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