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Transcript
ANALYSIS OF PROTEIN-PROTEIN INTERACTIONS BY MASS SPECTROMETRY Israeli Society for Mass Spectrometry Protein-Protein interaction workshop Dr. Yishai Levin Head of the de Botton Institute for Protein Profiling Introduction WHY IS STUDY OF INTERACTOME IMPORTANT? • Proteins (like most humans) are social creatures. From DNA replication to protein degradation, the work of the cell is accomplished mostly by macromolecular complexes. • Finding interaction partners for a protein can reveal its function. • The interactome is highly dynamic. STABLE: these are the interactions which are associated with proteins that are purified as multi-subunit complexes. TRANSIENT: these are on/off and require a specific set of conditions for the interaction to take place. (expected to control majority of cellular processes). Introduction Affinity Purification Mass Spectrometry (AP-MS) Bate Step 1 Interaction partner Step 2 Other proteins Step 3 Step 4 Introduction Affinity Purification Mass Spectrometry (AP-MS) Co-IP or pulldown Grow cells & lyse Mass spec Interacting partners What are the proper Type of beads? Elution or on-bead How to decide what is an controls? How much bead slurry? digestion? interaction? How many replicates? How much protein to Gel or no gel? How to refer to controls? Lysis buffer? load? Sequence database? What is the typical Tagged or untagged? How to wash the beads? Qualitative or quantitative? background? Do you expect PTMs? How to interpret the Isotopic labeling or label free? results? Grow cells & lyse What are the proper controls? How many replicates? Lysis buffer? Tagged or untagged? Isotopic labeling or label free? Solubilize the proteins Maintain interactions What is the epitope recognition of the antibody raised agianst the bait? Lysis of Cultured Cells for Immunoprecipitation, Hong Ji http://intl-cshprotocols.cshlp.org/content/2010/8/pdb.prot5466.full Grow cells & lyse What are the proper controls? How many replicates? Lysis buffer? Tagged or untagged? Isotopic labeling or label free? Solubilize the proteins Maintain interactions 4% SDS, 100mM DTT, 100mM Tris/HCl What is the epitope recognition of the antibody raised agianst the bait? Linear peptide Conformational Grow cells & lyse What are the proper controls? How many replicates? Lysis buffer? Tagged or untagged? Isotopic labeling or label free? Solubilize the proteins Note: RIPA lysis buffer disrupts most weak noncovalent proteinprotein interactions. 150mM NaCl, 1% NP-40, 0.5% Na-deoxycholate, Maintain interactions Strong 0.1% SDS, 50mM TrisHCl What is the epitope recognition of the antibody raised agianst the bait? Linear peptide Conformational Weak Grow cells & lyse What are the proper controls? How many replicates? Lysis buffer? Tagged or untagged? Isotopic labeling or label free? Solubilize the proteins Note: this is probably the most widely used lysis buffer. It relies on the nonionic detergent NP-40 as the major solubilizing agent, which can be replaced by Triton X-100 with similar results. Variations include lowering the detergent concentration or using alternate detergents such as digitonin, saponin, or CHAPS. 150mM NaCl, 1% NP-40, 50mM Tris-HCl Maintain interactions Strong What is the epitope recognition of the antibody raised agianst the bait? Linear peptide Conformational Weak Grow cells & lyse What are the proper controls? How many replicates? Lysis buffer? Tagged or untagged? Isotopic labeling or label free? Performing AP-MS, where the bait protein is tagged (fused to a protein or peptide) usually yields better results less background, high bait yield His, GFP, FLAG, HA, Biotin-Strep, GST and others Note: you should verify that introducing the tag will not alter the function/localization of the bait protein. Grow cells & lyse What are the proper controls? How many replicates? Lysis buffer? Tagged or untagged? Isotopic labeling or label free? Disclaimer: the information below is based on empirical observations over a wide range of different biological systems. The recommended tag should be evaluated on a case per case basis. Speak with the experts before designing the experiment Type Bait Yield Background Wash strength FLAG Excellent Low Moderate GFP Good Moderate Moderate Biotin Excellent Low High Endogenous Excellent to poor Moderate Low His Good High Moderate Co-IP or pulldown AP-MS What you think you’re doing Co-IP or pulldown What you’re actually doing Co-IP or pulldown Bate Interaction partners Non-specific binders Cross-reactive What would be the proper control? Co-IP or pulldown Bate Interaction partners Non-specific binders Cross-reactive A different antibody would be the proper control or knockout of the bait Co-IP or pulldown Bate Tag Interaction partners Non-specific binders Cross-reactive What would be the proper control? Co-IP or pulldown Bate Tag Interaction partners Non-specific binders Cross-reactive Cell line expressing the untagged protein Co-IP or pulldown Type of beads? How much bead slurry? How much protein