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Proteomics Informatics –
Protein Characterization II:
Protein Interactions (Week 12)
Discovering New Protein Interactions with
Affinity Capture Mass Spectrometry
E
A
A
D
C
B
Digestion
Mass spectrometry
Identification
F
Affinity Capture Optimization Screen
Cell extraction
More / better quality
interactions
+
Filtration
Lysate clearance/
Batch Binding
SDS-PAGE
Binding/Washing/Eluting
Hakhverdyan, et. al., "Rapid Optimized Screening of the Cellular Interactome", Nature Methods 2015.
Affinity Capture Optimization Screen
Hakhverdyan, et. al.,
"Rapid Optimized
Screening of the Cellular
Interactome", Nature
Methods 2015.
Analysis of Non-Covalent Protein Complexes
Taverner et al., Acc Chem Res 2008
Non-Covalent Protein Complexes
Schreiber et al., Nature 2011
Molecular Architecture of the NPC
Over 20 different extraction and washing
conditions ~ 10 years or art.
(41 pullouts are shown)
Actual model
Alber F. et al. Nature (450) 683-694. 2007
Alber F. et al. Nature (450) 695-700. 2007
Interaction Map of Histone Deacetylaces
Joshi et al. Molecular Systems Biology 9:672
Protein Complexes – specific/non-specific binding
Sowa et al., Cell 2009
Protein Complexes – specific/non-specific binding
Choi et al., Nature Methods 2010
Protein Complexes – specific/non-specific binding
Tackett et al. JPR 2005
Interaction Partners by
Chemical Cross-Linking
Protein
Complex
Chemical Cross-Linking
Cross-Linked
Protein Complex
Enzymatic Digestion
MS
Proteolytic
Peptides
Isolation
Peptides
Fragments
Fragmentation
MS/MS
M/Z
Protein Crosslinking by Formaldehyde
~1% w/v Fal
20 – 60 min
~0.3% w/v Fal
5 – 20 min
1/100 the volume
LaCava
Protein Crosslinking by Formaldehyde
RED: Formaldehyde crosslinking
BLACK: No crosslinking
SCORE: Log Ion Current / Log protein abundance
Interaction Sites by
Chemical Cross-Linking
Protein
Complex
Chemical Cross-Linking
Cross-Linked
Protein Complex
Enzymatic Digestion
MS
Proteolytic
Peptides
Isolation
Peptides
Fragments
Fragmentation
MS/MS
M/Z
Cross-linking
protein
n peptides with reactive groups
(n-1)n/2 potential ways to cross-link peptides pairwise
+ many additional uninformative forms
Protein A + IgG heavy chain 990 possible peptide pairs
Yeast NPC 106 possible peptide pairs
Cross-linking
Mass spectrometers have a limited dynamic range
and it therefore important to limit the number of
possible reactions not to dilute the cross-linked
peptides.
For identification of a cross-linked peptide pair, both
peptides have to be sufficiently long and required to
give informative fragmentation.
High mass accuracy MS/MS is recommended because
the spectrum will be a mixture of fragment ions from
two peptides.
Because the cross-linked peptides are often large,
CAD is not ideal, but instead ETD is recommended.
