Novel labeling technologies on proteins

＋

hnProtein 1

Protein 2

FRET

Fluorescence

hn

Fluorescence

BFP

GFP

a)

CFP

YFP

14-3-3 t

Substratepeptide

433 nm

476 nmPKA + ATP

phosphatase527 nm

433 nm

FRETpS

b)

c)

CN

Protein or peptide

GFP

CN

Fig.1 Fluorescent protein labeling (Gene fusion approaches)a Protein-protein interaction is detected by

FRET using two different GFP analoguesb Detection of kinase activity using GFP-fusion

proteinc Application of domain insertion for functional

switching

Aminoacyl-tRNA

Translated protein

Ribosome

mRNA withoutstop codon

P site A sitePuromycin-fluorophore conjugate

C-terminus labeled protein

a)

OHO O

CO2 -

HNO O P

O

O -O O N

OH

N

N

N

NH3C CH3

NH

NH2

OCH3O

b)

Florpuro

N N+

- O3S SO3

-

O OOPO OO - O N

N

O

NH2

PO OO - O N

OH

N

N

N

NH3C CH3

NH

NH2

OCH3O

NH

Cy5-puro

FLASHNon-fluorescent

fluorescent

c)

OHO O

CO2 -

As AsSS SS

O

O

HO

COOHAs

As

S

SS

S

α-helical CCxCC domain

d)OHO O

CO2 -

O NH

N

CO2HCO2H

HO2C

protein

Oligo-histidine tagNi2+

OHO O

CO2 -

O NH

N- O2C

CO2 -CO2

-

Quantum dots

OP C8

C8C8

OP

C8

C8

C8

OPC8

C8C8

SCH2CH2CO2H

R

R

RR ＝

QD1) DMAP

2) H2O

SCH2CH2CO2 -

R

R

R

HS-

R ＝

DNA

a)

OP C8

C8C8

OP

C8

C8

C8

OPC8

C8C8

QDDTT S S

HOOH

SS

OH

OH

SS

HO

HO

S S

HOO

O

N

N

SS

OHO

O

NN

SS

HO

O

ON

N

N N N

O

NDNA

H2N-

Surface modification of quantum dots

Making of Barcode by encapuslating Q-dots in polymer beads

Protein array

Cell lysate(protein mixture)

Ligand array

Each protein is detected bydirect 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)

Sample protein

Various proteins are spotted on a membrane

a)b)

PEG

Protein (adsorption)Protein (covalently immobilized)

a) Immobilization using SAM

Glass plate

Polyacrylamide gel padImmobilized protein

c)

Protein immobilization in gel pad

CHO C

=NO

OH

NH

BSA

NHO=C

Glass plate

d)

Protein is immobilized covalently onGlass slide

Ligand (Antibody)

Target protein

Protein complex

(a)

Ligand (Antibody)

Protein complex

(b)

Peptide Array

a)

N

O

OO

O

O

OH

NH

HN

Opeptide

NH

NO

O

O

Si O SiO

NH2

O Si O

NHO

NO

Ob)

peptide SH N

O

O

S

NO

O

S

O

S

O

O

O

O

( ) n

O

S

O

OH

O

S

O

OH HN

OO

O NH

OS

NH

H2N

O

peptide O

S

O

O

O

OHNO

OO

NHO

S

O

NH2

NH

O

S

O

OH

O

S

O

OH

c)

Si

NH

O OO

O

HO

Si

NH

O OO

O

HO

d)

H2N O

peptide Si

NH

O OO

O

NO

H

Si

NH

O OO

O

NO

H

e)

Si

NH

O OO

O

HO

Si

NH

O OO

O

HO

RHN

NH2O

NH

O

NH2

SH

peptide Si

NH

O OO

OS

RNH

NH2

OHN

O

HN

Si

NH

O OO

OS

RNH

NH2

OHN

O

HN

SPOT synthesis antibody

POD

SPOT synthesis antibody

POD

SPOT synthesis and epitope array for antibody-screening

Protein kinase

P

Target substrate is phosphorylated

P

Fluorescence-labeled antibody

Substrate array

Peptide-array for the detection of protein kinase activity

Proteomics & Mass Spectrometry

MALDI TOF MSMatrix assisted laser desorption ionization – Time of flightMass spectroscopy

α-cyano-4-hydroxycinnamic acidCHCA

MS fingerprint

Electrophoresis gel Protein Peptide fragments

Trypsin digestion MS analysis

Detecting MS profileof fragments

comparison

Database of protein sequences

Simulation of trypsin digesting pattern

Database of predicted MS fingerprintsof each known proteins

control experimental

Denature and reduce

N

N

N

N

C

C

C

C

Proteolysis using H216O

Proteolysis using H218O

A

B

combine

LC-MS

LC-MS

m/z

A1 A2 A3

B1

B2

B3

B4

m/z

aa4 aa3 aa2 aa1

Enzymatic labeling of stable isotope coding of proteomicsProteins 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.

Peptide MS fingerprint and Peptide sequence Tag

fmol level is needed to keep practical sped

control

Cells caltured using 1H/12C/14N -coded amnio acids or 15N-minimal media

experimental

Cells caltured using 2H/13C/15N -coded amnio acids or 15N-enriched media

Harvest cells

Combine and cell lysis

Proteolysis after denaturation and reduction

LC-MS

m/z

A1 A2 A3

B1

B2

B3

B4

in vivo stable isotope labeling of proteome sampleCells 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.

Quantified Proteome （ Labeling of stable iostope ）SILAC (Stable Isotope Labeling by Amino acids in Cell Culture)

d3Leu, d3Met, d2Tyr) , d3 Ser, 13C6Arg, 13C6Lys

Can’t be applied to animals

a)

Expression vector incorporated target gene combined with epitope tag gene

Trasfection to cell sample

Cell lysis Immunoprecipitation with

anti-epitope antibody

MS analysis

After immuno-precipitation, the complex is crosslinked with this cross-linker, then was digested with protease to make fragment couple linked with the reagent.

a) Immuno-precipitation General strategy for investigating intracellular protein interaction with MS analysis.

N

NaO3SO

O

O

O X2C (CH2)n

X2C

O

O N

SO3NaO

O

b)

X= H or D

b) Identification of interacting regions in protein-protein interaction

Stable isotope coded cross-linker

Isotope-coded Affinity Tag (ICAT) 法

Purified with Avidin column

Isotope-coded Affinity Tag (ICAT)Comparison of protein expression levels between two samples

by using fragments containing cysteine residue

Pa)

Biotin

Biotin

NH

O

R'R

PO3H2

O

baseNH

CH2

R'R

O

HSSH

NH

CH2

R'R

O

S

SH

S

NHHN

O

NH

O

OO

NH

O

N

O

O

NH

CH2

R'R

O

S

SN

O

O

S

NHHN

O

H2O

NH

CH2

R'R

O

S

SNH

HO2C

O

S

NHHN

O

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.

Application of ICAT to phosphoproteome