????? ????????? ???????? ?? ??? ??????? ???????? ????? MS

1
??????????? ??? ???????
??? ???? ????? ????
2
??? ???? ????? ??????????? ??? ????????

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

3
?????? ????-???????
4
????? ????????? ???????? ?? ??? ??????? ????????
????? MS
Co-immunoprecipitation

• ?????? ????????? ????? ????? ????? ?????? ??
  ????? ????? ????? ?'??????'
• ???? (?????????) ?? ???? ????? ???????
• ????? ?????? ???? ????????, ??????? ??????? ????
  ?????? ??? ??? ???????, ???? ???????? ??????
• ???????? ?????? ???? ??? ?????? ???????? ???????
  ????? ?? ??????
• ???????? ???????? ???? ?????? ????? MS

5
Co-immunoprecipitation
6
Mass Spectrometry

• ????? ??????? ?????? ?? ???? ????? ????
• ???? ??? ?????
• (Peptide Mass Fingerprinting) PMF
• ?????? ?????????? ???? ??? ???? ????? ?? ??? ????
• ?????? (?? ????????) ????? ?"? ????? ??????????
  ???????? ??????? ??????? ?????
• ???????? ?????? ??'? ??? ??????? ??? ???
• ?? ????? ???? ????? ??????? ????? ???? ??? -
  '????? ????'
• ?????? ????? ?? ?? ???????? ?????? ?????? in
  silico ????, ??????? ?????? ??????

7
????? ?????? Epitope Tagging

• ????? ?? Co-immunoprecipitation
• ?????? ??????? ????? ?? ????? ???? ??????
• ??? ????? ??? ???? ?-cDNA ????? fusion protein
  ????? ???? ??? ????? ????????? ????????
• ?????? ??? ???? ????? ????? ?????? ??? ?????
  ?????? ????? - ???? ??????? ?????? ?????? ?? ???
  ???? ?????

?????
8
????? ????????? ???????? ?? ??? ??????? ????????
????? MS ???????

• ????? ????? ?????? ??????? ?????? ???????????
• ????? ??????????? ?? ????????? ????? ??? ??
  ??????????? ??? ????? ???????
• ???? ????? ??? ??????? ?? ???? ???????, ???? ???
  ??? ????? ??? ?????? ????? ?? ??????

9
????? ????????? ???????? ?? ??? ??????? ????????
????? MS ???????

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

10
??? ??? ??????? ???? ???
11
?????? ??????
TAP Tandem Affinity purification (Gavin, A.C.
et al. Functional organization of the yeast
proteome by systematic analysis of protein
complexes. Nature 415, 141-147 (2002))

• epitope tagging ?? ??? ???? ????? ???????
• ???????? ???? ????? ????? ?????
• ??????? ????? ?????????? ????? ???? ???? ???
  ?????
• ???????? ???? ????? ???
• ?? ??????? ?????? ????? ????? ?? ????????

12
TAP Tandem Affinity purification
?????????
????? ?? ??????
13
?????? ??????
HMS-PCI High-Throughput Mass-Spectrometric
Protein Complex Identification (Ho, Y. et al.
Systematic identification of protein complex in
Saccharomyces cerevisiae by mass spectrometry.
Nature 415, 180-183 (2002))

• ??? ???? ?? epitope tagging
• ????? ??????? ????? ???? ????? ????? ??? ??? -
  inducible overexpression
• ?? ?????? ????? ?? ???? ??????? ????? ??????? ???
  ????? ??????

14
TAP ?- HMS-PCI - ??????
TAP
HMS-PCI
False positives
???? (??? ????)
???? (?? ???, ????? ???)
False negatives
???? (?? ????? ????? ??????)
???? (????? ???????)
15
Protein Chips

• SELDI protein chips
• ???? ????? ?????? ??????, ?????? ?? ?????? ?????,
  ????? ???? ??????? ??? ?????????
• ?????? ???? ???? ?? ????
• ???????? ??????????? ??????? ?? ?????? ????? MS
  ????? SELDI
• (Surface Enhanced Laser Desorption/Ionization)

• Protein Chips
• proteome scanning
• ??? DNA chip, ?? ????? ???????
• ???? ????? ?????? ??? ???????? ???????? ??
  ???????? ?? ???? ?????
• ?????? ???? ????? ?????, ?????? ?? ??? ???????
  ??? ????

