Proteomics Informatics Protein Characterization II: Protein - - PowerPoint PPT Presentation

Proteomics Informatics – Protein Characterization II: Protein Interactions (Week 11)

Discovering New Protein Interactions with Affinity Capture Mass Spectrometry

A B A C D

Digestion Mass spectrometry

E F

Identification

More / better quality interactions

Affinity Capture Optimization Screen

+

Cell extraction Lysate clearance/ Batch Binding Binding/Washing/Eluting SDS-PAGE Filtration

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

Peptides Fragments Fragmentation Proteolytic Peptides Enzymatic Digestion Protein Complex Chemical Cross-Linking MS MS/MS Isolation Cross-Linked Protein Complex

Interaction Partners by Chemical Cross-Linking

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

M/Z

Peptides Fragments Fragmentation Proteolytic Peptides Enzymatic Digestion Protein Complex Chemical Cross-Linking MS MS/MS Isolation Cross-Linked Protein 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 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.

Cloning nanobodies for GFP pullouts

• Atypical heavy chain-only IgG antibody produced in camelid family – retain

high affinity for antigen without light chain

• Aimed to clone individual single-domain VHH antibodies against GFP – only

~15 kDa, can be recombinantly expressed, used as bait for pullouts, etc.

• To identify full repertoire, will identify GFP binders through combination of

high-throughput DNA sequencing and mass spectrometry

VHH clone for recombinant expression

Cloning llamabodies for GFP pullouts

Identifying full-length sequences from peptides

Sequence diversity of 26 verified anti-GFP nanobodies

• Of ~200 positive sequence hits, 44 high confidence clones were synthesized

and tested for expression and GFP binding: 26 were confirmed GFP binders.

• Sequences have characteristic conserved VHH residues, but significant

diversity in CDR regions.

FR1 CDR1 FR2 CDR2 CDR3 FR3 FR4

HIV-1

gp120 Lipid Bilayer gp41 MA CA NC PR IN RT RNA

Particle Genome

env rev vpu tat nef 3’ LTR 5’ LTR vif gag pol vpr

CA MA NC p6 PR RT IN gp41 gp120

9,200 nucleotides

Digestion & Ligation

R7/3

+

Kanr PmeI Site Kanr

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 200 400 600 800

Fitness Landscape of Targeted Viral Segment

1 10 100 1000 10000 200 400 600 800 1 10 100 1000 10000 200 400 600 800 1 10 100 1000 200 400 600 800 1 10 100 1000 200 400 600 800 1 10 100 1000 200 400 600 800 1 10 100 1000 200 400 600 800 1 10 100 1000 10000 200 400 600 800 1 10 100 1000 10000 200 400 600 800 1 10 100 1000 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.

Specific and Non-Specific Interactors

Env-3xFLAG Vif-3xFLAG

300 nm 3 nm

Limitation of Light Microscopy

Fluorescent Imaging with One Nanometer Accuracy (FIONA)

Yildiz et al, Science 2003. 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 Microscopy

Using fluorescence proteins (mEOS, etc)

Using two lasers for interchangeable activation and excitation of probes

Molecular Organization

• f the Intercalated Disc

Saffitz, Heart Rhythm (2009)

Molecular Organization

• f 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 Cx43

Effect AnkG Silencing on Cx43

AnkG silencing results in increase of Cx43 cluster size and loss of circularity.

AnkG Sil

Monte-Carlo Simulations

Monte-Carlo Simulations

Experiment Simulation Experiment Simulation

Cx43 PKP2

Is the Observed Overlap Random?

Untreated AnkG Silencing

Cx43 Area Colocalization Area Cx43 Area Colocalization Area

Untreated AnkG Silencing

Uniform Non-uniform Experiment Experiment Experiment Experiment

Proteomics Informatics – Protein Characterization II: Protein Interactions (Week 11)