Novel labeling technologies on proteins. + h Protein 1 Protein 2 FRET Fluorescence h BFP GFP a)...
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Transcript of Novel labeling technologies on proteins. + h Protein 1 Protein 2 FRET Fluorescence h BFP GFP a)...
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
phosphatase
527 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 -
OO
O
PO O
O - O N
N
O
NH2
PO O
O - 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
CO2H
CO2H
HO2C
protein
Oligo-histidine tagNi2+
OHO O
CO2 -
O NH
N- O2C
CO2 -CO2
-
Quantum dots
OP C8
C8C8
OP
C8
C8
C8
OP
C8
C8
C8
SCH2CH2CO2H
R
R
R
R =
QD1) DMAP
2) H2O
SCH2CH2CO2 -
R
R
R
HS-
R =
DNA
a)
OP C8
C8C8
OP
C8
C8
C8
OP
C8
C8
C8
QDDTT S S
HOOH
SS
OH
OH
SS
HO
HO
S S
HOO
O
N
N
SS
OHO
O
N
N
SS
HO
O
O
NN
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 pad
Immobilized 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
NO
O
S
NO
O
S
O
S
O
O
O
O
( ) n
O
S
O
OH
O
S
O
OH HN
O
OO
NH
OS
NH
H2N
O
peptide
O
S
O
O
O
OHN
OO
O
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
mLC-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
OX2C (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
base
NH
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