proteomics informatics – protein characterization ii: protein interactions (week 12)
TRANSCRIPT
Proteomics Informatics – Protein Characterization II:
Protein Interactions (Week 12)
Discovering New Protein Interactions withAffinity Capture Mass Spectrometry
AB
A
CD
Digestion
Mass spectrometry
EF
Identification
More / better quality interactions
Affinity Capture Optimization Screen
+
Cell extraction
Lysate clearance/Batch Binding
Binding/Washing/Eluting
SDS-PAGE
Filtration
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
Over 20 different extraction and washing conditions ~ 10 years or art.(41 pullouts are shown)
Molecular Architecture of the NPC
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
Sowa et al., Cell 2009
Protein Complexes – specific/non-specific binding
Protein Complexes – specific/non-specific binding
Choi et al., Nature Methods 2010
Tackett et al. JPR 2005
Protein Complexes – specific/non-specific binding
M/Z
PeptidesFragments
Fragmentation
ProteolyticPeptides
Enzymatic Digestion
ProteinComplex
Chemical Cross-Linking
MS
MS/MS
Isolation
Cross-LinkedProtein Complex
Interaction Partners by Chemical Cross-Linking
Protein Crosslinking by Formaldehyde
~1% w/v Fal20 – 60 min
~0.3% w/v Fal5 – 20 min1/100 the volume
LaCava
Protein Crosslinking by Formaldehyde
RED: Formaldehyde crosslinkingBLACK: No crosslinking
SCORE: Log Ion Current / Log protein abundance
M/Z
PeptidesFragments
Fragmentation
ProteolyticPeptides
Enzymatic Digestion
ProteinComplex
Chemical Cross-Linking
MS
MS/MS
Isolation
Cross-LinkedProtein Complex
Interaction Sites by Chemical Cross-Linking
Cross-linking
protein
n peptides with reactive groups
(n-1)n/2 potential ways to cross-link peptides pairwise
+ many additional uninformative formsProtein 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
V2 Vn J1 JnD1 DnV1
Variable heavy-chain domain
CDR1 CDR2 CDR3 (Fingerprint)
……… J2 …
VDJ Recombination
Somatic hypermutation
CDR1 CDR2 CDR3
B cell Affinity SelectionSanger-Seq
Sequence DatabaseWith Paired Light-heavy Chain
Single Cell PCR
Serum IgG
Affinity Selection /MS
HIV-binding IgG
Spectra
Digest
Sorting AGTCCGATCGGATCCGTCCGATCGGATCCAAGTCCGATCGGATCCTCCGATCGGATCCCC
An MS-based Approach for Antibody Discovery
Scheid J, Mouquet H*, Ueberheide T*, Diskin R*, et al. Science, 2011
HIV Carrier
~500 Sequences
HIV-binding IgGs
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
Standard IgG
VH
CH 1
CH 2
CH 3
VL
CL
Light
Heavy
=
A Functional IgG Requires Paired Light and Heavy Chains
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
VH H
CH 2
CH 3
Single-chain IgG
Nanobody
Standard IgG
New MS-based Nanobody Discovery
New MS-based Nanobody Discovery
DNA Library ConstructionRead 1: 301 bp
Read 2: 301 bpOverlap: ~200 bp
Trim
Trim
Read 2 Quality
15
10-
14
30-
34
50-
59
150
-19
92
50-2
99
Read 1 Quality
1 5 10-1
4
30-3
4
50-5
9
150
-19
92
50-2
99
DNA Library ConstructionRead 1: 301 bp
Read 2: 301 bpOverlap: ~200 bp
Trim
Trim
Merged read length
Merged read quality
1 5 10-14
30-34
50-59
150-199250-299
Me
rgin
go
f read
s
Identifying peptides
Identifying full-length sequences from peptides
Identified Peptides
Nanobody Primary Sequences with
CDR Regions Annotated
Mapping
Annotated Nanobody Sequences with
MS coverage
Ranked Nanobody Lists
Ranking
Grouping
Ranked Nanobody Groups
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.
