A new technology called PEP was developed to analyze protein function at proteome scale. This technology can be used for biomarker discovery, cancer diagnostics development, drug target identification and validation, study protein variants and one-step functional protein/enzyme purification.

1. Discovery of Functional Protein Biomarkers
from Cancer Serum
Xing Wang, Ph.D.
Array Bridge Inc. USA
Molecular Med TRI-CON, February 11-16, 2018
San Francisco, CA
1

2. Topics Covered:
1. What is PEP Technology?
2. PEP Development
3. PEP for Biomarker Discovery.
4. Conclusions
2

3. 1. What is PEP Technology?
• PEP stands for Protein Elution Plate. It is a plastic plate with either
384 wells (small format) or 1536 wells (large format) with semi-
permeable membrane that retains proteins large than 10,000 Dalton
but allow salt and other small molecules to pass through in an electric
field.
• The Technology involves the modification of gel electrophoresis
conditions (no reducing reagent, lower SDS, no heat treatment etc.).
• Specific transfer and recovery solution to elute proteins from the gel
for enzyme analysis and prevent protein diffusion in the PEP.
• PEP Technology allows fast protein purification and systematic analysis
of enzyme activities in a proteome.
3

4. Modified
2-D Gel
Proteome
of interest
PEP transfer
Enzyme Assay
Samples recovered in
Four 384-Well Plates
3-D Enzyme Landscape
MS Protein Identification
PEP: A Functional Proteomics
Platform
4
For demonstration, actual gel not stained

5. 5
2. PEP Technology Development

6. Large Format PEP Plates for Complex Proteome ----- 1536 wells.
The PEP Plate is fitting with 4 standard 384-well microplates for easy processing.

7. Small Format PEP Plates for Simple Proteome ----- 383 wells.
The PEP Plate is fitting with standard 384-well microplates for easy processing.

8. pg enzyme
1 10 100 1000 10000 1e5 1e6
rate(deltaODpermin)
-0.2
-0.1
0
0.1
0.2
0.3
0.4
0.5
ADH
GDH
HRP
Many Enzymes and Functional Proteins
Have Sensitive Assays

9. minutes
0 2 4 6 8 10 12 14 16
A652
0
0.2
0.4
0.6
w/o SDS
w/ SDS
HRP activity - effect
of 0.1% SDS
minutes
0 2 4 6 8 10 12 14 16
A340
0
0.02
0.04
0.06
0.08
0.1
0.12
0.14
0.16
0.18
0.2 w/o SDS
w/ SDS
GST activity - effect
of 0.1% SDS
Many Enzymes can even Survive Low SDS Concentration

10. 8 4 2 1 0.5 0.25 0.125 0.063 µg/lane
Before Transfer
After Transfer
ADH
Proteins can be Eluted into the PEP Plates Effectively

11. PEP Plate Has Good Recovery
0
10
20
30
40
50
60
70
80
4 ng/well 400 ng/well 4000 ng/well
% Recovery
% Recovery

12. Polypropylene 384-plate Has Good Recovery
0
10
20
30
40
50
60
70
80
90
10 ng/well 100 ng/well 1000 ng/well
% Recovery
% Recovery

13. Effect of Urea and SDS on NAD Reductase ActivityNADReductaseActivity
NAD Reductase is stable in 3 M urea plus 0.1% SDS

14. Most NAD Reductase were Transferred and Recovered
in the PEP Plate
Gel stained after PEP Transfer Gel stained before PEP Transfer
10 µg 1 µg 0.1 µg 10 µg 1 µg 0.1 µg

15. NAD Reductase in SDS-PAGE and PEP ---
Most of Active Reductase Recovered in One Well.
Promega Reductase at 5.6 mg/mL was prepared with 3 M Urea and 2% CHAPS to a final
concentration of 1 mg/mL. After room temperature incubation for 30 min, the protein
was loaded on SDS-PAGE at 10 µg/lane, 1 µg/lane and 0.1 µg/lane.
NADReductaseActivity
Reductase
Reductase

