ProteinChip Technology® in Research

and Clinical proteomics

Ciphergen Biosystems, Inc
Leaders in ProteinChip® System and Array Technologies,
Products and Services

Consumables:
ƒ ProteinChip Array and Sorbent Chemistries
ƒ Reagents
ƒ Kits
Instrumentation:
ƒ ProteinChip Systems
ƒFully Automatable

ƒ ProteinChip - LDI – MS/MS Interface

ƒAdvanced Ion Cooling
Software:
ƒ ProteinChip Software
ƒ Biomarker Patterns™ Software

Services:
ƒ Biomarker Discovery Centers™ Worldwide
 Ciphergen Principle Locations

Corporate Facilities in Fremont Ciphergen’s Process Division (BioSepra

S.A.) Facility in Paris

Biomarker Discovery Centers in

ƒ Fremont
ƒ Malvern
ƒ Copenhagen
 ProteinChip® Arrays – Chemically Derivatized
Surfaces

Chromatography Coupling Chemistry

(Hydrophobic [C16], WCX [carboxylate], SAX [quaternary (PS-I [Carbonyldiimidazole, amine], PS-II
ammonium], IMAC [nitrilotriacetic acid], Normal [silica oxide]) [Epoxy, amine or thiol])

Hydrophobic
Barrier

Functionalized
Hydrogel or Latex

Aluminium Base

Hydrophobic [C16]
PS-10 [Carbonyldiimidazole]
 ProteinChip® Technology: Array Preparation

1. Apply Crude Sample

Proteins within the sample bind
to chemical or biological “docking
sites” on the ProteinChip surface
through an affinity interaction.

2. Wash ProteinChip Array

Proteins that bind non-
specifically or buffer
contaminants are washed
away, eliminating sample
“noise”.

3. Add Energy Absorbing Molecules

After sample processing the chip is
dried and EAM is applied to each spot
to facilitate desorption and ionization in
the TOF-MS.
 ProteinChip® Technology: SELDI-TOF-Mass
Spectrometry Detection
ƒ Retained proteins are “eluted” from the ProteinChip® Array by Laser Desorption
and Ionization

ƒ Ionized proteins are detected and their mass accurately determined by Surface
Enhanced Laser Desorption/Ionization-Time-of-Flight Mass Spectrometry (SELDI-
TOF-MS)

N-Laser

Detector
Spectral View

TOF-MS

Map View

Gel View
 Clinical Proteomics

Answering Clinical Questions via Biomarker Discovery

with SELDI ProteinChip® Technology
 SELDI ProteinChip® Technology
Clinical Proteomics Applications

„ Protein Based Predictive Medicine

„ Early detection of disease via biomarker discovery
„ Serum proteome profiling (Lancet)

„ Tissue proteome profiling

„ Early detection of toxicity via biomarker discovery

„ Protein Based Therapeutic Drug Discovery

„ Therapeutic drugs via biomarker discovery
„ Inhibitors of AIDS replication (Science)

„ Small molecule Inhibitors

„ β-amyloid assay (secretase inhibitors)

„ Signal transduction assay (kinase inhibitors)

Limitations of Current Cancer Diagnostics

„ Most of current diagnostics detect cancer after it has already spread

to other parts of the body

„ Currently single biomarkers are used for cancer diagnosis which

have lower sensitivity and specificity

„ Early detection of cancer improves treatment options and survival

rates

„ Novel high value multi-marker diagnsotics can be discovered by

profiling of serum and tissue samples
SELDI Applications in Cancer: Protein Based Predictive Medicine
via Biomarker Discovery
Identification of Protein Fingerprints representative
of Disease
15
10 Tumor Tissue
5 (Patient A)
0
10
5
Tumor Tissue
(Patient B)
0
40
Tumor Tissue
20
Signal (Patient C)
0
Intensity 15
10 Control Tissue
5 (Patient A)
0
75
50 Control Tissue
25 (Patient B)
0
15
10 Control Tissue
5 (Patient C)
0
10000 11000 12000 13000
Da
Molecular mass (M/z)
 Protein Based Predictive Medicine
Serum Proteome Profiling for Early Detection of Cancer

