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Focus on right sp
ots using
EttanDIG
E
GEH
C is
pa
rt o
f a
fa
mily
of
bu
sin
esse
s a
lign
ed w
ith
ou
r cu
sto
mer
s’ n
eed
s a
nd
act
ing
as
on
e co
mp
an
y to
dri
ve
gro
wth
.
Co
mm
erci
al F
ina
nce
Hea
lth
care
NB
C U
niv
ersa
l
Ind
ust
ria
lIn
fra
stru
ctu
re
Co
nsu
mer
Fin
an
ce
Pro
tein
Sci
ence
s P
rod
uct
s
Samplepreparation
Protein chracterisation
>Et
tan
DIG
E>
Bia
core
(SP
R)
>C
alo
rim
etry
, Mic
roca
l
Protein Purifica
tion -ÄKTA
>Ä
KTA
>Se
ph
ad
ex, H
iTra
p, S
OU
RC
E>
µg
-kg
2
0
100
46
8hours
% blocked target
0
Wh
at´s
diff
eren
t?
Diff
eren
t m
eth
od
s d
evel
op
diff
eren
t su
bse
ts o
f th
e p
rote
om
e w
ith
su
rpri
sin
gly
litt
le o
verl
ap
s.
Even
on
e a
nd
th
e sa
me
sam
ple
a
na
lyze
d o
n d
iffer
ent
bra
nd
s o
f ES
I or
MA
LDI i
nst
rum
ents
iden
tify
p
red
om
ina
ntl
y n
on
-red
un
da
nt
pro
tein
s.
Even
tua
lly, a
na
lyzi
ng
a s
tati
stic
ally
su
ffic
ien
t n
um
ber
of
rep
lica
tes
mig
ht
imp
rove
, bu
t n
ot
elim
ina
te t
his
d
ilem
ma
.
The
per
fect
wo
rkflo
w is
no
t in
sig
ht,
yet.
2DE-MALD
I
2DE-ES
I
PPF
LC-M
ALD
I
(sch
ema
tic
Ven
n d
iag
ram
)
Pro
teomics workflows
Differe
ntial a
nalysis: considera
tions
2-D
Ele
ctro
ph
ore
sis
-St
ren
gth
s
Ph
ysic
o-c
hem
ica
lpa
ram
eter
so
f p
rote
ins
mea
sure
d
Iso
form
sa
nd
po
st-t
ran
sla
tio
na
lmo
difi
cati
on
sd
isp
laye
d,
Hig
h r
eso
luti
on
, pa
rtic
ula
rly
aft
er p
refr
act
ion
ati
on
Hig
h t
hro
ug
hp
ut,
pa
ralle
l ru
ns
Cru
de
sam
ple
s to
lera
nce
Mu
ltip
le d
etec
tio
n, b
lott
ing
, ap
plic
ab
le
Effic
ien
t fr
act
ion
co
llect
or
No
n-s
ho
tgu
n m
eth
od
Mu
ltip
lexi
ng
, DIG
E
Rep
rod
uci
bili
ty, i
nte
rna
l sta
nd
ard
(DIG
E)
Diff
eren
tia
l an
aly
sis
usi
ng
2D
ele
ctro
ph
ore
sis
Diffe
rences d
ue t
o d
isease s
tate
, dru
g t
reatm
ent,
life
cycle
sta
ge
Induced b
iolo
gic
al
chang
e -
wh
at
we
wa
nt
to m
easu
re
Syste
m v
ari
ation
(gel-to
-gel vari
ation)
Exp
erim
enta
l fa
cto
rs
Diffe
rences in I
EF
/SD
S-P
AG
E c
onditio
ns
Gel dis
tort
ions
user-
to-u
ser
va
riation
Data
ana
lysis
user-
specific
editin
g a
nd inte
rpre
tation
Inhere
nt
bio
log
ical
variatio
n
Intr
insic
diffe
rences t
hat
occur
with
in p
opula
tions
eg
anim
al-to
-anim
al, p
lant-
to-p
lant
or
culture
-to-c
ulture
,
subje
cte
d t
o id
entical co
nditio
ns
DIGE –internalstandard
Fact
s a
bo
ut
the
ori
gin
s o
f ex
per
imen
tal v
ari
an
ces
in b
ioa
na
lyti
cs
Tech
nic
al v
ari
an
ces
Sa
mp
le p
rep
ara
tio
n (S
OP
)> 5
-20
%
MS-
mea
sure
men
t va
ria
nce
s< 5
-10
%
Err
ors
in d
ata
eva
lua
tio
n (F
PR
)> 5
%??