Antibodies
V1
VDJ Recombination
V2……
Vn
D1…Dn
J1 J2 …Jn
Variable heavychain domain
CDR1
CDR2
CDR3 (Fingerprint)
Somatic hypermutation
CDR1
CDR2
CDR3
An MS-based Approach for Antibody Discovery
Sequence Database
With Paired
Light-heavy Chain
Single Cell PCR
B cell Affinity Selection
SangerAGTCCGATCGGATCC
Seq
GTCCGATCGGATCCA
Sorting
AGTCCGATCGGATCC
TCCGATCGGATCCCC
~500 Sequences
Serum IgG
HIV Carrier
HIV-binding
IgG
Affinity
Selection
Spectra
HIV-binding
IgGs
Digest
/MS
Scheid J, Mouquet H*, Ueberheide T*, Diskin R*, et al. Science, 2011
HIV Antibodies
J.F. Scheid et al,
“Sequence and structural
convergence of broad
and potent HIV antibodies
that mimic CD4 binding”,
Science, 333 (2011) 1633-1637
A Functional IgG Requires
Paired Light and Heavy Chains
CH2
=
Light
CH3
Standard IgG
Heavy
Cloning Single-Chain Llama Antibodies
Single-Chain IgG from Llama
• Atypical single-chain IgG antibody produced in camelid family (e.g. llama)
• Retain high affinity for antigen without light chain
• Antigen binding domain can be cloned and expressed to make “Nanobodies”:
- Extremely Cheap & Unlimited Amounts
- Tiny (~15 kDa) , Fold well & Stable in Solution
- Easily Engineered for Special Needs
CH2
CH3
Single-chain IgG
Standard IgG
New MS-based
Nanobody Discovery
New MS-based Nanobody Discovery
DNA Library Construction
Trim
Read 1: 301 bp
Overlap: ~200 bp
Read 2: 301 bp
Trim
1
5
10-14
30-34
250-299
150-199
250-299
150-199
50-59
30-34
10-14
5
1
50-59
Read 2 Quality
Read 1 Quality
DNA Library Construction
Trim
Read 1: 301 bp
Overlap: ~200 bp
Merging
of reads
Trim
Read 2: 301 bp
Merged read length
Merged read quality
250-299
150-199
50-59
30-34
10-14
5
1
Identifying peptides
Identifying full-length sequences from peptides
Nanobody Primary
Sequences with
CDR Regions
Annotated
1. CDR regions are identified based on
approximate position in the sequence
and the presence of specific leading
and trailing amino acids.
2. Nanobody sequences ranked based
on: MS coverage and length of
individual CDR regions with CDR3
carrying highest weight; overall
coverage including scaffold region;
HT-Seq counts.
3.
Nanobody sequences grouped by
CDR3. One sequence is assigned to
a group where its hamming distance
to an existing member is 1.
Mapping
Annotated Nanobody
Sequences with
MS coverage
Ranking
Ranked
Nanobody Lists
Grouping
Ranked Nanobody
Groups
Identified
Peptides
Identifying full-length sequences from peptides
Nanobody Production Scheme
Sequence of Discovered
Nanobody Candidates
Gene synthesis &
Codon optimization
Expression Vector
Cloning
MAQVQLVESGGGLVQAGGSLRLSCVASGR
TFSGYAMGWFRQTPGREREAVAAITWSAH
STYYSDSVKDRFTISIDNTRNTGYLQMNS
LKPEDTAVYYCTVRHGTWFTTSRYWTDWG
QGTQVTVS
~ $100 / sequence
Transformation
One-Step
Purification
~ 2 mg / 1 L
E.coli Expression
Application of Anti-GFP Nanobodies in
Immunofluorescence Microscopy
GFP
Homemade
Nanobody
Creating Super-high-affinity Reagent Against GFP
GFP:
Clone A
KD = 0.7 nM
Clone B
KD = 16 nM
Nano
Nano
GFP
Overlay
Super-high-affinity
KD = 0.03 nM
HIV-1
Lipid Bilayer
gp120
gp41
MA
RT
IN
PR
NC
MA CA NC p6
CA
Genome
gp120
vpu
RNA
pol
5’ LTR
Particle
vif
gag
PR
RT
vpr
IN
9,200 nucleotides
gp41
nef
env
tat
rev
3’ LTR
Random Insertion of 5 Amino Acids in
Proviral DNA Clone
r
Kan
+
r
Kan
PmeI Site
R7/3
1000
Digestion &
Ligation
Random insertion of 5 amino acids (PmeI)
within specific viral coding region
100
10
1
0
200
400
600
800
Fitness Landscape of
Targeted Viral Segment
10000
10000
1000
1000
1000
100
Day 1
100
100
10
10
10
1
1
0
Day 3
200
400
600
800
1
0
200
400
600
800
1000
1000
1000
100
100
100
10
10
10
1
1
0
200
400
600
800
10000
1000
1000
100
100
10
10
200
400
600
800
0
200
400
600
800
0
200
400
600
800
1
0
10000
0
200
400
600
800
1000
100
Day 6
10
1
1
0
200
400
600
800
1
0
200
400
600
800
Specific and Non-Specific Interactors
I-DIRT = Isotopic Differentiation of Interactions as Random or Targeted
3xFLAG Tagged HIV-1
WT HIV-1
Infection
Light
Heavy
(13C labeled Lys, Arg)
1:1 Mix
Immunoisolation
MS
Lys
Arg
(+6 daltons)
(+6 daltons)
Modified from Tackett AJ et al., J
Proteome Res. (2005) 4, 1752-6.
