CONFIDENTIAL: FOR INTERNAL USE ONLY
Detection of Known and Unique Prostate Cancer Biomarkers by
SELDI Mass Spectrometry
Bao-Ling Adam, Ph.D.
Research Assistant Professor
Department of Microbiology and Molecular Cell Biology, Virginia Prostate Center, Eastern Virginia Medical School, Norfolk, VA 23501
CONFIDENTIAL: FOR INTERNAL USE ONLY
Objective
• Evaluate the ProteinChipTM SELDI-TOF-MS system for:
• Simultaneous detection of multiple known prostate cancer-associated biomarkers.
• Discovery of unique prostate cancer-associated biomarkers.
CONFIDENTIAL: FOR INTERNAL USE ONLY
Goals
• Identify PCA Signature Protein Biomarkers for:
• Early Detection of PCA
• Diagnosis
• Prognosis
• Therapeutic Targets
CONFIDENTIAL: FOR INTERNAL USE ONLY
Methods
• ProteinChipTM SELDI-TOF-MS:
• Chemically Defined Chip Arrays
• Lysates of LCM Procured Cells
• Serum and Seminal Plasma
• SELDI Immunoassay
• Single
• Multiplex
CONFIDENTIAL: FOR INTERNAL USE ONLY
SO4
SO4 SO4Me(II)
Me(II)Me(II)
NRNR
NR
Hydrophobic Anionic, Cationic IMAC Normal phase
SELDI ProteinChipTM ArraysBy surface “chemistries”
SELDI Analysis of Protein Bound to Chemical Defined Chip
CONFIDENTIAL: FOR INTERNAL USE ONLY
20000 30000 40000 50000 60000
00.511.5223995.3 47228.8
10000 15000 20000 25000 30000
020406010709.310949.711659.3 21558.3
25000 50000 75000 100000 125000
00.10.20.30.447684.1 99456.6
M/Z
Inte
nsit
y
SELDI ProteinChipTM Analysis of Pure Prostate Cancer Biomarkers Bound to H4 Chip.
PSP
PAP
PSMA
CONFIDENTIAL: FOR INTERNAL USE ONLY
1 0 0 0 0 2 0 0 0 0 3 0 0 0 0
05101510000 20000 3000014150.5 28235.6
a
b
c
d
Inte
nsit
y
10000 20000 30000
M/Z
Comparison Spectra of Pure PSA from Different Sources.
Biodesign
Medix
Sigma
EVMS
CONFIDENTIAL: FOR INTERNAL USE ONLYM/Z
Inte
nsit
y
5 0 0 0 0 7 5 0 0 0 1 0 0 0 0 0
Blank Cap
NPr
PCA
Stroma
M/Z50000 75000 100000
Inte
nsit
y
Analysis of LCM Procured Prostate Cell Lysates.
Candidate Biomarker
PSA PAP
PSMA
PSP
CONFIDENTIAL: FOR INTERNAL USE ONLY
90000 100000 110000
10000 15000 20000 25000
10831.911512.3
Seminal Plasma
20000 30000 40000 50000 60000
28437 47709.8
60000 80000 100000 120000
67474.481407.6
PSA complex
PSA PAPPSP
PSMA
M/Z
Identification of PSP, PSA, PAP and PSMA in Seminal Plasma from a PCA Patient.
CONFIDENTIAL: FOR INTERNAL USE ONLY
90000 100000 110000 120000
99850
Serum
20000 30000 40000 50000 60000
28423.933843
PSA
12500 15000 17500
10724.2
PSP
70000 80000 90000 100000 110000 120000
79664.1
PSA Complex
2
4PSMA
M/Z
Identification of PSP, PSA, PAP and PSMA in Serum from a PCA Patient.
CONFIDENTIAL: FOR INTERNAL USE ONLY
Identification of Potential Unique Prostate Cancer Biomarkers in Lysates of LCM Procured Prostate Cancer Cells.
