This document discusses several methods for studying protein-protein interactions, including mass spectrometry, yeast-two-hybrid systems, and phage display. It also describes online databases and information extraction techniques for mining literature to identify protein-protein interaction data not currently in databases. The yeast-two-hybrid system involves using bait and prey proteins to activate reporter genes if an interaction occurs, while phage display fuses proteins to bacteriophage for screening interactions. Mass spectrometry can analyze purified protein complexes. Databases like DIP and Suiseki aggregate interaction data, and information extraction applies rules to text to extract interaction information for curation.

1. Protein-protein Interactions

2. Outline
 Introduction
 The Study of Protein-protein Interaction
by Mass Spectrometry
 Yeast-Two-Hybrid
 Phage Display
 Protein-protein Interaction on the Web
 Information Extraction (IE)

3.  Introduction
 The Study of Protein-protein Interaction
by Mass Spectrometry
 Yeast-Two-Hybrid
 Phage Display
 Protein-protein Interaction on the Web
 Information Extraction (IE)

4. Introduction
Nature, 2000, 405:837-846.

5. Introduction
 Intrinsic to every cellular process
 Form the basis of phenomena
-DNA replication and transcription
-metabolism
-signal transduction
-cell cycle control

6.  Introduction
 The Study of Protein-protein
Interaction by Mass Spectrometry
 Yeast-Two-Hybrid
 Phage Display
 Protein-Protein Interaction on the Web
 Information Extraction (IE)

7. The Study of Protein-protein
Interaction by Mass Spectrometry
 A key question about a protein,in
addition to when and where it is
expressed, is with which other
proteins does it interact.
Interaction partners are an
immediate lead into biological
function and can potentially be
exploited for therapeutic
purposes. Creation of a protein–
protein interaction map of the cell
would be of immense value to
understanding the biology of the
cell.
Purification of protein complex
bait

8. The study of Protein-protein
Interaction by Mass Spectrometry
Nature Biotechnology, 1999, 17, 676-682.

9. The Study of Protein-protein
Interaction by Mass Spectrometry
Nature Biotechnology, 1999, 17, 676-682.

10. Generic Strategy to Analyze the Spatial
Organization of Multi-protein Complexes by
Cross-linking and Mass Spectrometry (1/2)
Anal. Chem., 2000,72, 267-275.

11. Generic Strategy to Analyze the Spatial
Organization of Multi-Protein Complexes by
Cross-Linking and Mass Spectrometry (2/2)
Anal. Chem., 2000,72, 267-275.

12.  Introduction
 The Study of Protein-protein Interaction
by Mass Spectrometry
 Yeast-Two-Hybrid
 Phage Display
 Protein-Protein Interaction on the Web
 Information Extraction (IE)

13. Yeast Two-hybrid
 Researchers insert a gene in yeast for a "bait" protein alongside DNA for
half of an "activator" protein. The other half of the activator DNA is then
inserted alongside DNA for random "prey" proteins. The yeast cells are
then grown up and the proteins are allowed to interact. If bait and prey
proteins bind, the two halves of the activator protein be close enough to
work together to turn on another yeast gene that turns the cell blue,
signaling a match.
ILLUSTRATION: C. SLAYDEN

14. Yeast Two-Hybrid System
 Useful in the study of various interactions
 The technology was originally developed during
the late 1980's in the laboratory Dr. Stanley
Fields (see Fields and Song, 1989, Nature).

15. Yeast Two-Hybrid Assay

16. Hybrid proteins
DNA-binding domain
Bait domain Activation domainFish domain
Hybrid 1 Hybrid 2

17. Transcriptional Activation by
Hybrid Protein in Yeast
UAS HIS gene
Transfect yeast cells with genes encoding
hybrid 1 and hybrid 2
Transcription-
initiation complex
//
HIS mRNA

18. Fishing for Proteins that
Interaction with Ras
ras gene
TRP
cDNA from library
LEU
Bait plasmid
Fish plasmid
Transfect into trp, leu, his mutant yeast cells
Select for cells that grow in absence of tryptophan
and leucine
Plate selected cells on medium lacking histidine

19. Fishing for Proteins that
Interaction with Ras
HIS
Ras hybrid
Bait
Fish
Ras-interacting
hybrid

20. Fishing for Proteins that
Interaction with Ras
Colony Formation

21. Fishing for Proteins that
Interaction with Ras
Ras hybrid
Bait
Fish
Noninteracting
hybrid
HIS

22. Fishing for Proteins that
Interaction with Ras
No Colony Formation

23.  Introduction
 The Study of Protein-protein Interaction
by Mass Spectrometry
 Yeast-Two-Hybrid
 Phage Display
 Protein-Protein Interaction on the Web
 Information Extraction (IE)

24. Phage Display
 A method where bacteriophage
particles are made to express either a
peptide or protein of interest fused to a
capsid or coat protein.
 It can be used for peptide epitopes,
peptide ligands, enzyme substrates or
single-chain antibody fragments.

25. Phage Display
Bacterially expressed GST-fusion proteins or domains
Immobilize individually into wells
Wash unbound phage particles
Amplify phage particles
Sequence the cDNA insert

26.  Introduction
 The Study of Protein-protein Interaction
by Mass Spectrometry
 Yeast-Two-Hybrid
 Phage Display
 Protein-Protein Interaction on the
Web
 Information Extraction (IE)

27. Protein-protein Interaction on
the Web
 DIP
 Suiseki
 Useful database

28. http://dip.doe-mbi.ucla.edu

29. DIP NEWS
January 24th, 2002
 Another release of the complete dataset. It
contains more than 11,000 individual protein-
protein interactions described in 1,800
research papers. Please, note that this is,
most probably, the last dataset released in
the old format as we anticipate the next one
(to be released witin ~3-4 months; we'll try to
prepare at least 3 updates a year) will be
XML formatted.

30. Database of Interacting
Proteins

35. Suiseki(1/3)
•DNA replication
•The Immune system
•The E2F transcription factor
•The talin/viniculin/actin system
is a system for the
extraction of protein-
protein interactions
from large
collections of
scientific text
http://www.pdg.cnb.uam.es/suiseki/

36. Suiseki (2/3)

37. Suiseki (3/3)
 Regulate
 Activate

40.  Introduction
 The Study of Protein-protein Interaction
by Mass Spectrometry
 Yeast-Two-Hybrid
 Phage Display
 Protein-protein Interaction on the Web
 Information Extraction (IE)

41. Information Extraction (IE)
 A vast amount of data on protein-
protein interactions residues in the
published literature, which never been
entered into databases.
 IE have been applied to gaining
information on protein-protein
interactions.

42. Mining Literature for Protein-
protein Interactions

43. Mining Literature for Protein-
protein Interactions

44. Mining Literature for Protein-
protein Interactions

47. Detection of Protein Names
 Protein Names are not standardized
and are often used in different forms in
free text style.

48. Creation of the Protein Name List
Comp Funct Genom 2001, 2, 196-206

49. Extraction of the interactions
 The nouns and verbs are taken from a
hand constructed list containing nouns
such as activation, phosphorylation or
interaction, and verbs such as
activates, binds or phosphorylates.
Rules are applied directly to the text by
string comparison.
Comp Funct Genom 2001, 2, 196-206

50. Extraction of the Interactions
 The sentence “The expressed p53
protein showed nuclear localization and
its expression was associated with an
induction of p21 and bax expression”
relates p53 with p21 and bax but does
not imply a physical interaction between
them.
Comp Funct Genom 2001, 2, 196-206

51. Distribution of the Protein
Interaction Universe
Comp Funct Genom 2001, 2, 196-206