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TRANSCRIPT
A N
ovel
Met
hod
For D
isco
very
of P
erip
hera
l Blo
od B
iom
arke
rs in
Idio
path
ic P
ulm
onar
y Fi
bros
is U
sing
Ext
ensi
ve D
eple
tion
and
TMTc
alib
rato
r™ T
issu
e-En
hanc
ed P
lasm
a Pr
oteo
mic
sI.
Pike
1 , M
. Bre
man
g1, P
.J. W
olte
rs2 ,
R. G
aste
r3, S
. Tur
ner3
, M. D
ecar
is3
1 –
Prot
eom
e Sc
ienc
es p
lc, H
amilt
on H
ouse
, Mab
ledo
nPl
ace,
Lon
don,
WC
1H 9
BB, U
K; 2
–U
nive
rsity
of C
alifo
rnia
at S
an F
ranc
isco
, San
Fra
ncis
co, U
SA;
3 –
PLIA
NT
Ther
apeu
tics,
700
Sag
inaw
Driv
e, S
uite
150
, Red
woo
d C
ity, C
A 94
063,
USA
Intr
oduc
tion
Perip
hera
lbi
omar
kers
rela
ted
toth
epa
thog
enes
isof
idio
path
icpu
lmon
ary
fibro
sis
(IPF)
are
urge
ntly
need
edto
impr
ove
diag
nosi
s,se
lect
ion
and
asse
ssm
ent
oftre
atm
ent,
parti
cula
rlyin
the
cont
exto
fnew
drug
deve
lopm
ent.
Rec
ent
impr
ovem
ents
inth
ede
plet
ion
ofhi
ghan
dm
ediu
mab
unda
ntpr
otei
nsan
dde
velo
pmen
tof
tissu
e-en
hanc
edflu
idpr
oteo
mic
sha
vein
crea
sed
the
brea
dth
and
dept
hof
plas
ma
prot
eom
eco
vera
ge.W
eha
veno
wco
mbi
ned
thes
em
etho
dsto
obta
inun
para
llele
dco
vera
geof
the
IPF
plas
ma
prot
eom
e.
Met
hod
Long
itudi
nal
arch
ival
plas
ma
sam
ples
(n=2
5)fro
msi
xin
divi
dual
sw
ithIP
Fan
dfiv
eco
ntro
lin
divi
dual
sw
ere
obta
ined
from
the
UC
SFBi
oban
kan
dpr
oces
sed
assh
own
inFi
gure
1.•
Supe
r-dep
letio
nof
the
top
~70
high
and
med
ium
abun
dant
prot
eins
usin
gSe
ppro
®Ig
Y14
and
Supe
rmix
®
colu
mns
(Mer
ck).
•Pl
ex#1
-5ea
chco
mpr
ised
6pl
asm
asa
mpl
esw
ith4
tissu
etri
gger
/cal
ibra
ntsa
mpl
es.
Plex
6co
mpr
ised
10pl
asm
asa
mpl
eson
ly.•
Each
TMT®
10pl
exw
asan
alys
edus
ing
30x
2ho
urgr
adie
nton
anO
rbitr
ap®
Fusi
onTr
ibrid
®m
ass
spec
trom
eter
with
in-li
neuH
PLC
.•
MS
files
wer
epr
oces
sed
with
ase
quen
tial
SEQ
UES
Tse
arch
stra
tegy
inPr
oteo
me
Dis
cove
rer
v1.4
(The
rmo
Scie
ntifi
c)w
itha
stan
dard
sear
chfo
llow
edby
abe
spok
em
etho
din
the
resi
dual
unm
atch
edsp
ectra
with
varia
ble
mod
ifica
tion
for
less
-com
mon
post
-tran
slat
iona
lm
odifi
catio
nssu
chas
hydr
oxyl
atio
nof
prol
ine
and
lysi
ne.
•D
ata
inte
grat
ion,
norm
alis
atio
n,qu
antit
atio
nan
dst
atis
tical
anal
yses
wer
epe
rform
edw
ithin
Prot
eom
eSc
ienc
es’i
n-ho
use
bioi
nfor
mat
ics
wor
kflo
ws.
•R
epor
ted
plas
ma
abun
danc
eof
sele
ctIP
F-re
late
dpr
otei
nsw
asob
tain
edth
roug
hth
ehu
man
plas
ma
prot
eom
eda
taba
sean
d/or
PubM
edse
arch
es.