Johns Hopkins
National Cancer Institute
Eastern Virginia Medical School
Study Design and Sample Preparation is Essential for
the Best Results

Dilute Sample in 9M urea, 2%CHAPS (pH 9)

Q-HyperD Fractionation

pH 9 Flow- pH 9 eluant pH 7 eluant pH 6 eluant pH 4 eluant isopropyl,

through (Q2) (Q3) (Q4) (Q5) acetonitrile, TFA
(Q1) (Q4)

Cu(II)-IMAC Array Hydrophobic Array Cationic Exchanger

Differential Protein Expression
Single Marker Discovery

Sensitivity Specificity

(“True (“True
Positives”) Negatives”)

Single Marker 65% 35%

 The Impact of Multiple Biomarkers & Biomarker
Patterns™ Software

Higher Sensitivity and

Specificity

Sensitivity Specificity

(“True (“True
Positives”) Negatives”)

Single Marker 65% 35%

Biomarker Pattern 90% 90%

Biomarker Patterns™ Classification of serum samples from high
grade prostate cancer and aged matched normal groups
(training set)

Node
Node
1 1
M447
N = 195

Node
Node
2 2 Node
Node4 4
M19 M59
N =N65
= 65 N = 130

Terminal Node
Node
3 3 Terminal Terminal
Node 1 M282 Node
Node
4 4 Node
Node5 5
N = 27 N =N38
= 38 N=N 127
= 127 N=N 3= 3

Terminal Terminal
Node 2 Node 3
N = 25 N = 13
Biomarker Patterns™ Classification of serum samples from high grade
prostate cancer and aged matched normal groups (training set)

Start off with all data (N =

194, 98 cancer, 96 normal)

• Rule at node 1: is P128

Node 1
(peak 128) <= 2.98?
P128
N = 194
• End up with N = 102, 88 Y N • End up with N = 92, 82
cancer, 14 normal at node 2 Node 2 Node 4 normal, 10 cancer at node 4
• Rule at node 2: is P297 P297 P73 • Rule at node 4: is P73 <=
<= 0.06? Y N = 102
N Y N = 92
N 0.25?
• End up with N = 92, 87 Node 3 Terminal Terminal Terminal
cancer, 5 normal at node 3 P109 Node 3 Node 4 Node 5
• Rule at node 3: is P109 N = 92 N = 10 N = 10 N = 82 1 misclassified in each
Y N
<= -0.22? terminal node
Terminal Terminal 1 cancer 9 cancer 1 cancer
Node 1
N=3
Node 2
N = 89
9 normal 1 normal 81 normal Disease
0 cancer 87 cancer
+ -
3 normal 2 normal
+ 96 3

Test
0 misclassified 2 misclassified
- 2 93
Each terminal node is classified by a
majority-rule vote: green = normal Sensitivity = 98%
node, red = cancer node Specificity = 97%
 Multi-Markers Have Higher Specificity &
Sensitivity than Single Markers

Markers Cancer Sensitivity Specificity

PSA Prostate 65% 35%

SELDI Multi-Marker
Prostate 83% 97%
Profile

CA15.3 Breast 23% 69%

SELDI Multi-Marker
Breast 93% 91%
Profile

CA125 Ovarian 35% 98%

SELDI Multi-Marker
Ovarian 100% 95%
Profile
 Multi-Markers in Serum for Early Detection of
Cancer

„ Prostate Cancer
„ 5 markers for prostate cancer (George Wright & colleagues)

„ Breast Cancer
„ 3 markers for breast Cancer (Daniel Chan & Colleagues)

„ Ovarian Cancer
„ A pattern of 5 markers in ovarian cancer (Liotta and Petricoin)
 Biomarker Patterns™ Software

„ Generates classification Generates clinically relevant

trees from SELDI data bioinformatics

Rapidly reduces complex spectral

data to critical biomarkers
information

Prioritizes protein ID projects

„ Generates trees via Generally provides higher confidence

“training” data set than “clustering” algorithms
A) Patient 1 in relapse B) Patient 2 in relapse
BN, LV & LV 2° BN 2° LV 2° LV 2° PR LV &
DX SP 2° PR PG PGDied DLN 2° & DLN 2° CR DLN 2° Died
45

