 2003, 422, 198-207