Download Mapping Protein Interactions and Topologies with

Mapping Protein Interactions and Topologies with
Protein Interaction Reporter Technology
Cascadia Proteomics Symposium 2011
Juan D. Chavez
Department of Genome Sciences
University of Washington
Protein Topology Enables Interactions
• Regulated protein networks are a product of
selection Kuriyan and Eisenberg, Nature, 2007
•
•
Colocalization
Adaptive mutation
• Selective adaptation - topologies that yield
advantage
• Genome evolution → protein topologies
• In vivo topological info the most critical
• Topologies known for very few interactions
Cascadia Proteomics Symposium 2011
Cross-linking strategy
Spacer arm
Reactive Groups
NHS-esters
Imidoester
Maleimide
Hydrazides
.
.
.
New covalent bonds contain information
• Protein interactions
• Topological features
• Regions of close proximity
Cascadia Proteomics Symposium 2011
Applications of in vitro Cross-Linking:
A Complimentary Approach
Revealing Protein Topology
Proc Natl Acad Sci U S A. 2011 Apr 19;108(16):6426-31
Cascadia Proteomics Symposium 2011
Quantifying Interactions
J Proteome Res. 2011 Apr 1;10(4):1528-37.
Challenges of Large-Scale Application
Log10(Possible cross-linked peptide combinations)
14
Homo sapiens~35,000 proteins
6.1 x 1012 combinations
Caenorhabditis elegans
~19,000 proteins
1.8 x 1012 combinations
13
Drosophila Melanogaster
~14,000 proteins
9.8 x 1011 combinations
12
Escherichia coli
~6,000 proteins
1.7 x 1011 combinations
11
10
9
Carsonella ruddi
Smallest Genome
~182 proteins
1.7 x 108 combinations
•Effectively, an N database becomes an N2 database
•Mass of cross-linked pair is non-unique
•MS/MS complex – peptide mass unknown
8
7
2
Cascadia Proteomics Symposium 2011
2.5
3
3.5
4
Log10(Total proteins in genome )
4.5
5
Protein Interaction Reporter – PIR Technology
Affinity Tag
Accurate MS of PIR-Labeled Peptides
Reporter
Cleavable
bonds
m/z
Cleave PIR
Bonds
Reactive Groups
m/z
Tang et al., Anal. Chem. 2005 77, 311.
Zhang et al., Mol. Cell. Proteomics 2009 8(3), 409.
Yang, et al. Anal. Chem. 2010 82, 3556.
Tang et al., Mol Biosyst. 2010 Jun;6(6):939-47
Cascadia Proteomics Symposium 2011
•Assign connectivity
•Reduces to N database
•Mass and/or MS/MS ID of proteins
PIR – Product Type Differentiation
Inter-cross-link
Mass
Relationships
=
Intra-cross-link
=
Dead-end
=
+
+
+
+
+
OH
OH
X-Links
Anderson et al., J. Proteome Res.
2007,6, 3412.
Cascadia Proteomics Symposium 2011
BLinks
Hoopmann et al., J. Proteome Res.
2010, 9, 6323.
Download Blinks @:
http://brucelab.gs.washington.edu/software.php
Protein Interaction Reporter – BRink
H
O
H
N
Biotin affinity tag
NH
H
S
NH
O
O
OH
NH
H 3 CO
O
NH
OCH 3
O
O
NH
O
O
O
NH
O
O
N
O
NH
Cleavable
RINK bonds
O
H 3 CO
O
O
NHS ester amine
Reactive groups
OCH 3
NH
O
O
N
O
O
Chemical Formula: C74H91N11O24S
Exact Mass: 1549.59591
Molecular Weight: 1550.63864
Elemental Analysis: C, 57.32; H, 5.92; N, 9.94; O, 24.76; S, 2.07
Tang et al., J Proteome Res. 2007 Feb;6(2):724-34.
Hoopmann et al. J Proteome Res. 2010 Dec 3;9(12):6323-33
Cascadia Proteomics Symposium 2011
Zheng et al. Mol Cell Proteomics. 2011 Jun 22.