Quantity of SAA by immunoassay

1200 45 750
CT

Normalized peak intensity

RT

(ProteinChip Array)
CT 600
RT 900
to BN
30 30

(µg/ml)
450
600
300
15 15
300 150
11.6 kDa 11.6 kDa
11.8 kDa 11.8 kDa
SAA SAA
0 0 0 0
0 100 200 300 400 500 0 1100 1200 1300 1400 1500 1600
No. of days No. of days

C) Patient 3 in relapse D) 11 Patients in remission

BN 2° BN 2°
RT
90 1 RT
st

Quantity of SAA by immunoassay

CT
7000 90

Normalized peak intensity

Normalized peak intensity

6000

(ProteinChip Array)
(ProteinChip Array)

75 75
5000
60 60
4000 (µg/ml)
45 45
3000
30 2000 30
15 11.6 kDa 1000 15
11.8 kDa
SAA 0
0 0
0 200 250 300 350 400 450 0 1000 2000 3000
No. of days No. of days
 Protein Based Predictive Medicine

Tissue Proteome Profiling for Early Detection of

Cancer

National Cancer Institute

Eastern Virginia Medical School
Ciphergen Biosystems
Laser Capture Microdissection –
Prostate Cancer

„ Clinical Question
„ Capture specific population of prostatic epithelium cells
„ Identify early predictors of prostate cancer

„ Advantages of ProteinChip® Technology for LCM procured cells

„ Sensitive (2,000 cells vs. 100,000 cells for 2D gel analysis)
„ Speed
„ No secondary label required
 Overview of Laser Capture Microdissection

Microscope LCM tissue section preparation

stage for protein analysis

„Frozen section

„Ethanol Fixation

„Stain

„Dehydration (ethanol and

xylene

„LCM

LCM (Laser Capture Microdissection) technology is

manufactured by ARCTURUS Engineering, Inc
 Laser Capture Microdissected Samples
Before LCM After LCM Captured cells
A B C

BPH

D E F

Cancer
LCM-captured Prostate Epithelium

3573.9+H PCA #1
7.5
Candidate
Intensity

5 Biomarker

2.5

PCA #1

PCA #2

PCA #3

NPR #1

NPR #2

NPR #3
2500 5000 7500 10000
3000 4000 5000 6000 7000

Molecular Weight (Da)

Identification Phase

„ Identification useful for

„ Drug target assessment
„ Biological research
„ Assay development

„ Strategy
„ Combination of on chip and column chromatography
„ Tryptic digest
„ Match tryptic fragment fingerprint against web database (e.g. ProFound)
„ ProteinChip-tandem MS interface available for CID and match against web
database (ProteinProspector)

„ Time required: depends on abundance and difficulty of purification for

each individual protein
Corporate Facilities in Fremont BioSepra S.A. Facilities in Paris

Continuum of Chemistries

Chromatography Surface Chromatography Beads

ƒ In 2001 Ciphergen purchased Biosepra, a chromatography sorbent

company based in France.

ƒ Matched Separation chemistries between Arrays and Sorbents

 ProteinChip™ Array assisted Purification Monitoring:

Designing Purification Strategies based on ProteinChip Binding

Step 1. Define first Purification conditions

WCX
pH3.5
pH3.5 - 10ul
1.2

0.8
pH4.5
pH4.5 - 10ul
0.6

0.4

0.2
pH5.5
pH5.5 - 10ul

0
pH3.5 pH4.5 pH5.5 pH6.5
10ul 10ul 10ul 10ul
pH6.5
pH6.5 - 10ul

15000 20000 25000 30000 35000 2

SAX 1.5
pH9.5

1
pH8.5
0.5

pH7.5
0
pH9.5 pH8.5 pH7.5 pH6.5
10ul 10ul 10ul 10ul
pH6.5
15000 20000 25000 30000 35000
 ProteinChip™ Array assisted Purification Monitoring:

Designing Purification Strategies based on ProteinChip Binding

Step 3. Optimize Elution of Contaminants from Strong Anionic Exchanger Resin (eg QHyperD)

15000 20000 25000 30000 35000

15

10

5
0mM
0mM NaCl Crude Sample
26171.6+H
0
15

10

5
26163.6+H
10mM
10mM NaCl
0
15

10

5
50mM 100mM Buffer pH6.5 (Binding)
26182.8+H 50mM NaCl
0
15

10

5
100mM
100mM NaCl
0
26163.2+H Optimal Elution of Contaminants
15 pH6.5, 400mM NaCl
10
200mM
5
26173.8+H
200mM NaCl
0
15

10
400mM
5

0
26184.3+H
400mM NaCl
15

10
600mM
5

0
26175.8+H 600mM NaCl
15

10

5
1M
1M NaCl
26161.2+H
0

15000 20000 25000 30000 35000

 ProteinChip™ Array assisted Purification Monitoring:

Designing Purification Strategies based on ProteinChip Binding

Step 4. Optimize Elution of protein from Strong Anionic Exchanger Resin (eg QHyperD)

5000 10000 15000 20000 20000 25000 30000 35000 40000 45000
60 2.5
2
40

20
pH6.5
1.5
1 26177.1+H
pH6.5
pH6.5 Wash Crude Sample
0.5
0 0
60 2.5
2
40
1.5

20
+400mM1NaCl
26205.5+H
pH6.5 +
+400mM NaCl

0.5
400mM NaCl
0
60
0
2.5 100mM Buffer
2
40
1.5 pH6.5 (Binding)
20
+1%TFA1
26208.8+H
1% TFA
+1%TFA

0.5
0 0
60 2.5
2
40 1.5 1% TFA +
+1%TFA + 10% IsoP.
+1%TFA1+ 10% IsoP.
20
0.5 26191.4+H
10% Isoprop
0 0
60 2.5
2
40 1.5
+1%TFA1+ 20% IsoP.
1% TFA +
+1%TFA + 20% IsoP.
Elution of Contaminants
20
0.5
26197.7+H
20% Isoprop pH6.5, 400mM NaCl
0 0
60 2.5
2
40 1.5 1% TFA +
+1%TFA + 30% IsoP.
+1%TFA1+ 30% IsoP.
20
0.5 26244.5+H 30% Isoprop
0
60
0
2.5
Elution of protein at pH6.5
40
2
1% TFA +
400mM NaCl, 1%TFA+30% isopropanol
1.5
+1%TFA1+ 40% IsoP. +1%TFA + 40% IsoP.
20
0.5
40% Isoprop
26181.6+H
0 0
60 2.5
2
40 1.5
+1%TFA1+ 50% IsoP.
1% TFA +
+1%TFA + 50% IsoP.
20
0.5
26195.1+H
50% Isoprop
0 0

5000 10000 15000 20000 20000 25000 30000 35000 40000 45000

Mass Range Mass Range

5-20kd 20-45kd
 ProteinChip™ Array assisted Purification Monitoring:

Designing Purification Strategies based on ProteinChip Binding

Step 5. Prepare Digest

Crude Sample

In Tube Enzyme 1D Gel Followed by

100mM Buffer Digestion In-Gel Digestion
pH6.5 (Binding)

Peptide Mass Fingerprinting

Elution of Contaminants And/or MSMS
pH6.5, 400mM NaCl

Elution of protein at pH6.5

400mM NaCl, 1%TFA+30% isopropanol
Protein Sequencing by Collision Induced Dissociation
Directly from the ProteinChip Surface
Collision
ProteinChip
Cell
Scanning Detector
Ion Guide Quadrupole Pulser
Microchannel
Plates