??
Bio
log
ica
l va
ria
nce
s
Cel
l cu
ltu
re s
am
ple
s >
5-2
0%
Sa
mp
les
fro
m in
bre
d m
ice>
20
-40
0%
Hu
ma
n t
issu
e sa
mp
les>
4-5
-fo
ld
Hu
ma
n c
an
cer
tiss
ue
sam
ple
s >
2-2
0-f
old
fro
m H
elm
ut
E.M
eyer
, Bo
chu
m, s
cien
tific
ta
lk, H
am
bu
rg 2
00
8
Tra
ckingof (repro
ducible?) pro
tein
loss
es
Tra
ckingof (repro
ducible?) pro
tein
loss
es
TF
(fals
en
ega
tive
)
Pro
tein
isreally
diff
eren
tia
lly e
xpre
ssed
F(fa
lse
po
siti
ve)
TP
rote
in is not
diff
eren
tia
lly e
xpre
ssed
Pro
tein
isd
etec
ted
as
diff
eren
tia
lly e
xpre
ssed
P
rote
in is
not
det
ecte
d a
s d
iffer
enti
ally
exp
ress
ed
Differe
ntial a
nalysis using 2D electrophoresis
T = tru
e resu
ltF = false resu
lt
•W
hat re
sults ca
n you exp
ect?
Statistics
H0
H1
Differe
ntial a
nalysis using 2D electrophoresis
inducedvariance
Statistics
H0
H1
Ω²
Differe
ntial a
nalysis using 2D electrophoresis
Not differe
ntlyexp
ress
ed, b
ut
detectedas differe
ntlyexp
ress
ed
= falsepositive
Differe
ntlyexp
ress
ed, b
utnot
detectedas differe
ntlyexp
ress
ed
= falsenegative
Detectedas differe
ntly
exp
ress
ed, e
xcluding
falsenegatives
Ettan D
IGE
Ettan D
IGE is
an in
tegra
ted solution for
the detection and quantification
of real b
iological d
iffere
nces in pro
tein
exp
ress
ion.
DIG
E co
mp
on
ents
DIG
E D
yes
Ima
gin
g h
ard
wa
re
DIG
E a
na
lysi
s so
ftw
are
CyD
yeD
IGE
Flu
or
dye
sM
inim
al l
ab
ellin
gd
yes
Lab
el 5
0 µ
g o
f pro
tein
3 c
olo
rs: C
y™2
, Cy3
, Cy5
MW
ma
tch
ed (~
45
0D
a)
Ch
arg
e m
atc
hed
(po
siti
ve)
Lab
el ε
-am
ino
gro
up
of
lysi
ne
Sen
siti
vity
-0
.02
5 n
g
Lin
ear
dyn
am
icra
ng
eo
ver
4 o
rder
s o
f
ma
gn
itud
e
NH
S r
eactive g
roup
N
N+
N
OO
O
O
Lab
ellin
g C
hem
istr
yMinim
al Dyes Dye+
N+
N
ON H
pH
8.5
pro
tein
+H
3N
-pro
tein
Dye+
A p
ositiv
ely
ch
arg
ed d
ye m
ole
cule
re
pla
ces t
he p
ositiv
e
charg
ed lysin
e o
n t
he p
rote
in -
no n
et
chang
e in p
I
Sen
siti
vity
Take
nfr
om
:Proteomicsin Drug Research
Edite
db
yK
. Ma
rcu
s, K
. Stü
hle
r, A
. va
n H
all,
M. H
am
ach
er, B
. Wa
rsch
eid
, H. E
. Mey
erC
op
yrig
ht
© 2
006
Wile
y-V
CH
Ver
lag
Gm
bH
& C
o. K
Ga
A, W
ein
hei
m IS
BN
: 3-5
27-3
122
6-9
DIG
Ew
ork
flow
Multiplexing2-D
E w
ithan in
tern
als
tandard
Ove
rla
y o
f n
orm
al a
nd
tra
nsg
enic
mo
use
im
ag
es
Normal mouse = CyTM3 labelled -Blue
Transgenic = Cy5 Labelled -Red
Increased abundance
Equal abundance
Reduced abundance
Co-d