20000 30000 40000 50000
20000 30000 40000 50000
NPr #1
NPr #2
NPR #3
PCA #1
PCA #2
PCA #3
M/Z
PCA #1
17970.8
20000 25000 30000 35000
0
2
4
6In
tens
ity
PCA #1
PCA #2
PCA #3
NPR #1
NPR #2
NPR #3
CandidateBiomarker
CONFIDENTIAL: FOR INTERNAL USE ONLY
2500 5000 7500 10000
M/Z
PCA #1
PCA #2
PCA #3
NPR #1
NPR #2
NPR #3
PCA #1
Inte
nsit
y
3000 4000 5000 6000 7000
Identification of Potential Unique Prostate Cancer Biomarkers in Lysates of LCM Procured Prostate Cancer Cells.
CandidateBiomarker
CONFIDENTIAL: FOR INTERNAL USE ONLY
0
0.05
0.1
0.15
30000 40000 50000 60000
0
0.05
0.1
0.15
0
0.025
0.05
0.075
30000 40000 50000 60000
0
0.025
0.05
0.075
0
0.5
1
1.5
2
30000 40000 50000 60000
0
0.5
1
1.5
2
0
2
4
30000 40000 50000 60000
0
2
4
# 3776 NPr
# 3776 CaP
# 4 NPr
# 4 CaP
# 3778 NPr
# 3778 CaP
# 3 NPr
# 5 CaP
33563
49038
28693
36753
4852628602
33679
48488
48192
28581
33492
48837
33311
28695
Mass/Charge
Inte
nsit
yIdentification of a 33 KDa PCA Protein Upregulate in PCA Cells.
CONFIDENTIAL: FOR INTERNAL USE ONLY
Mass/Charge (± 1%)
Normal
Ave
rage
In
ten
sity
PCA
0
1
2
1
2
Composite Map of Unique Proteins in Seminal Plasma from Normal and PCA patients.
CONFIDENTIAL: FOR INTERNAL USE ONLY
NPr #1
NPr #2
NPr #3
PCA #1
PCA #2
PCA #3
100000 125000 150000 175000
0
0.05
0.1
0.15
0.2
PCA #1
Inte
nsity
Candidate Biomarker
Identification of Potential Unique Prostate Cancer Biomarkers in Seminal Plasma from Normal and PCA Patients.
CONFIDENTIAL: FOR INTERNAL USE ONLY
Identification of Potential Unique Prostate Cancer Biomarkers in Seminal Plasma from Normal and PCA Patients.
2000 3000 4000 5000
2000 3000 4000 5000
9 Q5 1+2(2)
9 Q1 1+2
107 Q4 1+2
9 Q5 1+2
100 Q6 1+2
108 Q4 3+4
9 Q6 1+2
NPr #1
NPr #2
NPr #3
PCA #1
PCA #2
PCA #3
PCA #2
Inte
nsit
y CandidateBiomarker
CONFIDENTIAL: FOR INTERNAL USE ONLY
20000 40000 60000 80000
20000 40000 60000 80000
20000 40000 60000 80000
Day 1
Day 2
Day 3
67280.3 40Da
67365.8 33Da
67243.4 37Da
28445.4 31Da
47354.4 43Da
28468.0 22Da
47472.9 62Da
28448.8 18Da
47322.0 48Da
M/Z
10
20
30
40
0
10
20
30
40
0
10
20
30
40
0
Inte
nsity
Intra and Inter-assay Reproducibility of Spectra Obtained by SELDI ProteinChipTM Analysis of Seminal Plasma.
Free-PSA
Free-PSA
Free-PSA
PAP
PAP
PAP
Albumin
Albumin
Albumin
CONFIDENTIAL: FOR INTERNAL USE ONLY
+ EAM
Put in mass reader
+ sample
wash M/Z
Inte
nsi
ty
SELDI ProteinChipTM Immunoassay1 2 3 4 6 7 85
1 2 3 4 6 7 85
1 2 3 4 6 7 85
Control Ab Bait Ab
Sample
Control
1. Capture/retention
2. Detection
3. Data analysis
CONFIDENTIAL: FOR INTERNAL USE ONLY
20000 25000 30000 35000
10
30
50
0
1
Free-PSA
M/Z
Inte
nsity
0
2
4
6
8
10
PSA concentration (ng)
1 10 100
PS
A P
eak
Are
a
1000
10000
r2=0.999
B
Generation of PSA Standard Curve for the SELDI ProteinChipTM Immunoassay.
