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Transcript of Wall PROTEOMICS spear METABOLOMICS snake VENOMICS tree GENOMICS rope GLYCOMICS fan PHENOMICS...
wallPROTEOMICS
spearMETABOLOMICS
snakeVENOMICStree
GENOMICS
ropeGLYCOMICS
fanPHENOMICS
FluorescenceBringing Life to Proteins and Proteins to Life
FABLS Network LaunchFeb 2005
Australian Proteome Analysis Facility Ltd (APAF)
• birthplace of the word “proteomics” in 1995
• 1st medium:high-throughput proteomics Facility in 1996
• wholly-owned public company in 2002
• Major National Research Facility until 2006
• world class “one-stop” proteomics discovery facility with 40 EFTS located in 4 purpose-built centres of excellence that work synergistically
• service provider, discovery partner, educator, collaborator & proteomics technology developer
Australian Proteome Analysis Facility Ltd (APAF)
Discovery, development & utilisation of sensitive, accurate & cheap fluorescence proteomics probes (1DE, 2DE, IPG, native gel, blot) for :
1. Protein
2. Enzymatic activity
3. Post-translational modification (PTM)
4. Peptide bioactives
• Client-based Contract Research & Life Sciences Collaborations
• Linkages with other MNRFs
• Technology E,R&D
• Education & Training Program
• In-house Scientific Programs• Biomarker Discovery• Invasive Cancer Biology• Agricultural Productivity Traits• Bacterial Pathogenesis
APAF’S MAJOR ACTIVITIES
BIOLOGICAL SAMPLEFRACTIONATION
ORHIGH ABUNDANCE
PROTEIN REMOVAL
DATAANALYSESARCHIVINGREPORTING
PROTEINIDENTIFICATION
SEQUENCING
COLLECTION OF PROTEOMIC ARRAY
DATA(e.g., IMAGING)
BIOLOGICAL SAMPLEPROTEOMICARRAYING
PROTEINIDENTIFICATION
SEQUENCING
Using Fluorescence to Turn Proteomic Bottlenecks into Development Opportunities
Removal of 99.9% most
abundant human biofluid proteins
& from other biological sources
Improved sensitivity via
high throughput sample
processing direct on biochip technology
Development of new improved fluorescent all protein stains
Development of fluorescent spot cutter/digestion
robot MS/MS capability direct
off-chip
MS/MS capable affinity chips
Development of less operator intensive HTS
imaging systems
Proteomic LIMS development
Development of 2D-LC HTS
Fractionation of relevant proteins
hemopexin
HSAfibrinogen
IgG heavy chain
IgG heavy chain
Ig light chainApoA1
transferrin
a1-antitrypsin
haptoglobin leucine-rich
a2 glycoprotein
A2 HS-glycoproteinfetuin
transthyretin
a2 M
fibrinogen
Drilling deeper into the plasma proteome by removal of high abundance
proteins
Plasma Protein Medium Abundance
1E-10
1E-09
1E-08
1E-07
1E-06
1E-05
0.0001
0.001
0.01
0.1
1
10
100
albu
min IgG
hapt
oglo
bin
2-1
type
a-lip
opro
tein
(H
DL)
b-lip
opro
tein
(LD
L)fib
rinog
entr
ansf
errin
a1-a
ntitr
ypsi
na2
-mac
rogl
obul
in IgA
hapt
oglo
bbin
1 IgM
a1 a
cid
glyc
opro
tein
c3b-
com
pone
ntce
rulo
plas
min
hem
opex
ina2
HS
-gly
copr
otei
nin
ter-
a 1
-try
psin
inhi
bito
ra1
ant
ichy
mot
ryps
inG
C-g
lobu
linC
1s in
hibi
tor
a1 b
-gly
copr
otei
n IgD
His
tidin
e ric
h gl
ycop
rote
ina1
T g
lyco
prot
ein
prot
hrom
bin
a1 M
gly
copr
otei
nre
tinol
-bin
ding
pro
tein
a3 g
lyco
prot
ein
Apo
C-I
IIa2
ant
ipla
smin
tran
sfer
rin R
ferr
itin
myo
glob
inla
ctof
errin
b2-m
icro
glob
bulin
C-r
eact
ive
prot
ein
insu
linP
SA
(as
ympt
omat
ic)
com
plem
ent C
4 pr
ecur
sor
CE
A (
smok
er, n
on c
ance
r)pr
osta
tic a
cid
phos
phat
ase
TN
Fa
(non
infla
mm
ator
y)
Protein
Log
(mg/
mL)
PLASMA PROTEINS NEEDING TO BE VISUALISED FOR BIOMARKER DISCOVERY
VISUALISATION REQUIRED TO DISCOVER NOVEL PLASMA PROTEIN
DISEASE BIOMARKERS
SarASarASarASarA
D C B A
IsaA
IsaA
IsaA
IsaA
IsaA
IsaA
IsaA
IsaA
D
B
C
A
Identifying S. aureus proteins associated with methicillin resistance
Meth sensitive 8325
8325 +TX Meth resistant COL
COL +TX
New fluorescent stainsfor deeper drilling of the
proteome
250ng 30ng 4ng
0
2
4
6
8
10
12
14
16
300 400 500 600 700 800
Wavelength (nm)
Inte
nsit
y
Em 610 (BSA)
Ex 520 (BSA)
UVB302 nm
UVA365 nm
Argon488nm
YAG532 nm
He-Ne543 nm
More sensitive detection meansmore proteins of interest
WHAT’S HOT IN PROTOEMICS !!!
APAF and LCI STS Biochip R&D program
AIM
To develop the new STS biochip platform for the rapid affinity capture,
concentration and TOF/TOF identification of peptides and proteins
MALDI, AnchorChips, SELDI & STS biochips
ApplySample
DrySample
STS Biochip
BrukerAnchorChip
DrySample
ApplySample
ApplySample
ABI 4700 Opti-TOFMALDI Plate
ApplySample
Wash Sample& Dry
SELDIWash SampleFocus & Dry
Diffuse SampleOver Large Area
Diffuse SampleOver Large Area
Selective Binding
Selective Binding
Non-selective Binding
Non-selective Binding ConcentrateSample
ConcentrateSelected Sample
Chemical proteomics: active site directed probes
Leung, D.; Hardouin, C.; Boger, D. L.; Cravatt, B. F. Nat. Biotech. 2003, 687-71.
Serine proteases
Discovery of known and unknown serine hydrolase activity
TGR’s SureFireTM TechnologyRemoving a Bottleneck in drug / bioactive discovery
High throughput, Sensitive, Quantitative Assays
Thanks from all the APAF team