1. Novel labeling technologies on proteins

2. ＋ a) hn b) c) Fluorescence Protein 1 BFP FRET GFP Protein 2 433 nm CFP
YFP 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

3. 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

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

5. Quantum dots

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

7. Protein array

8. (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)

9. 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

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

11. Peptide Array

13. SPOT synthesis and epitope array for antibody-screening

14. Peptide-array for the detection of protein kinase activity

15. Proteomics & Mass Spectrometry

16. MALDI TOF MS
Matrix assisted laser desorption ionization – Time of flight
Mass spectroscopy
α-cyano-4-hydroxycinnamic acid
CHCA

17. 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

18. 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

19. 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.

20. 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

21. 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

22. 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.