Res
ults
&C
oncl
usio
n•
Supe
r-dep
letio
nre
mov
ed~9
9%of
tota
lpla
sma
prot
ein
(Fig
ure
2).
•D
etec
tion
limit
inm
id-p
g/m
lra
nge
inpl
asm
ach
anne
lsw
hen
usin
gth
etis
sue
trigg
er.
•~4
,000
prot
eins
quan
tifie
dw
ithSu
per-d
eple
tion
only,
mor
eth
ando
uble
dto
~9,0
00w
hen
tissu
etri
gger
adde
d(T
able
1).
•Ke
ypo
st-tr
ansl
atio
nally
mod
ified
prot
eins
only
dete
cted
whe
nus
ing
tissu
etri
gger
(Tab
le1.
)•
Impr
oved
cove
rage
ofse
lect
IPF-
rela
ted
prot
eins
(Tab
le2)
.•
Dis
tribu
tions
for
GO
cellu
lar
com
pone
nts
(Fig
ure
3A)
orbi
olog
ical
proc
esse
s(F
igur
e3B
)w
ere
sim
ilarw
ithor
with
outt
issu
etri
gger
.•
Prop
ortio
nally
grea
ter
impa
cton
dete
ctio
nof
cellu
lar
vsse
cret
edpr
otei
ncl
asse
s(F
igur
e4A
)an
dfo
rlu
ng-a
ssoc
iate
dpr
otei
nsw
ithTM
Tcal
ibra
tor™
than
inSu
per-d
eple
ted
plas
ma
alon
e(F
igur
e4B
).•
TMTc
alib
rato
r™w
ithSu
per-d
eple
tion
prov
ides
outs
tand
ing
prot
eom
eco
vera
gefo
rpl
asm
abi
omar
ker
disc
over
yof
ferin
gde
eper
insi
ghts
into
IPF
dise
ase
proc
esse
san
dre
spon
seto
treat
men
t.•
Larg
erst
udie
sre
quire
dfo
rrep
licat
ion/
valid
atio
nof
nove
lcan
dida
tebi
omar
kers
.
Figu
re 1
–TM
Tcal
ibra
tor™
wor
kflo
w w
ith p
lasm
a Su
per-d
eple
tion
and
inco
rpor
atio
n of
four
-poi
nt tr
igge
r/cal
ibra
nt. T
MT1
26 –
131N
= T
ande
m M
ass
Tag
repo
rter i
on m
ass-
to-c
harg
e.
Tabl
e 2
–Pe
ptid
e co
vera
ge o
f sel
ect I
PF-re
late
d pr
otei
ns. T
MTc
al+S
D=
Supe
r-dep
lete
d pl
asm
a w
ith fo
ur-c
hann
el tr
igge
r/cal
ibra
nt; S
D =
Sup
er-d
eple
tion
only
; C
once
ntra
tions
from
plasmaproteomedatabase.org;
NR
= n
ot re
porte
d
ww
w.pr
oteo
mic
s.co
m
TMTC
alibrator™
with
Sup
er-dep
letio
n
TMT126
TMT127N
TMT127C
TMT128N
TMT128C
TMT129N
TMT129C
TMT130N
TMT130C
TMT131N
TMT10plexReagentLabelling&Mixing
bRPFractionation
F1
F2
F3
F4
F5
F6
F7
F8
……
F30
LungTissueTrigger/Calibrant
1 4 6 10
0.000
1.000
2.000
3.000
4.000
5.000
6.000
7.000
IPFT0
IPF
T3
IPFT6
IPF
T9
IPFT1
2
CTL
CAL1CA
L2CA
L3CA
L4
Integrin
αV
LongitudinalPlasmaSamples
ProteinExtraction&TrypsinDigestion
Figu
re 4
–In
crea
sed
cove
rage
of t
he p
rote
ome
usin
g TM
Tcal
ibra
tor™
with
Su
per-d
eple
tion.