etection and m
atching
Image 1:P
ooled in
tern
al s
tandard
Boundaries transferred
to im
age 2 and 3
Image 2: S
ample 1
Image 3: S
ample 2
Gel 1
Gel 2
•Boundaries are use
d for
quantita
tionrelative to pooled
intern
al s
tandard
•Matching betw
een gels via
pooled in
tern
al s
tandard
Ettan D
IGE in
tern
al s
tandard
Sample 2, g
el 2
Sample 1, g
el1
Sample 3, g
el 3
Sample 4, g
el 4
Conclusion
exp
ress
ion in
crease
in samples 3 and 4
Is this system
variation or induce
d
biological c
hange?
Sample 2 (C
y5)
Sample 1 (C
y3)
Gel 1
Sample 3 (C
y3)
Sample 4 (C
y5)
Gel 2
Sta
ndard
(Cy2)
Sta
ndard
(Cy2)
Conclusion
exp
ress
ion decrease
in sample 3
Ettan D
IGE in
tern
al s
tandard
Typhoon,
DIG
E lysis
buff
er
incl. P
IS
tandard
ly
sis
buff
er
inc. P
I
Caco-2
cells
Cy5
Cy5
Cy5
Cy5
Cy5
Cy5
Cy3
6 ×
50 µ
g300 µ
g
Cy5
Cy5
Cy5
Cy5
Cy5
Cy5
Cy3
Cy3
Cy3
Cy3
Cy3
Cy3
6 ×
50 µ
g
6 S
YP
RO
Rub
y g
els
6 D
IGE
gels
(2 ×
50 µ
g /gel)
Analy
se b
y P
DQ
uest
Rem
ark
:
Influence o
f diffe
rence in
lysis
conditio
ns is n
ot
taken into
account in
this
set-
up
Analy
se b
y D
eC
yder
0
50
10
0
15
0
20
0
25
0
30
0
35
0
40
0
Distribution of ratios
Mean values gel 1, 2, 3 vs. gel 4, 5, 6
0.28
0.4
0.52
0.64
0.76
0.88
1
1.12
1.24
1.36
1.48
1.6
1.72
1.84
1.96
2.08
2.2
2.32
2.44
SY
PR
O R
ub
y,
PD
Qu
est
DIG
E,
De
Cyde
r
Range
Number of spots in bin
Another wayto compare
overall system perform
ance
Mea
suri
ng
ind
uce
d b
iolo
gic
al c
ha
ng
e
Diffe
rences d
ue t
o d
isease s
tate
, dru
g t
reatm
ent,
life
cycle
sta
ge
Induced b
iolo
gic
al
chang
e -
wh
at
we
wa
nt
to m
easu
re
Syste
m v
ari
ation
(gel-to
-gel vari
ation)
Exp
erim
enta
l fa
cto
rs
Diffe
rences in I
EF
/SD
S-P
AG
E c
onditio
ns
Gel dis
tort
ions
user-
to-u
ser
va
riation
Data
ana
lysis
user-
specific
editin
g a
nd inte
rpre
tation
Inhere
nt
bio
log
ical
variatio
n
Intr
insic
diffe
rences t
hat
occur
with
in p
opula
tions
eg
anim
al-to
-anim
al, p
lant-
to-p
lant
or
culture
-to-c
ulture
,
subje
cte
d t
o id
entical co
nditio
ns
DIGE –internalstandard
Statistics
H0
H1
Ω²
Differe
ntial a
nalysis using 2D electrophoresis
Not differe
ntlyexp
ress
ed, b
ut
detectedas differe
ntlyexp
ress
ed
= falsepositive
Differe
ntlyexp
ress
ed, b
utnot
detectedas differe
ntlyexp
ress
ed
= falsenegative
Detectedas differe
ntly
exp
ress
ed, e
xcluding
falsenegatives
Ho
w m
an
y re
plic
ate
s d
o I
nee
d t
o
det
ect
a s
elec
ted
exp
ress
ion
ch
an
ge?