In vivo Application of PIR to cultured cells
Full MS
694.946
z=5
In vivo cell labeling
Relative Abundance
Cell lysis and digestion
100
75
50
0300 400 500
600 700 800
Precursor scan
50
0
400
25
800
m/z
ISCID
25
500
600 700 800
100
MS/MS
for
1200
800
m/z
693.19
561.95
1085.73
1264.70
758.73
351.35
m/z
450.35
869.25
790.54
1165.65
1411.88
645.39
492.97
272.31
300
MS/MS of
100
970.71
0
Cascadia Proteomics Symposium 2011
m/z
MS/MS of
50
time
50
0
MS/MS for 500
Relative Abundance
Relative Abundance
Intensity
LC-MS or LC-MS/MS
z=1
900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900
Bead
Affinity purification
Relative Abundance
C)
100
50
PIR mass
relationship
807.921
ISCID
561.748
z=2 733.387z=2
75
0
300 400
1200
900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900
100
SCX cleanup
868.430
z=4
m/z 900
1600
Relative Abundance
B)
Relative Abundance
A)
Relative Abundance
100
716.48
562.05
50
m/z
698.51
492.94
619.49
466.35
0
400
535.31
742.85583
550
m/z
750
Confocal Images of PIR-Labeled HeLa Cells
Neutravidin green
Propidium Iodide
3D overlaid image
Subcellular Distribution of PIR Reactive Proteins in E. Coli
1503 Peptides / 416 Proteins
Unannotated, 118
Cytoplasmic, 212
Outer Membrane, 13
Cytoplasmic
Membrane, 35
Periplasmic, 38
65 cross-linked peptide pairs have been identified from cells.
Cascadia Proteomics Symposium 2011
Mol Cell Proteomics. 2011 Jun 22.
Subcellular Distribution of PIR Reactive Proteins in HeLa
Cells
1383 peptides / 684 proteins
nucleoplasm
Mitochondrio Cell projection
2%
2%
n inner
membrane
Mitochondrion
2%
Single-pass
2%
type I
membrane
protein
Membrane
2%
3%
Lipid-anchor
1%
Endoplasmic reticulum
lumen
1%
Endoplasmic
reticulum
membrane
3%
Cytoplasmic
side
Cytoplasm
26%
3%
Melanosome
3%
Peripheral
membrane protein
4%
Cell membrane
4%
nucleolus
4%
Chromosome
4%
Multi-pass
membrane protein cytoskeleton
4%
5%
Nucleus
24%
Secreted
1%
Outer Membrane Protein A - OmpA
• Structure of N-terminal 171 AA (out of 345)
• 8 Strand Beta barrel
• Structural protein
• Ion channel?
• C-terminal domain resides in periplasm
• Resistant to crystal formation
• Structural model of C-terminal domain
Pautsch and Schulz, J. Mol. Biol. 2000
Walton et al.., PNAS 2009
Cascadia Proteomics Symposium 2011
Docking Model (SymmDock) of OmpA Dimer
OUTER MEMBRANE
26.1Å
10Å
14.3Å
28.6Å
Predicted disorder
Filter top 100 models with observed cross-link constraints
Only 2 models were consistent with observed sites (<35Å)
Cascadia Proteomics Symposium 2011
K213
K294
K338
Peptidylprolyl isomerase A (cyclophilin A)
K151
K125
8.3Å
6.7Å
K125
K151
Catalyze cis/trans isomerization of proline
Overexpressed in cancer
Important role in immune response in organ transplants
Dimer in complex w/Sanglifehrin A is immunosuppressive form
Cascadia Proteomics Symposium 2011
Identification of cross-linked site in GAPDH
Cascadia Proteomics Symposium 2011
Superimposition of PIR-Cross-Linked GAPDH in HeLa
and E. coli cells
NAD Binding Pocket
K122
NAD Binding Pocket
K191
• Cytoplasmic protein
• Glycolysis, apoptosis,
transcription activation
• Known to form
multimer
Cascadia Proteomics Symposium 2011
8.6 Å
K107
K107
K194
GAPDH Disorder Prediction
1
K122/K107
K191/K194
0.9
Predicied Disorder
0.8
0.7
0.6
E.coli
0.5
Rabbit
0.4
Human
0.3
0.2
0.1
0
0
50
100
150
200
250
Residue Number 
Cascadia Proteomics Symposium 2011
300
350
400
Summary
• PIR technology: Identification of peptides that were cross-linked in cells
• Accurate mass measurements allow connectivity assignment
• Intact peptide MS & MS/MS enable confident protein identification
• Chemical method for in vivo protein interaction identification
• In vivo topological information on proteins
• Protein complexes
• Allows insight on the function of disordered protein regions.
Cascadia Proteomics Symposium 2011
On the Horizon
• Explore conservation of topologies and interactions between species
• Relationship between protein disorder and cross-linked sites
• Quantify changes in protein interactions & topologies
• Drug resistance in cancer
• PLoS One. 2011;6(5):e19892. Epub 2011 May 26
• New PIR design
Acknowledgements
Bruce Lab Members
Juan Chavez
Chad Weisbrod
Jake Zheng
Former members
Li Yang
Chang Xue
Mike Hoopmann
UWPR
Jimmy Eng
Priska von Haller
Eva Baker
Funding
Department of Energy
National Institutes of Health