16. Stability of Promega Reductase after 24 Hrs. Treatment in 3 M
Urea 2% CHAPS and 0.1% SDS
Reductase mg/mL
NADReductaseActivity

17. Kenneth M, Bischoff et. al.
Analytical Biochemistry 260, 1-17, 1998
Enzymes Active in SDS-PAGE Reported in the Literature
17

18. Enzymes Active in SDS-PAGE Reported in the Literature (cont.)
18

19. Enzyme Activity Profiling
PEP can be used to build 3-D enzyme function
landscape for large enzyme families such as:
Protein Kinases (>500 in the human genome)
Protein Phosphatases (>150 in the human genome)
Proteinases (>1,000 in the human genome)
Oxido reductases (>600 in the human genome)
CYP Monooxygenases (57 in the human genome)
Methytransferases (epigenetics)
Any enzyme families with multiple isoforms.
The Platform can be used in basic research, drug target
identification and validation, drug safety studies,
biomarker discovery and diagnostics development.
19

20. NADPH Oxidase Activities from Beef Liver
ΔOD340nm
20

21. NADH Oxidase Activities from Beef Liver
ΔOD340nm
21

22. Selected Proteins from a Proteome with
NADH-Dependent Oxidase Activities Recovered in PEP.
22

23. 3. PEP for Functional Biomarker
Discovery from Human Serum
23

24. Biomarker Area
Enzyme Activity-based Functional Proteomics for Biomarker
Discovery Could Push the Sensitivity Further than Protein
Staining and Mass Spectrometry
PEP Technology: A New Dimension and Could be More Sensitive
New Area of
Discovery with PEP
Technology and
Functional Assay

25. 25
Enrichment of Low Abundance Proteins from Human Serum
Without
Enrichment
With
Enrichment

26. 26
Time-dependent Hexokinase Activity Analysis from
AlbuVoid-enriched Normal Serum
Hexokinase activity was measured by NADP reduction at 340 nm at 0 (A), 60 min (B).
and overnight (C) incubation.

27. 27
Time-dependent Hexokinase Activity Analysis from
AlbuVoid-enriched Lung Cancer Patient Serum
Hexokinase activity was measured by NADP reduction at 340 nm at 0 (A), 60 min (B).
and overnight (C) incubation.

28. 28
Comparison of Normal and Lung Cancer Patient
Serum Hexokinase Activity Pattern
Hexokinase activities from both normal and lung cancer patient serum were displayed
in a heat-map for comparison and subsequent process for mass spectrometry identification.