16
???? ???? ????? ?? ????
17
Yeast two-hybrid assay

• ??? ??????? ??????? ?fusion proteins
  (hybrids) ??????
• ??? ???????? (?-'??????') ????? ????? ????? DNA
  ?????? ????? (??"? Gal4)
• ???? (?-'??') ????? ????? ?????? ????? ?? ????
  ?????, ?????? ?????? ????? DNA

18
Yeast two-hybrid assay

• ????????? ??????? ?? ???? ?-DNA ???????? ???????
  ??????? ?????? ?????? ???
• ??????? ??? ??????? ?????? ????
• ????? ?????? ???????? ???? ??? ?? ???? ??????
  ?????? ?????? ???? ??? ?????? ???
• ?? ?????????? ??? ???????? ???? ?????? ?????
  ????? ????, ???? ?????? ?? ?- mRNA

19
Yeast two-hybrid assay
20
Yeast two-hybrid assay
??
AD
?????
DBD
?? ?????
21
Yeast two-hybrid assay ???????

• ???? ????? in vivo
• ?? ????? ?????? ??? ?????? ??????
• ?? ????? ???? ?????? ?????? ?? ????????

22
Yeast two-hybrid assay ???????

• ?????? ?-fusion protein ???? ?????? ????????????
  ??????? ??????????? ?????? ????
• ?? 2 ??????? ?????? ?? ????? - ?? ?????? ?????
  ??????? ?????????????
• ??????????? ?????? ?????? ??????? ???? ?? ????
  ??????? ??????
• ???? ????? ??????? ??? ??? ???? ??????????

23
?????? ????????
24
Correlated mRNA expression (Synexpression)

• ?????? ?-mRNA ?????? ??? ???? ????? ?????? ?????
  DNA chips
• ???? ?????? ????? ????? ????, ??????? ????? ?????
  clustering
• ???? ????? cluster ?????? ??????? ?????? ?????
  ??? ?????? ??????????? ??????

25
Synexpression???????

• ???? ?????? in vivo
• ???? ???? ???? ????? ??????????? ???? ?? ????
  ????
• ???? ????? ????

26
Synexpression ???????

• ????? ???? ????? ?????? ????? ?????? ????? ??
  ?????????? ?????
• ???? ????? ?????? ??????? ?????? clustering ????
  ??????? ?? ???????

27
Genetic Interactions(Synthetic lethality)

• ??? ??????? ?? ??????? ????? ?? ??????? ????
  ????? ?????? ?? synthetically lethal
• ??????? ???? ???? ??? ?????? ???????, ???????
  ???? ?????? ?????????? ??????
• ?????? ???? ?????? ?? ?????? ??? ??????
  ???????????
• ?????? ?????? ?????? ??????????? ??????

28
Genetic Interactions(Synthetic lethality)
LIVES
LIVES
LIVES
DEAD
29
Synthetic lethality ???????

• ???? ?????? in vivo
• ????? ????? ?? ?? ????? ??? ????? ????? ???????
  ?? ????? ????????

30
Synthetic lethality ???????

• ????? ???? ????? ?????? ????? ?????? ????? ??
  ?????????? ?????
• ??? ???? ?? ????? ????? ????????, ???? ??? ??????
  ??? ?? ????? ????????

31
Phage display

• ???? DNA ????????? ??????? ????? ?? ?????? ?????
  ?? ?????????'??
• ?? ?? ??? ???'?? ??????? ?? ?????? fusion
  proteins ?? ??????? ???????
• ???'?? ??? ?????? ?????, ???? ??? ?????? ??? ???
  ?-DNA ?????? ????'
• ?????? ?? ????'?? ?? ???? ????? ????? ?????
  ??????
• ???'?? ?? ??????? ??????? ?????? ??????? ??????
  ???? ?????
• ?? ?????? ???? ????? ?????? ???? ???? ?????

32
Phage display
33
Phage display ???????

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

34
Phage display ???????

• ?????? ?-fusion protein ???? ?????? ????????????
  ??????? ??????????? ?????? ????
• ???? ?? ????? ?? ????? ????????? ??? ???????
  ??????? ???

35
In Silico Predictions Through Genome Analysis

• ?????? ????? ?????? ????? ????? ???? ?????
  ??????????? ??? ???????
• ???????? ????? ?????? - ???????????
• ??????? ?? ?????? ????? ?????? - ?????? ????????
  ????
• ??????? ???????? ????? ??? ???? ?????? fusion
  protein ????? ????

36
In Silico Predictions ???????

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

37
In Silico Predictions ???????

• ????? ?? ????? ???? ????? ????????? ????? ??????
  ?? ?????????? ?????
• ?????? ????? ????? ???????????, ?????? ????
  ?????? ???????????? ???? ??????
• ??????? ?????? ????? ???????????

38
????? ???? - ?????

• ????? ??????? ?????????? ?????
• Yeast two-hybrid assay
• MS of purified complexes
• Protein chips
• Phage display
• ????? ??????
• Synexpression
• Synthetic lethality
• In silico predictions

39
????? ???? - ?????

• In vivo
• Yeast two-hybrid assay
• Synexpression
• Synthetic lethality
• In vitro
• MS of purified complexes
• Protein chips
• Phage display
• In silico
• In silico predictions

40
????? ???? - ?????