Identifying full-length sequences from peptides
Gene synthesis & Codon optimization
Cloning
Expression Vector
Transformation
E.coli ExpressionOne-Step Purification
~ $100 / sequence
Nanobody Production Scheme
MAQVQLVESGGGLVQAGGSLRLSCVASGRTFSGYAMGWFRQTPGREREAVAAITWSAHSTYYSDSVKDRFTISIDNTRNTGYLQMNSLKPEDTAVYYCTVRHGTWFTTSRYWTDWGQGTQVTVS
Sequence of Discovered Nanobody Candidates
~ 2 mg / 1 L
GFP
HomemadeNanobody
Application of Anti-GFP Nanobodies in Immunofluorescence Microscopy
Super-high-affinity
KD = 0.03 nM
Clone A
Creating Super-high-affinity Reagent Against GFP
GFP:
Nan
o
Nan
oGFP
Overlay KD = 0.7 nM
Clone B KD = 16 nM
HIV-1
gp120Lipid Bilayer gp41
MA
CA
NC
PR
IN
RT
RNA
Particle
Genome
env
rev
vpu
tat
nef
3’ LTR5’ LTR
vif gagpol
vpr
CAMA NC p6
PR RT IN
gp41gp120
9,200 nucleotides
Digestion & Ligation
R7/3
+
Kanr
PmeI SiteKanr
Random insertion of 5 amino acids (PmeI)within specific viral coding region
Random Insertion of 5 Amino Acids in Proviral DNA Clone
1
10
100
1000
0 200 400 600 800
Fitness Landscape of Targeted Viral Segment
1
10
100
1000
10000
0 200 400 600 8001
10
100
1000
10000
0 200 400 600 8001
10
100
1000
0 200 400 600 800
1
10
100
1000
0 200 400 600 8001
10
100
1000
0 200 400 600 8001
10
100
1000
0 200 400 600 800
1
10
100
1000
10000
0 200 400 600 8001
10
100
1000
10000
0 200 400 600 8001
10
100
1000
0 200 400 600 800
Day 1
Day 3
Day 6
Specific and Non-Specific Interactors
3xFLAG Tagged HIV-1 WT HIV-1
Infection
Light Heavy (13C labeled Lys, Arg)
1:1 Mix
Immunoisolation
MS
I-DIRT = Isotopic Differentiation of Interactions as Random or Targeted
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
300 nm
3 nm
Limitation of Light Microscopy
Fluorescent Imaging with One Nanometer Accuracy (FIONA)
Yildiz et al, Science 2003.X axis
Y axis
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)
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
Huang, Annu. Rev. Biochem, 2009
Bates, 2007 Science
STORM: STochastic Optical Reconstruction Microscopy Using doubly labeled (Cy3-Cy5) Ab
Betzig, 2006 Science
PALM: PhotoActivation Localization MicroscopyUsing fluorescence proteins (mEOS, etc)
Using two lasers for interchangeable activation and excitation of probes
Molecular Organization of the Intercalated Disc
Saffitz, Heart Rhythm (2009)
Molecular Organizationof the Intercalated Disc
Connexin43 (Cx43) Gap junctions
Plakophilin-2 (PKP2) Desmosome
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
Cx43
PKP2
Regular Microscopy v. Super-Resolution
Cx43
PKP2
Regular Microscopy v. Super-Resolution
What Do We Mean by Colocalization?
Characterization of Cx43 Clusters
Two distinct size populations corresponding to hemi-channels and full channels.
Predominantly circular
Scale =200 nm
Cx43-PKP2 Overlap Analysis
A correlation between overlap and Cx43 cluster area
100%
overla
p
50%
overlap
Cx43
Effect AnkG Silencing on Cx43
AnkG silencing results in increase of Cx43 cluster size and loss of circularity.
AnkG Sil
100% o
verla
p
50% overlap
Monte-Carlo Simulations
Monte-Carlo Simulations
Experiment
Simulation
Experiment
Simulation
Cx43
PKP2
Is the Observed Overlap Random?
Untreated AnkG Silencing
Cx43 Area
Colo
caliz
ati
on
Are
a
Cx43 Area
Colo
caliz
ati
on
Are
a
Untreated AnkG Silencing
Uniform
Non-uniform
Experiment
Experiment
Experiment
Experiment
Proteomics Informatics – Protein Characterization II:
Protein Interactions (Week 12)