Novel labeling technologies on proteins a) hn Protein 1 hn BFP FRET Fluorescence ＋ Fluorescence GFP Protein 2 b) 433 nm 433 nm CFP PKA + ATP 14-3-3 t 476 nm pS FRET phosphatase Substrate peptide YFP 527 nm Fig.1 Fluorescent protein labeling (Gene fusion approaches) aProtein-protein interaction is detected by FRET using two different GFP analogues bDetection of kinase activity using GFP-fusion protein cApplication of domain insertion for functional switching c) Protein or peptide GFP N C N C a) Translated protein Puromycin-fluorophore conjugate P site A site mRNA without stop codon Aminoacyl-tRNA Ribosome b) C-terminus labeled protein - O3S HO SO3 - O O H3C CO2 - O HN N+ N O O P O O- N NH2 N O N CH3 N O Florpuro NHOH OCHCy5-puro 3 O O N H N O O P O O- N O N O H3C N CH3 N O P O O- NH2 O N N N NHOH O OCH3 NH2 c) α-helical CCxCC domain S As HO S S As S S O O O As CO2 S S - fluorescent O COOH As S HO FLASH Non-fluorescent d) HO O O HO O O CO2 - CO2 - protein CO2H O N H N HO2C O CO2H N H Ni2+ N - O2C -2 CO CO 2 Oligo-histidine tag Quantum dots Surface modification of quantum dots a) QD O C8 P C8 C8 R C8 C 8 P C 8 O R 1) DMAP R R O P C8 C8 C8 2) H2O HSR R R＝ SCH2CH2CO2H R＝ O QD O C8 P C8 C8 O P C8 C8 C8 O OH DTT N N N S S N DNA H2N- S S S HO N N S S HO S S O O N O HO SCH2CH2CO2 HO N C8 C 8 P C 8 O DNA S S S OH N OH HO OH O N O Making of Barcode by encapuslating Q-dots in polymer beads Protein array (a) Cell lysate (protein mixture) Each protein is detected by direct labeling, labeled antibody, mass spectrometry or SPR Ligand array (b) Protein or other molecule, that is interested Fluorescent label Protein array Molecular interaction is detected by direct labeling, labeled antibody, mass spectrometry or SPR b) a) Protein (covalently immobilized) Protein (adsorption) Sample protein PEG Various proteins are spotted on a membrane a) Immobilization using SAM d) c) Immobilized protein Polyacrylamide gel pad N = CHO C Glass plate Protein immobilization in gel pad BSA NH O=C NH O OH Glass plate Protein is immobilized covalently on Glass slide (a) (b) Target protein Protein complex Protein complex Ligand (Antibody) Ligand (Antibody) Peptide Array a) O b) O O O O N O N N O O O S NH O OH peptide HN O NH2 NH NH SH peptide O N O O O Si O Si O Si O O c) NH O O S O NH2 N H O O O HN O ( OH OH H N n ) O O O N H H N 2 O peptide O O OH S N H O O O S S S d) H O O NH Si Si O O O O O O H H O NH O N O N H O O O H2N O NH NH peptide Si O O O Si O O O e) R R NH O O NH2 O H O O O O NH Si O O O NH Si O O O O O O S H OH O O O S O O O O O H R N O S O N H NH2 peptide HN O NH2 HN HN SH NH2 NH O HN S O S O NH Si O O O NH Si O O O S N O POD SPOT synthesis antibody SPOT synthesis and epitope array for antibody-screening Protein kinase P Target substrate is phosphorylated Substrate array Fluorescence-labeled antibody P Peptide-array for the detection of protein kinase activity Proteomics & Mass Spectrometry MALDI TOF MS Matrix assisted laser desorption ionization – Time of flight Mass spectroscopy α-cyano-4hydroxycinnamic acid CHCA MS fingerprint Electrophoresis gel Peptide fragments Protein Trypsin digestion Detecting MS profile of fragments MS analysis comparison Database of protein sequences Database of predicted MS fingerprints of each known proteins Simulation of trypsin digesting pattern Peptide MS fingerprint and Peptide sequence Tag A B control A1 experimental LC-MS B1 B3 A2 B2 A3 B4 Denature and reduce m/z N N C N C N Proteolysis using H216O aa4 C aa3 aa2 aa1 C Proteolysis using H218O m/z combine LC-MS fmol level is needed to keep practical sped Enzymatic labeling of stable isotope coding of proteomics Proteins from two distinct proteome are digested with protease in normal water or isotopically labeled water. Isotobe code is labeled in every C-terminus of the digested peptides. Then, two samples are combined and analyzed by LC-MS/MS. Expression level of proteins between two states can be estimated. Amino acid sequence of selected peptide fragment can be identified, too. Quantified Proteome（Labeling of stable iostope） SILAC (Stable Isotope Labeling by Amino acids in Cell Culture) control Cells caltured using 1H/12C/14N -coded amnio acids or 15N-minimal media Harvest cells experimental Combine and cell lysis Cells caltured using 2H/13C/15N -coded amnio acids or 15N-enriched media A1 Proteolysis after and reduction B1 d3Leu, d3Met, d2Tyr) , d3 13 13 LC-MS Ser, C6Arg, C6Lys B3 A2 B2 denaturation A3 B4 Can’t be applied to animals m/z in vivo stable isotope labeling of proteome sample Cells are grown in normal media or isotopically labeled media. Mass tags are incorporated into every protein. An equivalent number of cells for each sample are combined and processed for MS. a) Immuno-precipitation General strategy for investigating intracellular protein interaction with MS analysis. a) Cell lysis Immunoprecipitation with anti-epitope antibody Trasfection to cell sample Expression vector incorporated target gene combined with epitope tag gene MS analysis b) Identification of interacting regions in protein-protein interaction b) O O NaO3S O N O O X2 C (CH2)n X= H or D X2 C SO3Na O O N O Stable isotope coded cross-linker After immuno-precipitation, the complex is crosslinked with this cross-linker, then was digested with protease to make fragment couple linked with the reagent. Isotope-coded Affinity Tag (ICAT) Comparison of protein expression levels between two samples by using fragments containing cysteine residue Purified with Avidin column Isotope-coded Affinity Tag (ICAT)法 Application of ICAT to phosphoproteome a) P Biotin Biotin b) SH PO3H2 O R CH2 base R R' N H R' N H SH HS S R O O N H O HN NH O O N H S O O CH2 R' O O N H N O O HN a) O HN NH NH O S N H O S N S S HO2C H2O m/z=446 fragment S R N H O S CH2 R' O R N H CH2 R' O Scheme of isolating phosphorylated peptide b) Reaction scheme of the chemical conversion of phosphoserine residue to a biotinylated moiety.