• Top-down - proteomic
• Yeast two-hybrid assay
• MS of purified complexes
• Protein chips
• Bottom-up - genomic
• Phage display
• Synexpression
• Synthetic lethality
• In silico predictions

41
????? ????!!!
42
Comparative assesment of large-scale data sets of
protein-protein interactions
?????? ??? ??????

• Von Mering C, Krause R, Snel B, Cornell M, Oliver
  SG, Fields S, Bork P (Germany)
• (Nature 2002)

43
?????? ??????? ??? ??????

• ?- 80,000 ??????????? ????? ?? ?? (2002) ??????
  ?- high throughput ??????
• ?? 2,400 ??? ?????? ?? ???? ????? ???!
• ?????? ??????? ???
• ?????? ??? ?? ????? ?????? (?? ????? ??
  ?????????)
• ?????? ????? false positives ????
• ???? ?????? ?? ???? ?????? ??????????? ??????
  ???????? false negatives

44
??????? ????? ?????

• ?????? ???????? ?? ????????? ??????? ???? ?????
  ????? ???? ?? ??????? ?????????
• ????? ????? ???? ???? ????? ????
• Yeast two-hybrid ?? ???? ????? ???? ??? ???????
  ??????? ?????? ??????
• ????? ????? ??????? ???? ?????? ?????? ????

45
??????? ??????? ??? ??????
Glycine decarboxylase

• ??????? ??????? ?? 4 ??????? ????? ???? ?????
  ????? ????? ??? ??????
• ?? ??? ?? ???? ????? ?"? MS purifications
• ????? ?? ??? ???? ????? ?????? ?????? ?? ??? ????
• Synexpression ?- in silico predictions ????
  ??????????? ??? 3 ???? 4 ???????? ????????

46
??????? ??????? ??? ??????
????? ????? PPH3

• protein phosphatase ??? ???? ???? ?? ??????
• Synexpression ?- in silico predictions ?? ??????
  ?? ????????? ???????? ?????
• ?-4 ?????? MS purifications ???? ?????? ?????
  ??????? ?????? ???? ????? ??????? ?????? ??
  ??????? ??????

47
??????? ??????? ??? ??????
48
????? ?????

• ????? ?????? ????? ?????? ?? ??????????? ??????
  ??? ??????? (???? ?-10,000 ???????????)
• ????? ?????? ?? ???? ??????? ???? ?? ??? ?????
  ??
• ???? ??????????? ????? ?????? ????? ??? ??????
  ????? ??????? (?? ????? ???? ?????
  false positive)
• ???? ?????? ????? ?? ????? ?? ???????? ????????
  ????? (?? ????? ???? ????? false
  negative)

49
????? ????? tradeoff????? ??????

• ?-MS of purified complexes ???? ?????? ?? ??????
  ?? ????? ??????? ??? ?????? ??? ??? ??????
  ????????
• ?? ??????? ?? ????? ?-2 ?-6.8 ?-HMS-PCI
  ??-12.5 ?-27.8 ?-TAP
• ?????, ?? ???? ?? ????? ????? ?????? ?? ??????

50
????? ????? tradeoff????? ???????

• ?-synexpression
• ????? ??????? ???? clustering ?? ????
• ?-in silico predictions
• ?? ?? ???? ????? ????? ????? ??????????? ???
  ????

51
????? ?????
52
????? ?????
53
?????? ?? ????? ?-High Througput ??????? ??????
???? ????? ????? ???????????
54
?????? ?? ????? ?-High Througput ??? ???? ??
???? ????? ???? ??????????? ??????? ????? ???
55
????? ????? ?? ??????

• ????? ????? (Von Mering et al) ??????? ????? ????
  ??? ??????? ???????? ?????? ?-High Throughput
  ?????? ?? false positives
• ????? ?? ?????? ?? ?? ?????? ???? ??? ???????????
  ??? ??????? ???????? ????????? ????? ?? ?????,
  ??? ??? ??????? ??????? ??????? ????? ???

56
??? ???? ?? ??? ??????

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

57
??? ??? ??????????? ????? ???? ????? ?????

• ???????????? ?????? ?? ??? ???? ????? ??? ??
  ????? true positives
• ?? 20 ???? ???? ???? ????? ?? ????? ??? ??? ?????
  ?????? ?????????
• ??????? ????? ???????????? ??? ??????? ???? ?????
  ???? ???????? ????? ????? ???
• ???? ????? ??? ?????? ????
• ?-10,000 ??????????? ?????? ???? ????
• ????? ?????? ??? ?-30,000 ??????????? ????

58
A comprehensive analysis of protein-protein
interactions in Saccharomyces cerevisiaeUetz P,
Giot L et al (USA)Nature 2000 403623-627
??????? ??????? ??????