Fitness Landscape of HIV with
random 15 bp insertions in ENV
HIV interactome
Limitation of Light Microscopy
300 nm
3 nm
Fluorescent Imaging with
One Nanometer Accuracy (FIONA)
CCD image of a single Cy3 molecule:
Width ~ 250nm
Center is localized within width/(S/N)
(S/N)2 ~ N
N = total # photon
(for N ~ 104 center within ~ 1.3 nm)
Yildiz et al, Science 2003.
Paul Selvin
Limitation of Light Microscopy
3 nm
3 nm
3 nm
3 nm
3 nm
3 nm
3 nm
3 nm
3 nm
Limitation of Light Microscopy
3 nm
3 nm
3 nm
3 nm
3 nm
3 nm
3 nm
3 nm
3 nm
Limitation of Light Microscopy
3 nm
3 nm
3 nm
3 nm
3 nm
3 nm
3 nm
3 nm
3 nm
Limitation of Light Microscopy
3 nm
3 nm
3 nm
3 nm
3 nm
3 nm
3 nm
3 nm
3 nm
Limitation of Light Microscopy
20 nm
20 nm
20 nm
20 nm
20 nm
20 nm
20 nm
20 nm
20 nm
Super-Resolution Localization Microscopy
Using two lasers for
interchangeable
activation and
excitation of probes
PALM: PhotoActivation Localization Microscopy
Using fluorescence proteins (mEOS, etc)
Betzig, 2006
Science
STORM: STochastic Optical Reconstruction Microscopy
Using doubly labeled (Cy3-Cy5) Ab
Bates, 2007 Science
Huang, Annu. Rev. Biochem, 2009
Molecular Organization
of the Intercalated Disc
Saffitz, Heart Rhythm (2009)
Molecular Organization
of the Intercalated Disc
Plakophilin-2 (PKP2)
Desmosome
Connexin43 (Cx43)
Gap junctions
What is the interaction map of ID proteins?
Agullo-Pascual E, Reid DA, Keegan S, Sidhu M, Fenyö D, Rothenberg E, Delmar M.
"Super-resolution fluorescence microscopy of the cardiac connexome reveals
plakophilin-2 inside the connexin43 plaque“, Cardiovasc Res. 2013
Regular Microscopy v.
Super-Resolution
Cx43
PKP2
Regular Microscopy v.
Super-Resolution
Cx43
PKP2
Regular Microscopy v.
Super-Resolution
Cx43
PKP2
What Do We Mean by Colocalization?
Characterization of Cx43 Clusters
A
Cx43
PKP2
C
Area (nm2x103)
Area (nm2x103)
Two distinct size populations
corresponding to hemichannels and full channels.
F
Intensity
(nm x103)
Circularity
Circularity
F
Predominantly circular
sity
x103)
rimeter
E
Counts
E
Counts
(nm x103)
Perimeter
D
Counts
Area (nm2x103)
Log 10 perimeter
B
C
Counts
Scale =200 nm
B
D
Cx43-PKP2 Overlap Analysis
Cx43
A correlation between overlap and Cx43 cluster area
B
Counts
Counts
C
Area (nm2x103)
E
Circularity
F
AnkG Sil
Intensity
(nm x103)
Log 10 perimeter
x43
KP2
Effect AnkG Silencing on Cx43
Circularity
Distance (nm)
AnkG silencing results in increase of Cx43 cluster size and loss of
circularity.
Monte-Carlo Simulations
Monte-Carlo Simulations
Experiment
Simulation
Cx43
Experiment
Simulation
PKP2
Is the Observed Overlap Random?
Experiment
AnkG Silencing
Experiment
Colocalization
Area
Untreated
Cx43 Area
Uniform
Experiment
Experiment
Non-uniform
AnkG Silencing
Colocalization
Area
Untreated
Cx43 Area
Proteomics Informatics –
Protein Characterization II:
Protein Interactions (Week 12)
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