Stro
ng
ef
fect
1
00
%
med
ium
ef
fect
5
0%
sma
ll ef
fect
2
5%
(%)
34
81
0D
IGE
10
23
68
30
Syp
ro
Ru
by
52
13
3*
41
6*
80
Silv
er
no
. of
rep
lica
tes
nee
ded
to
d
etec
t a
n e
xpre
ssio
n
cha
ng
e o
f
Va
ria
tio
nsy
stem
+
bio
log
ica
l
Sta
in o
r la
bel
* Exp
ress
ion changes sm
alle
r th
an 2-fold not easy
to publis
h
Pro
teo
mic
s: N
o M
ista
kes
Allo
wed
!
Ettan D
IGE
Applic
ations
Ettan D
IGE public
ations: to June 09
0
50
100
150
200
250
300
350
400
2000200120022003200420052006200720082009
Review
Research
0
200
400
600
800
1000
1200
1400
1600
1800
1997199819992000200120022003200420052006200720082009
Review
Research
•Healthy and tumor sa
mples
from 6 patients = 6 gels
•52 unique pro
teins identified
with significant changes
•42 of th
ese
were
only evident
when in
cluding the in
tern
al
standard
in the analysis
DIGE;
42
excl. Int
Std; 10 Fr
ied
ma
n e
t a
l. Proteomics
2004
; 4(3
): 79
3-8
11
Ettan D
IGE for analysis of human colon
cancer
No
n-
fra
ctio
na
ted
Mem
bra
ne
fra
ctio
nC
yto
solic
fra
ctio
n
Cy™
3C
ell s
urf
ace
Silv
er s
tain
ed
Cel
l su
rfa
cela
bel
ling
spec
ifici
ty
.Ettan D
IGE & Blue N
ative Electrophoresis
•Pro
tein complexe
s of
Ara
bidopsis mitoch
ondria
•(Cy3, g
reen) resp
irato
ry
system and chloro
plasts
•(Cy5, red) p
hoto
synth
esis
system.