CONFIDENTIAL: FOR INTERNAL USE ONLY
PSP Concentration (ng)
1 10
PS
P P
eak
Are
a
100
1000
10000
r2=0.994
C
PSP Standard Curve for the SELDI ProteinChipTM Immunoassay.
CONFIDENTIAL: FOR INTERNAL USE ONLY
1 0 0 0 0 1 5 0 0 0 2 0 0 0 0 2 5 0 0 0 3 0 0 0 0
M/Z
Inte
nsity
10000 15000 20000 25000 30000
Detection of PSP and PSA in Seminal Plasma by SELDI ProteinChipTM Immunoassay.
PSP
PSA
PSP + PSA
IgG
PSP
PSP
PSA
PSA
CONFIDENTIAL: FOR INTERNAL USE ONLY
40000 60000 80000 100000
M/Z
Inte
nsity
40000 60000 80000 10000
Detection of PSMA and PSA in Seminal Plasma by SELDI ProteinChipTM Immunoassay.
PSMA
PSMA
PSA
PSA
PSMA
PSA
PSMA + PSA
IgG
CONFIDENTIAL: FOR INTERNAL USE ONLY
40000 60000 80000
(PSA+ACT)+
(PSA+ACT)++
Free PSA
Dimer
a
b
M/Z
Inte
nsity
SELDI ProteinChipTM Analysis of Purified Free-PSA and Complexed PSA (PSA-ACT).
CONFIDENTIAL: FOR INTERNAL USE ONLY
20000 25000 30000 35000 70000 75000 80000 85000 90000 95000
free PSA
PSA complex
Total PSA
IgG
==
==
Inte
nsit
y
A
20000 30000 75000 85000M/Z
20000 25000 30000 35000
free PSA
PSA complex
Total PSA
IgG
70000 75000 80000 85000 90000 95000
=B
==
=
Inte
nsit
y
20000 30000 75000 85000M/Z
Detection of Free-PSA and Complexed PSA (PSA-ACT) by SELDI ProteinChipTM Immunoassay.
Free-PSA
PSA-ACT
CONFIDENTIAL: FOR INTERNAL USE ONLY
70000 80000 90000 10000025000 30000 35000 40000
M/Z
Inte
nsit
y
a
b
==
25000 30000 35000 80000 90000
Detection of Free-PSA and Complexed PSA (PSA-ACT) in Seminal Plasma and Serum by SELDI ProteinChipTM Immunoassay.
Seminal Plasma
Serum
Free-PSA
Free-PSA
PSA-ACT
PSA-ACT
CONFIDENTIAL: FOR INTERNAL USE ONLY
Conclusions• Four known prostate cancer biomarkers (PSA, PAP, PSP, and
PSMA) were successfully identified by SELDI analysis in LCM procured cell lysates, serum, and seminal plasma bound to chemically defined protein chips.
• These prostate cancer associated proteins were also successfully identified by single and multiplex SELDI immunoassays.
• Both free-PSA and complex (PSA-ACT) forms of PSA were specifically identified by SELDI immunoassays.
• The potential of SELDI for the discovery of unique prostate cancer biomarkers was demonstrated.
CONFIDENTIAL: FOR INTERNAL USE ONLY
Collaborators• Eastern Virginia Medical School
– Lisa H. Cazares
– Lei Gong
– Shuhail Nasim, M.D.
– Paul F. Schellhammer, M.D.
– Antonia Vlahou, Ph.D.
– George L. Wright, Jr., Ph.D.
• Ciphergen Biosystems, Inc.
– Sau-Mei Leung, Ph.D.
– Brad Thacher, Ph.D.
– Tai-Taung Yip, Ph.D.