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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)