• A combined experimental and computational
  strategy to define protein interaction networks
  for peptide recognition modules
• Tong AH et al (Canada,USA,Italy)
• Science 2002 295321-324

59
???? 1 analysis yeast two-hybrid
???? ?? 6000 ?????? ????? ????????? ??? ???
????? ?? ??? ?- ORFs ?? ???? ???? (????? ?? Gal4
AD) ???? ??? ?????? ?????? ?????? ?????
???????????
?????? ?-screen ?? ????? ?? ??? Pcf11 (????? ??
Gal4 DBD) ???? ??? ???????? ?????? ??????
????? ???? ??? ?????? ?? HIS3 ??? ??????
60
??????? ????????

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

???????? ????? ?? ??????? ?????? ???? ???, ??
??????? ??????????? ????????? ??????? ?????????
????????? ??? ????? ??????? ??????? ?? ????
?????? ??????????? ?????? ???? APG13 ????? ??????
?????????
61
??????? ????????

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

Cdc28 ??? ???? ?????? ??????? ?????? ????? ??
????? ??? ???? ?? ????? ???? ?????? ?? ???
???????? Clbs ??? ????? ?????? ??????????? ???
?? ?-Clbs ???? Cks1, ????? ?????? ?? ?? ??????
????? ?????? ???
62
??????? ????????

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

Msh5 ???? ??????? ?? crossovers ??? ??????????
?????? Mre11 ??? ??-synaptonemal complex ??????
??????DSBs (double stranded
breaks) Tid3 ???? ?????? ?-spindle pole body,
???? ?????????? ?? Dmc1 Dmc1 ???? ?????? ?-
synaptonemal complex ??? ????? ???????
?????????? ??? Msh5 ???? Tid3 ??? ???? Mre11, ??
????? ?-Msh5 ???? ??? DSBs ?-crossovers
63
???? 2 phage display yeast two-hybrid

1. ????? ?????? ??????? ???????? ?? ??? phage
   display ?????? ??? ????????? ????? ???? ??? ????
   ?????
2. ????? ??? ??????????? ?????? ?? ??? ???? ???
   ???????? ??????? ?? ??? ?????????
3. ????? ??? ????? ??? ?? ???????? ???????? ?? ???
   yeast two-hybrid screen
4. ????? ?????? ??? ??? ????? ????????????, ??????
   ?????????? ????????? ?? ??????????? ???

64
????? SH3

• ????? ?????? ??????? ???????? ?? ??? phage
  display ?????? ??? ????????? ????? ???? ??? ????
  ?????

65
????? SH3

1. ????? ??? ??????????? ?????? ?? ??? ???? ???
   ???????? ??????? ?? ??? ?????????

• ?? ?? ???? ????????? ?????? ???? ??????? PSSM
• ?? ???????? ?????? ?? ???? ????????? ????? ???
  ?-PSSM, ??-20 ?????? ?? ?????? ??? ???? ?????
  ???? ????????????

66
????? SH3

1. ????? ??? ????? ??? ?? ???????? ???????? ?? ???
   yeast two-hybrid screen

67
????? SH3

1. ????? ?????? ??? ??? ????? ????????????, ??????
   ?????????? ????????? ?? ??????????? ???

68
???????? ?? ???? ?????

• phage display ?-yeast two-hybrid ?? ??? ?????
  ???????? ?? ??????? ????? ???? ??????? ?? ?? ??
• Phage display ?????? in vitro ??????? ????????
  ????????? ?????
• Yeast two-hybrid ?????? in vivo ??????? ????????
  ??????
• ????? ????? ?????? ?????? ??? ??? ???? ????
  ???? ?? ???? ?????
• ????? ????? ???? ???? ????????

69
references

• www.biologicalprocedures.com
• Yeast Two-hybrid State of the Art
• Wim van Criekinge and Rudi Beyaert
• European Journal of Biochemistry Volume 270 Issue
  4 Page 570  - February 2003
• Affinity purification-mass spectrometry -
  Powerful tools for the characterization of
  protein complexes Andreas Bauer and Bernhard
  Kuster
•  
• www.pubmedcentral.nih.gov
• Metabolic networks of microbial systems -
  methods for determination of ensemble of
  interacting partners
• Sumana Bhattacharya, Subhra Chakrabarti, Amiya
  Nayak and Sanjoy K Bhattacharya
• von Mering C, Krause R, Snel B, Cornell M, Oliver
  SG, Fields S, Bork P
• Comparative assessment of large-scale data sets
  of protein-protein interactions
• Nature. 2002 417399-403
•  
• Uetz P, Giot L et al
• A comprehensive analysis of protein-protein
  interactions in Saccharomyces cerevisiae
• Nature. 2000 403623-627
•