Integrating Biotechnology into Translational Cancer 

Research

O. John Semmes, Ph.D.

Director

Cancer Biology and Infectious Disease Research Center

Eastern Virginia Medical School 

Norfolk, VA

Eastern Virginia Medical School

Discovery Laboratory
Research Laboratory Pathologist

Basic Scientist Physician/Scientist

Cancer

Biorespository

BIOMARKERS

DIAGNOSTICS

THERAPEUTICS

TRANSLATIONAL RESEARCH PROGRAM
“Fishing Expedition”

Unbiased Discovery “Maybe Peyton Manning is good at baseball”

“Maybe my favorite gene is a biomarker”

Targeted Discovery

Rational Design

BIOMARKER DISCOVERY
“Integromics”

“Data Analysis” “Systems Biology”

“Orthoganol Confirmation”

“Death by Validation”
Proteomics

Metabolomics

Genomics

CBIDRC Biotechnology Expertise
Example Technologies

 Glycoproteomics

 Imaging Mass Spectrometry

 Quality Human Tissues for PRoBE compliant study
Rasman, R. Nature methods 2, 817 (2005) 

Bertozzi, CR. Science 291, 2357 (2001) 

Fuster MM, Nature review cancer 5, 526 (2005)

Glycoproteomics
Metabolic labeling

N3

AC4ManNAz Biotin-alkyne Streptavidin-FITC

Streptavidin beads

Click reaction Fluorescent detection

Affinity purification

Can we identify a cell surface glycoprotein involved 

in metastatsis?

N2 versus ML2

1. Metabolic labeling and chemoselective conjugation “CLICK”

2. Affinity detection and enrichment of labeled cell-surface sialylated proteins.

3. Streptavidin-captured protein subjected to quantitative LC-MS/MS. 
FL1-H: Alexa 488 FL1-H: Alexa 488

N2 ML2

Imaging cell-surface sialylation with ManNaz and dcfb-65 

x80 x80

x80 x80

ManNAz

ManNAc

A. Flow cytometry

analysis of fluorescent 

labeling. grey 

histogram, untreated 

cells; yellow, SAv-A488 

only; green, control 

ManNAc treated cells; 

red, ManNAz treated 

cells.

B. Microscopic 

analysis of fluorescent 

labeling on live 

cells.TO-RPO-3 was 

used to stain nuclei. 
Tumor gland

Normal gland

x10 x104 x104

Green: CDCP1

Red: PI

CDCP1 mAb 41-2

CDCP1 Subcellular Localization in PCa tissue 

x10 x88 x88

Green: CDCP1

Red: PI
Characterization and Quantitation of 

Target Glycopeptides

How to overcome the “either peptides or sugars” 

technology barrier?

Application of MRM and QqQ technology

Peptide bonds are stronger and more uniform than 

glycanyl/glycyl bonds.
4000 Q-TRAPTM System

Source

Q0 High Pressure Cell Q1 Q2 Q3 Detector

Ions can be trapped 

while Q3 (LIT) is 

scanning ions during 

MS/MS and MS3

Transmits and/or 

selects parent ions

Fragments parent 

/Precursor ions

(Hybrid Quadrupole/Linear 

Ion Trap) Transmits/ Scans 

for preselected ion 

fragments and MS/MS 

scans

Modified from ABI/MDS Sciex Proteomics Technology Note
Discovery and Diagnostics
Application of Imaging Mass Spectrometry to 

the Clinical Management of Prostate Cancer

MALDI Target

m/z

Intensity
13999.0

* 100 Prostate cells\0_83\1\1SLin, Smoothed, "Baseline subt."

0

200

400

600

800

1000

10088.3

* Prostate4631-1OCT041307\0_R00X122Y032\1\1SLin, Smoothed, "Baseline subt."

0

100

200

300

4000 6000 8000 10000 12000 14000 16000 18000 20000 22000

A.

Comparison of MALDI spectra acquired from LCM cells versus in-tact tissue. A) Representative spectra generated from 100 

prostate adenocarcinoma cells microdissected and mounted onto the MALDI target. B) Representative spectra from the same tissue 

in an area of prostate adenocarcinoma collected via direct analysis of intact tissue.

