Transcript Click-IPx
Chemical proteomics lab project Click chemistry and target identification via pulldown enrichment Fabio Socciarelli, Julia Steinmetz, Elena Eyre Sánchez, Oihana Basabe Burgos and Tatiana Álvarez Giovannucci Aim: enrichment of serine hydrolases Features of ActivX Azido-FP Serine Hydrolase Probe — covalently binds and labels the reactive site of active serine hydrolases Applications: • Determine serine hydrolase enzyme activity in cells and lysates • Mapping the active-site serine of functionally diverse serine hydrolase family members (e.g. proteases, lipases, esterases) • Screen for small molecule binding affinities and active-site inhibition • Profile serine hydrolases using fluorescent, Western blot or mass spectrometry workflows Because many of the proteolytic enzymes in the serine hydrolase family are expressed as inactive proenzymes (zymogens), the ActivX FP probes selective enrichment only those enzymes that are functionally active and biologically relevant at the time of labeling. 1. Cell lysate (freeze-thaw) 2. Incubation with FP-azido probe (2 µM) or DMSO as control FP-azido probe - Specific binding to target proteins DMSO – negative control 3. Copper-catalyzed click chemistry reaction Biotinalkyne Azido-FP probe Biotinalkyne + CuSO4 (source of Cu(II)) + Sodium ascorbate (reducing agent) + THPTA (ligand) Azido-FP probe Conjugate covalently linked via a triazole moiety 4. Proteins are precipitated 1:4 acetone o/n 2) Click biotin-alkyne Remove unbound biotin Solubilize unbound biotin Click reaction Control Centrifuge Collect pellets Add ice-cold ethanol Pool the pellets Discard unbound biotin Centrifuge Discard supernatant Click reaction Control Bind to beads and wash Add avidin beads Avidin-Biotin Centrifuge Collect pellets 2X Add PBS Centrifuge Collect pellets 2X Add Urea Centrifuge Collect pellets 2X Add digestion buffer Centrifuge Collect pellets Protein denaturation Trypsin is active at pH8 https://www.thermofisher.com/se/en/home/life-science/protein-biology/protein-biology-learning-center/protein-biology-resource-library/pierce-protein-methods/avidin-biotin-interaction.html Reduction + alkylation • Add TCEP (tris(2-carboxyethyl)phosphine) http://www.biosyn.com/tew/instruction-of-reduction-reaction-using-tcep.aspx • Add CAA (chloroacetamide) Digestion • Add Trypsin – Trypsin cleaves peptide chains mainly at the carboxyl side of the amino acids lysine or arginine, except when either is followed by proline. http://worldofbiochemistry.blogspot.se/2014/09/trypsin.html Reduction, Alkylation and Digestion Avidin-Biotin Complex Avidin Beads B B B B Probe Serine hydrolases • • • • Denaturated: Urea Reduced: TCEP Alkylated: CAA Digested: Trypsin Supernatant collection and pH correction • After an overnight incubation supernatant from cltrl and Click is collected; • Beads are resuspended, washed with Phase A solution (3% acetonitrile, 0,1% formic acid in MQ water) and supernatant recollected for two times; • Supernatant pH is corrected to <3 with 10% formic acid Cation exchange (SCX cleaning) Cation exchange Wash buffer: 30% methanol, 0,1%, MQ water Preparation of samples for MS • Samples were dried with a speed vac device, resuspended in MS solvent and 6ml were injected in the MS system for reversed-phase liquid chromatography Reversed-phase liquid chromatography • Hydrophobic stationary phase surface • Molecules will bond depending on their hydrophobicity • Needed for obtaining a good separation at the chromatogram • For obtaining narrow and high peaks at the chromatogram: right column lenght and internal diameter, consider beads dimension, column flow and LC pump pressure (HPLC). • 4 hour gradient • 0,4 ml/min flow rate Data search using PD 1.4 • PD application identifies proteins from the mass spectra of digested fragmented peptides. It compares the raw data from mass spectrometry to the information from a selected FASTA database • Sequest HT search Engine: calculates XCorr scores for peptide matches and provides the peptide matches having the best XCorr score for each spectrum • Percolator: algorithm that improves the discrimination between correct and incorrect spectrum identifications • Quantification: PD offers both isotopically labeled precursor ion quantification and isobarically labeled reporter ion quantification methods Data search using PD 1.4 Spectrum selector: m/z 350-700Da Precursor Mass Tolerance: 10ppm Fragment Mass Tolerance: 0.02Da Sequest Hit (scoring algorithm): Database: Human_Uniprot_fasta Enzyme: Trypsin NO Missed Clevages: 2 Peptide length: 6-14 Variable Modification: Oxidation (M), Phosphorylation (S,T,Y) Static Modification: Carbamidomethylation (C) Percolator (Target decoy search): Target FDR (Strict): 0.01 Target FDR: 0.05 Data analysis Control Click Protein groups 439 659 Merged Proteins 1111 1638 Peptides 1709 2703 PSMs 2642 4053 Serine containing proteins in the two data sets: Control: 15 Click: 22 Phosphorylated peptides in the two data sets: Control: 50 Click: 56 Venny 2.1 overlap analysis Protein accession numbers Gene IDs Highest abundant proteins PSM filtered Control: Click: Analysis of “Click” unique gene IDs KEGG by Enrichr Genes unique for “Click” pull down http://amp.pharm.mssm.edu/Enrichr/ Genes common in Ctrl and click, negative control KEGG’s table output by Enrichr http://amp.pharm.mssm.edu/Enrichr/ Gene IDs: http://amp.pharm.mssm.edu/Enrichr/ PSMB6;PSMD12;PSMD6;PSMA4;PSMD11;PSMC3;PSME3;PSMC1;PSMC2;PSMD3;PSME2;PSMD1 http://www.kegg.jp/kegg/pathway.html Enrichment analysis Gene Onthology using Enrichr http://amp.pharm.mssm.edu/Enrichr/ Enrichment analysis of gene ontology by Gorilla Unique “click” gene IDs http://cbl-gorilla.cs.technion.ac.il/ What could have gone wrong?! • Insufficient amount of probes use or degradation of probe • Lysis was incomplete • Oxygen in tube, click-reaction slows down • Proteins were not fully denatured after labeling • Amount of beads vs protein amount • Too little amount of biotin reagent/biotin reagent bad status (degraded)