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)