to load? How to wash the beads? Lamond, JCB, 2008 – The bead proteomes Agarose Sapharose Magnetic Each type of bead will generate more or less non-specific binding http://jcb.rupress.org/content/183/2/223.short http://www.crapome.org https://tools.thermofisher.com/content/sfs/brochures/1601945-Protein-Interactions-Handbook.pdf Co-IP or pulldown Type of beads? How much bead slurry? How much protein to load? How to wash the beads? Cytoskeletal/Structural Heat shock proteins Ribosomal proteins Lamond, JCB, 2008 – The bead proteomes Agarose Sapharose Magnetic actin, cofilin, desmin, desmoplakin, epiplakin, filamin, myosin, peripherin, plectin, tropomyosin, tubulin, vimentin Co-IP or pulldown Type of beads? How much bead slurry? How much protein to load? How to wash the beads? More protein More non-specific binding Less protein Less enrichment Total protein: 0.5mg to 10mg Beads: 15 to 50uL slurry Co-IP or pulldown Type of beads? How much bead slurry? How much protein to load? How to wash the beads? Bate Interaction partners Non-specific binders Cross-reactive • • • • Don’t want to loose our bait Don’t want to loose the interacting partners Do want to eliminate the non-specific binders Do want to keep proteins solubilized Co-IP or pulldown Type of beads? How much bead slurry? How much protein to load? How to wash the beads? • 1-2 washes with lysis buffer • For MS analysis (not gel based), two washes with PBS Co-IP or pulldown Bate Interaction partners Non-specific binders Cross-reactive • Good QC to make sure the IP worked • No always a good indication for MS analysis Western Blotting Sample buffer Co-IP or pulldown Bate Interaction partners Non-specific binders Cross-reactive MS Mass Spec Elution or on-bead digestion? Gel or no gel? Targeted or discovery? Sequence database? Qualitative or quantitative? Do you expect PTMs? Low-pH elution (0.1M glycine) Peptide elution Low-pH elution (0.1M glycine) Sample buffer elution YPYDVPDYA DYKDDDDK On-bead digestion In-solution digestion In-gel digestion Quantification Isotopic Labeling Label-free Case 1 Heavy Light Lysis Case 2 Lysis Control Lysis m/z Relative abundance Relative abundance Elute and mix 1:1 m/z m/z m/z Quantification Label-free Case 1 Quantification method Spectral counting Number of spectra matched to a protein Peptide Intensity (MS1 intensity) Averaged peptide intensity (all, Hi-3) Lysis Relative abundance Type Case 2 m/z Control Lysis m/z m/z Workflow Mass Spec Co-IP Statistics nUPLC-MS/MS Data processing Workflow Mass Spec nUPLC-MS/MS Liquid chromatography Mass spectrometry Data Acquisition Mass Spec Data Dependent Acquisition Peptides off MS scan: Selection of peptides to fragment on MS/MS of peptide 1 on MS/MS of peptide 2 on MS/MS of peptide 3 Quadrupole Collision cell Second analyzer Workflow Mass Spec nUPLC-MS/MS MS MS/MS Sequence database searching Sequence database K R R Sample K K In silico digestion R R K Digestion K R R K LC-MS/MS analysis and sequencing K R R K K R K Filter results for max. false Discovery rate (FDR) (not adjustment for multiple hypothesis testing) Assigned incorrect Assigned correct Incorrect distribution Correct distribution Grow cells & lyse Co-IP or pulldown • We search against a specie specific database + common lab proteins. • If your bait was expressed in one organism and incubated with proteins from another organism – the database should include both. Mass spec Elution or on-bead digestion? Gel or no gel? Targeted or discovery? Sequence database? Qualitative or quantitative? Do you expect PTMs? Interacting partners Grow cells & lyse Co-IP or pulldown • If the IP/pulldown resulted in low background we may be able to detect PTMs (phosphorylation, acetylation, methylation). Mass spec Elution or on-bead digestion? Gel or no gel? Targeted or discovery? Sequence database? Qualitative or quantitative? Do you expect PTMs? Interacting partners Sequence database searching Sequence database K R R Sample K K In silico digestion R R K Digestion K R R K LC-MS/MS analysis and sequencing K S p K R K R T p R Y p K K R K Data alignment, protein quantification & identification Raw data Raw data filtering Run-to-run alignment Processed data Feature and isotopic detection Protein/Peptide identification Real data demonstration Experiment 1: FLAG Co-IP vs control (untagged) Experiment 2: BioID pulldown (+/- biotin) Experiment 1: FLAG Co-IP vs control (untagged) Peptide intensities of the bait Experiment 1: FLAG Co-IP vs control (untagged) Intensity Protein m/z Experiment 1: FLAG Co-IP vs control (untagged) Peptide intensities of the bait GroupA_1 GroupA_2 GroupA_3 Control_1 Control_2 Control_3 BioID: proximity-dependent biotin identification Kyle J. Roux et al. J Cell Biol 2012;196:801-810 • Expression of a promiscuous biotin–ligase fusion protein in live cells leads to the selective biotinylation of proteins proximate to that fusion protein. • After stringent cell lysis and protein denaturation, biotinylated proteins are affinity purified. • Subjected to mass spectrometry or immunoblot analysis.
