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.