A –
Prot
ein
clas
s; B
–Lu
ng p
rote
ins
(bas
ed o
n 18
6 ge
nes
defin
ed a
s ov
erex
pres
sed
in lu
ng a
t proteinatlas.org)
. TM
Tcal
+SD
= Su
per-
depl
eted
pla
sma
with
four
-cha
nnel
trig
ger/c
alib
rant
; SD
= S
uper
-dep
lete
d pl
asm
a
Figu
re 2
–SD
S-PA
GE
gels
sho
win
g co
nsis
tent
pla
sma
depl
etio
n –
Left
pane
l = Ig
Y14
depl
etio
n; R
ight
pan
el =
IgY1
4 +
Supe
rmix
depl
etio
n
0
20
40
60
80
100
120
All'Lung'MS4A15
SFTA2
SFTPA1
LRRN4SCGB3A2SFTPC
SFTPD
NAPSA
SFTB
AGER
SLC34A2SFTPA2
NumberofPeptides
GeneName
SD
TMTcal+SD
050
100
150
200
250
300
350
Storageprotein
Extracellularmatrixprotein
Surfactant
Defen
se/immunityprotein
Signalingmolecule
Cellad
hesionm
olecule
Chap
erone
Hydrolase
Cytoskeletalprotein
Isomerase
Recep
tor
Enzymemodulator
Calcium-bindingprotein
Lyase
Structuralprotein
Mem
branetrafficprotein
Tran
sfer/carrierprotein
Ligase
Oxidoreductase
Celljunctionprotein
Tran
sferase
Viralprotein
Tran
smem
branereceptor
Tran
sporter
Nucleicacidbinding
Tran
scriptionfactor
%Change
Percentagechan
geinProteinClassdetectedwithTMTcal+SDvsSD
A B
1 –
Mol
ecul
ar w
eigh
t mar
kers
2 –
Cru
de p
lasm
a, 4
µg3
–D
eple
tion
D1-
1, 2
µg4
–D
eple
tion
D1-
2, 2
µg5
–D
eple
tion
D1-
3, 2
µg
1 –
Mol
ecul
ar w
eigh
t mar
kers
2 –
Cru
de p
lasm
a, 4
µg3
–D
eple
tion
D1-
1, 2
µg4
–D
eple
tion
D1-
2, 2
µg5
–D
eple
tion
D1-
3, 2
µg6
–D
eple
tion
D2,
2µg
IgY1
4Ig
Y14
+ Su
perm
ix1
2
3
4 5
6
1
2
3
4 5
Gen
e Sy
mbo
lTM
Tcal
+SD
(# p
eptid
es)
SD(#
pep
tides
)Pl
asm
a C
oncn
POST
N19
516
220
0 ng
/ml
Col
lage
nI/I
V12
4037
1~1
00 n
g/m
lC
HI3
L110
1140
ng/
ml
CC
L18
30
31 n
g/m
lSP
-A70
3130
ng/
ml
TGFB
18
205
ng/m
lKR
T18
240
4 ng
/ml
MM
P72
14
ng/m
lKR
T842
13
ng/m
lTG
FB2
20
0.2
ng/m
lLO
XL2
10
0.1
ng/m
lTG
M2
301
NR
ITG
AV55
6N
RIT
GB6
220
NR
MU
C1
192
NR
TGFB
R1
12
NR
Figu
re 3
–Bi
olog
ical
cov
erag
e of
TM
Tcal
ibra
tor™
vs
Supe
r-dep
letio
n al
one.
A
–G
O C
ellu
lar C
ompa
rtmen
t; B
–G
O B
iolo
gica
l Pro
cess
. TM
Tcal
+SD
= Su
per-
depl
eted
pla
sma
with
four
-cha
nnel
trig
ger/c
alib
rant
; SD
= S
uper
-dep
lete
d pl
asm
a on
ly
A B
Tabl
e 1
–O
utpu
t met
rics
for T
MTc
alib
rato
r™ a
nd T
MT®
MS2
ana
lysi
s of
IPF
plas
ma
–St
anda
rd S
earc
h (u
pper
pan
el);
Sequ
entia
l PTM
sea
rch
(low
er
pane
l)
IP &
MB
are
paid
em
ploy
ees
and
hold
sto
ck a
nd/o
r sto
ck o
ptio
ns in
Pro
teom
e Sc
ienc
es p
lc. I
P is
an
inve
ntor
on
pate
nts
cove
ring
the
TMTc
alib
rato
r™ te
chno
logy
ST &
MD
are
paid
em
ploy
ees
and
hold
sto
ck a
nd/o
r sto
ck o
ptio
ns in
Plia
nt
Ther
apeu
tics
Inc.
who
fund
ed th
e st
udy