UV Beam

Ion Mirror
SELDI Tandem MS CID Sequencing of Biomarker Tryptic Peptides

800

1912.014: (K)QATVGDVNTDRPGLLDLK(G)
700 911.5577

40

35

600 30

25
1911.9899
1340.7613
652.3794
20
755.4781
496.2708

500 15
1652.8333

268.1700
10
1081.5957
1783.9330

400 0
200 400 600 800 1000
m/z
1200 1400 1600 1800

300

Diazepam Binding Inhibitor Diazepam Binding Inhibitor

200
1351.7783 1912.0587

100

0
1000 1200 1400 1600 1800 2000 2200 2400 2600 2800
M/z
 Interaction Difference Mapping (IDM™)
A Novel Discovery and Assay Platform for in
vitro Protein Interactions
 ProteinChip® Arrays – Chemically Derivatized
Surfaces

Chromatography Coupling Chemistry

(Hydrophobic [C16], WCX [carboxylate], SAX [quaternary (PS-I [Carbonyldiimidazole, amine], PS-II
ammonium], IMAC [nitrilotriacetic acid], Normal [silica oxide]) [Epoxy, amine or thiol])

Hydrophobic
Barrier

Functionalized
Hydrogel or Latex

Aluminium Base

Hydrophobic [C16]
PS-10 [Carbonyldiimidazole]
 Interaction Difference Mapping (IDM™) Platform

ƒ Reactive surface
ƒ IMAC surface ƒ Reactive sorbents
ƒ Ionic surfaces ƒ IMAC sorbents
ƒ Ionic sorbents
Continuum of Matched
Chemistries

Affinity ProteinChip Arrays Affinity Sorbents

P13K

PDK1

AKT

GSK3 Biological States Activity Assays

ƒ Apoptosis Discover Interactors
ƒ Differentiation
ƒ Replication

Multivariate Software for

Biological State Prediction
 PS-10 or PS-20 Couple target Block and wash surface

Incubate with complex mixture Wash away non-specific binders Dry, add EAM

7.5
5
2.5
0
6000 8000 10000 12000

Native Mass

Surface Enhanced Laser Desorption and Ionisation Time of Flight Mass Spectrometry (SELDI-TOF-MS)
 Signal Intensity

0
20
40
60
1828.1+H 1-15

1955.9+H 1-16

2000
2068.2+H 1-17
2165.7+H 1-18
CV =12-15%
LOD = 1 ng/ml

2314.5+H 1-19

2460.3+H 1-20

3000

Molecular Mass (M/z)

3673.7+H 1-33
4000

4074.4+H 1-37
4131.1+H 1-38
ƒ Protein Interaction Assay of Amyloid β Peptide Variants

4230.9+H 1-39

4329.4+H 1-40
Simple Stable Interactions - Alzheimer Research

4514.1+H 1-42
Simple Transient Interactions
ƒPhosphorylation/Dephosphorylation
ƒ Kinase Assays P-

ƒGlycosylation/Deglycosylation
ƒ Glycosidase Assays
ƒProteolysis
ƒ Proteolysis Assays

Complex Transient Interactions (pathways) P-

ƒEnzyme Cascades
Simple Transient Interactions – Kinase Assays
Simple Transient Interactions – Kinase Assays

Panel C from Learning & Memory 5:429-445

Death Signals (eg TNFα, TNFβ, FasL) Survival Factors Present (eg IL-2)

Death Receptor

Ras
PI3K Raf
MAP PKC
Erk-1, 2
PKA
AKT cSRC
p90RSK

Stat-3

Bad

NF-κB JNK Bcl-2 Bcl-xL

Mitochondria

Cyto C
IAP
Caspase-9

Caspase- 8, 10 Caspase- 3, 6, 7

Cell Shrinkage
GSK3β Membrane blebbing
DNA Fragmentation

Apoptosis
Summary of Measured MAP, PKA, AKT, PKC and GSK3β
Kinase Activity (in solution) from Neuronal Cortical Cell
extracts stimulated with BDNF.

Control Wash
4
BDNF Treatment 5 min

3 BDNF Treatment 45 min

Fold Change

MAPK PKA AKT PKC GSK3β

3P
 Thank You
Ciphergen Biosystems Inc
Fremont, California, USA, 94555

www.ciphergen.com
„ This week the new Autoloader and the Full Auto Biomarker System
was debuted at the scientific meeting for the American Society of
Human Genetics.