Novel labeling technologies on proteins

＋ a) hn b) c) Fluorescence Protein 1 BFP FRET GFP Protein 2 433 nm CFP YFP 14-3-3 t Substrate peptide 433 nm 476 nm PKA + ATP phosphatase 527 nm FRET pS b) Fig.1 Fluorescent protein labeling (Gene fusion approaches) Protein-protein interaction is detected by FRET using two different GFP analogues Detection of kinase activity using GFP-fusion protein Application of domain insertion for functional switching c) C N Protein or peptide GFP

a) b) Aminoacyl-tRNA Translated protein Ribosome mRNA without stop codon P site A site Puromycin-fluorophore conjugate C-terminus labeled protein a) b) Florpuro Cy5-puro

c) d) α-helical CCxCC domain S fluorescent FLASH Non-fluorescent protein Oligo-histidine tag Ni2+

Quantum dots

Surface modification of quantum dots QD 1) DMAP 2) H2O HS- DNA a) DTT H2N- Making of Barcode by encapuslating Q-dots in polymer beads

Protein array

(a) (b) Cell lysate (protein mixture) Each protein is detected by Ligand array Each protein is detected by direct labeling, labeled antibody, mass spectrometry or SPR (a) Protein array Fluorescent label Protein or other molecule, that is interested Molecular interaction is detected by direct labeling, labeled antibody, mass spectrometry or SPR (b)

b) Sample protein Various proteins are spotted on a membrane a) PEG Protein (adsorption) Protein (covalently immobilized) a) Immobilization using SAM CHO C = N O OH NH BSA O=C Glass plate d) Protein is immobilized covalently on Glass slide Glass plate Polyacrylamide gel pad Immobilized protein c) Protein immobilization in gel pad

Ligand (Antibody) Target protein Protein complex (a) (b)

Peptide Array

SPOT synthesis and epitope array for antibody-screening

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-4-hydroxycinnamic acid CHCA

MS fingerprint Peptide fragments Protein Detecting MS profile of fragments Electrophoresis gel MS analysis Trypsin digestion 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 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. fmol level is needed to keep practical sped

Quantified Proteome（Labeling of stable iostope） SILAC (Stable Isotope Labeling by Amino acids in Cell Culture) Harvest cells Combine and cell lysis Proteolysis after denaturation and reduction d3Leu, d3Met, d2Tyr) , d3 Ser, 13C6Arg, 13C6Lys Can’t be applied to animals mLC-MS 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.

Immuno-precipitation General strategy for investigating intracellular protein interaction with MS analysis. Cell lysis Trasfection to cell sample MS analysis b) Identification of interacting regions in protein-protein interaction After immuno-precipitation, the complex is crosslinked with this cross-linker, then was digested with protease to make fragment couple linked with the reagent. Stable isotope coded cross-linker

Isotope-coded Affinity Tag (ICAT)法 Isotope-coded Affinity Tag (ICAT) Comparison of protein expression levels between two samples by using fragments containing cysteine residue Purified with Avidin column

Application of ICAT to phosphoproteome Biotin base H2O m/z=446 fragment b) a) Scheme of isolating phosphorylated peptide b) Reaction scheme of the chemical conversion of phosphoserine residue to a biotinylated moiety.