Blue N
ative PAGE course,
October 2006,
Institute for Plant Genetics
, University of Hannover
CyD
yeD
IGE
Flu
ors
Sca
rce
sam
ple
lab
ellin
gd
yes
Lab
el 5
µg
of
pro
tein
2 c
olo
rs: C
y™3
, Cy5
Size
ma
tch
ed (~
68
0D
a)
Ch
arg
e m
atc
hed
(neu
tra
l)
Lab
el t
hio
lgro
up
of
cyst
ein
e
NO
N H
O
O
NO
N H
O
OS
Male
imid
e r
eactive g
roup
Lab
ellin
g c
hem
istr
y -
2. C
ou
plin
gCyDye DIGE Fluor saturation dyes
Dye
37 °
C,
30 m
in
pH
8.0
pro
tein
HS
-pro
tein
Dye
Satu
ration labe
llin
g -
all
ava
ilable
cyste
ines
are
la
be
lled
Enable
s m
ore
sensitiv
e d
ete
ction f
or
scarc
e s
am
ple
s
Hs766
5.000 cells
+ Cy3
5.000 cells
+ Cy5
Silverstainof
10.000 cells
Cy5 stainof
5.000 cells
Co
urt
esy
of
Ka
i Stü
hle
r,R
U, B
och
um
, DE
Rel
ati
ve S
ensi
tivi
ty
Harvest
Flowthrough
2-D
DIG
E resu
lts
Eluate
MabSelectSuRe™
Affinity
chromatography
Chromatogram
1-D SDS PA
GE
Sep
ara
tion
acc
ord
ing
to
siz
eP
rote
ins
sta
ined
wit
h C
oo
ma
ssie
™ o
r Si
lver
Dra
wb
ack
s:Lo
w s
ensi
tivi
ty (C
oo
ma
ssie
)N
arr
ow
dyn
am
ic r
an
ge
Low
rep
rod
uci
bili
tyLi
mit
ed s
epa
ratio
n r
eso
luti
on
ELISA
Ba
sed
on
an
tig
en-a
ntib
od
y re
cog
nit
ion
Ove
rall
pro
tein
det
ectio
n w
ith
ou
t se
pa
rati
on
Dra
wb
ack
s:N
ot
all
HC
P a
re d
etec
ted
-a
ccu
racy
ba
sed
o
n q
ua
lity
of
po
lycl
on
al a
ntib
od
y a
ga
inst
H
CP
Ba
tch
to
ba
tch
va
ria
tion
s o
f a
nti
bo
dy
Spec
ifici
ty, s
ensi
tivi
ty a
nd
cr
oss
rea
ctiv
ity
mu
st b
e ve
rifie
d
FDA
rec
entl
y ex
pre
ssed
th
eir
con
cern
s re
ga
rdin
g t
he
qu
alit
y o
f EL
ISA
HC
P
an
tib
od
ies
Cu
rren
t te
chn
iqu
es f
or
HC
P a
na
lysi
s
Harvest
Flow through
Eluate
IgGhc
IgGlc
Harvest
Flow through
Eluate
A)
B)
Impro
ved analysis of HCP w
ith 2-D
DIG
E
2-D DIGE (50 µ µµµg)
1-D SDS PAGE (5µ µµµg)
Coomassie™ staining
Increase
d reso
lution (2
-D separa
tion)
Red
ox-DIGE using cysteine labeling
2D
DIG
ERed
spots:p
rote
ins
ha
veu
nd
erg
on
ere
do
x-se
nsi
tive
thio
l mo
d.
Yello
wYe
llow
spots
spots:p
rote
ins
con
tain
ing
occ
lud
edth
iols
(N-e
thyl
ma
leim
ide)
from:H
urd
TR ,
Pri
me
TA, H
arb
ou
rM
E, L
illey
KS,
Mu
rph
y M
P.
Det
ecti
on
of
rea
ctiv
e o
xyg
en s
pec
ies-
sen
siti
ve t
hio
l pro
tein
s b
y re
do
x
diff
eren
ce g
el e
lect
rop
ho
resi
s.
J B
iolC
hem
28
2 (2
00
7) 2
20
40
–22
05
1.
No
np
ho
sph
ory
late
da
nd
ph
osp
ho
ryla
ted
fo
rms
of
GSK
3β
cou
ld b
e d
etec
ted
in
PC
-3U
cel
ls w
ith
ou
t TG
F-β
act
iva
tio
n.
Ettan D
IGE & M
ultiplex W
estern
Blotting
Va
lue
of
Etta
nD
IGE
”…w
ith
ou
tth
e b
enef
ito
f th
e in
tern
als
tan
da
rd…
42
pro
tein
sw
ou
ldh
ave
bee
no
verl
oo
ked
du
eto
larg
ed
egre
eo
f va
ria
tio
n…
”
Frie
dm
an
et
al. Proteomics
20
04
, 5, 7
93
-81
1
Red
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nw
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