Detection of Known and Unique Prostate Cancer Biomarkers by SELDI Mass Spectrometry

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


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.


Goals

• Identify PCA Signature Protein Biomarkers for:

• Early Detection of PCA

• Diagnosis

• Prognosis

• Therapeutic Targets


Methods

• ProteinChipTM SELDI-TOF-MS:

• Chemically Defined Chip Arrays

• Lysates of LCM Procured Cells

• Serum and Seminal Plasma

• SELDI Immunoassay

• Single

• Multiplex


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


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


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


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.


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.


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


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


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.


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.


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.


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


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


+ 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


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.


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.


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


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


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).


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


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


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.


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.

Documents

Detection of Known and Unique Prostate Cancer Biomarkers by SELDI Mass Spectrometry