B.
100

%

0% 5mm 5mm

Registering Expression Levels of Selected m/z 

to Tissue Grid Position
m/z 4036.29 m/z 4365.23

4679-1

OCT

PCA

NPr

PCa PCa

Mass difference 328.94
H & E MEKK2 stain MALDI-MSI (m/z 4355)

Area of intense staining

Evaluating The Utility of MEKK2 IHC For PCa

Stage/Grade
Normalized Intensity

0

5

10

15

20

m/z 4355

1

2

3

4

5

6

7

8

m/z 5657

Normalized Intensity

0

2

4

6

8

10

12

14

16

18

2

3

4

5

6

7

Normalized Intensity

0

10

20

30

40

50

60

70

m/z 10096

Insignificant

0

1

2

3

4

5

6

Significant

m/z 9521

Significant Insignificant

m/z 6178 m/z 5361

47 Prostates

11 Insignificant Disease

36 Significant Disease

4355 sequence identified as MEKK2
Insignificant

Significant

MEKK2 is Over-expressed in Insignificant Disease 
MATRIX

TISSUE SECTION

GLASS SLIDE

CHARGED PEPTIDES

ENTER GAS PHASE

MALDI LASER

TIME OF FLIGHT

MASS DETECTOR

M/z 

MATRIX

TISSUE SECTION

GLASS SLIDE

CHARGED PEPTIDES

ENTER GAS PHASE

MALDI LASER

TIME OF FLIGHT

MASS DETECTOR

M/z 

PhotocleavableTag

Glass Slide

Tissue Section

Antigen

Tagged Antibody

PhotocleavableTag

Glass Slide

Tissue Section

Antigen

Tagged Antibody

Mass Tag

Glass Slide

Tissue Section

Antigen

Tagged Antibody

PhotocleavableTag

Glass Slide

Tissue Section

Antigen

Tagged Antibody

Mass Tag

Glass Slide

Tissue Section

Antigen

Tagged Antibody

MALDI LASER 

PhotocleavableTag

Glass Slide

Tissue Section

Antigen

Tagged Antibody

PhotocleavableTag

Glass Slide

Tissue Section

Antigen

Tagged Antibody

PhotocleavableTag

Glass Slide

Tissue Section

Antigen

Tagged Antibody

PhotocleavableTag

Glass Slide

Tissue Section

Antigen

Tagged Antibody

Mass Tag

Glass Slide

Tissue Section

Antigen

Tagged Antibody

PhotocleavableTag

Glass Slide

Tissue Section

Antigen

Tagged Antibody

Mass Tag

Glass Slide

Tissue Section

Antigen

Tagged Antibody

MALDI LASER 

Direct IMS Diagnostics or Standard IHC Diagnostics??
Slice frozen embedded 

tissue on cryostat 

at 10 μm

Serial sections acquired 

Spray 

with matrix

Acquire 

mass spectra

Molecular profile Molecular image

Adjacent section H & E stained

Compare molecular image to 

stained serial section

conductive 

slide for 

MALDI-MSI Pathologist defines 

areas of interest

PathologyDirected 

Discovery 

Process
Integration is Critical to Success in Translational Research

IMS

SNP

miRNA Tissue sample

CDE1

CDE2

CDE3

EMR to CAISIS

Genologics LIM System
Clinical 

Biorepository

Prostate, Breast, 

Bladder, Renal, 

H&N, Melanoma

36,000 serum

4,000 urine

1,700 plasma

7,000 frozen tissue

600 EPS

Informatics 

Infrastructure

CAISIS

Genologics

EMR Portals

CBIDRC RESOURCES

Study Compliance

35 Grant submissions*

26 approved IRB protocols

6 patents

Clinical Sites

Dermatology

Internal Medicine

Ob/Gyn Otolaryngology

Pathology

Pediatrics

Oncology

Urology

Surgery

Molecular 

Pathology

IHC/SlidePath

Tissue processing

LCM

Tissue Staging
Fresh Prostate

Frozen Cores

Routine Histology
Left Right

Base

Mid

Apex

Twelve Core Prostate Biopsy 

•Routine histology

•Two cores per block

•Six blocks per case
Paraffin 

Embedded Tissue

Frozen tissue

Tissue Microarray (TMA)

microRNA

ImmunoHistochemistry

Imaging MALDI

Proteomics/Glycomics
E

V

M

S

Cancer Biology and Infectious Disease 

Research Center

Investigators

Rick Drake, PhD

Aurora Kerscher, PhD

Julius Nyalwidhe, PhD

Raymond Lance, MD

Dean Troyer, MD

Staff

Barb Conyers

MaryAnn Clements

Jessica Abbate

Tarek Kandil

Brian Main

Michael Ward

Chris Wilkins

Paige Miller

Yvonne Oden

Elizabeth Smith

Fellows/Students

Cindy Guo, PhD

Lisa Cazares, PhD

Lifang Yang, MD

Tanya Burch, PhD

Shamina Mitchell

Krista White, PhD

Breanne Grigch

Tiffany Benzine

Clinical Research Activity

Jennifer Taylor, RN, Senatra

Laurie Jackson, RN, CRU, SMG

Director

John Semmes, PhD