29. 29
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
A 0.036 0.0447 0.0428 0.0484 0.0398 0.0287 0.0232 0.0415 0.0179 0.0333 0.0305 0.0394 0.0594 0.0565 0.0381 0.0427 0.025 0.0392 0.0579 0.0618 0.0413 0.0293 0.0263 0.0304
B 0.0408 0.063 0.0283 0.0656 0.0513 0.0513 0.0403 0.0553 0.0101 0.0609 0.0576 0.0476 0.0487 0.0585 0.0385 0.0346 0.0292 0.0281 0.0624 0.0665 0.0451 0.0323 0.0363 0.0261
C 0.0503 0.0565 0.0594 0.0296 0.0451 0.044 0.0346 0.0346 0.0475 0.0414 0.0592 0.0314 0.0482 0.0377 0.032 0.0365 0.0326 0.0465 0.0468 0.079 0.0593 0.0356 0.0284 0.0174
D 0.0313 0.0592 0.0145 0.0561 0.0389 0.0415 0.0606 0.0566 0.0208 0.057 0.0272 0.0687 0.0291 0.0298 0.0318 0.0344 0.033 0.0369 0.0703 0.0698 0.0561 0.043 0.0352 0.0167
E 0.0356 0 0.0318 0.0242 0.0251 0.0533 0.0291 0.0648 0.0394 0.0332 0.0262 0.0264 0.0245 0.0272 0.0255 0.0262 0.0264 0.0626 0.0568 0.0612 0.059 0.0494 0.032 0.0088
F 0 0.119 0.014 0.0436 0.0436 0.0321 0.0434 0.0743 0.041 0.0507 0.0353 0.0328 0.0238 0.0222 0.0244 0.0579 0.047 0.0607 0.0515 0.0409 0.0483 0.0344 0.0263 0.0197
G 0.002 0.0861 0.0347 0.0223 0.0266 0.027 0.042 0.04 0.0242 0.0369 0.0183 0.0229 0.0265 0.0333 0.035 0.0315 0.0346 0.0289 0.0347 0.0482 0.0378 0.0265 0.0209 0.0098
H 0.0204 0.0377 0.0172 0.0123 0.0227 0.0076 0.0253 0.0382 0.0141 0.0452 0.0253 0.0265 0.0256 0.0245 0.0208 0.0196 0.026 0.0269 0.0205 -0.001 0.0187 0.0158 0.0107 0.006
I 0.0057 0.0334 0.0165 0.01 0.0081 0.0052 0.0283 0.0197 0.0161 0.0398 0.0168 0.0183 0.0218 0.0022 0.0113 0.0233 0.0184 0.0261 0.018 0.0202 0.013 0.0112 0.0113 0.0084
J 0.0142 0.04 0.0103 0.007 0.0106 0.0133 0.0087 0.0184 0.0079 0.0418 0.0224 0.0171 0.0184 0.0196 0.0208 0.0196 0.0205 0.0252 0.0236 0.0132 0.032 0.0139 0.009 0.008
K 0.0487 0.0446 0.0059 0.0002 0.0167 0.0072 0.0197 0.0111 0.0114 0.0154 0.0162 0.0153 0.0185 0.0201 0.0161 0.016 0.0292 0.0229 0.0136 0.0176 0.0109 0.0193 0.0114 0.0096
L -0.0071 -0.0055 0.0105 0.0047 0.0057 0.0068 -0.007 0.0143 0.008 0.0087 0.0173 0.0091 0.0151 0.0251 0.0203 0.0149 0.0156 0.0367 0.0113 0.0053 0.0081 0.0152 0.0018 -0.0036
M 0.0198 0.0292 0.0094 0.0536 -0.0228 -0.0185 0.0189 0.0356 0.0199 0.0227 0.0244 0.0105 0.0133 0.0159 0.0178 0.0162 0.0381 0.0214 0.0212 0.0079 0.0118 0.0108 0.0153 -0.0004
N 0.0102 0.0417 0.0061 0.0116 0.0539 -0.0009 0.0223 0.0466 0.0353 0.0426 0.0269 0.0238 0.0232 0.0231 0.026 0.0172 0.0166 0.0295 0.0134 0.0147 0.0359 -0.0056 0.0199 0.0111
O 0.0701 0.0549 0.0366 0.035 0.0107 0.0101 -0.0021 0.0267 0.0052 0.0102 0.0016 0.0082 0.0066 0.0123 0.0071 0.0085 0.0156 0.0216 0.0259 0.0197 0.0114 0.0021 0.0168 0.0061
P 0.1498 0.0987 0.0719 0.0415 0.0463 0.0439 0.0375 0.0433 0.0634 0.0271 0.0112 0.0322 0.0528 0.0456 0.034 0.0567 0.0708 0.0688 0.082 0.0562 0.0701 0.0348 0.0384 -0.0043
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
A 0.0431 0.0205 0.0377 0.0477 0.0604 0.027 0.0649 0.0373 0.0311 0.0354 0.0389 0.0423 0.0517 0.0754 0.0533 0.0688 0.0185 0.0384 0.0447 0.0482 0.0296 0.0171 0.0135 0.0111
B 0.0237 0.0471 0.0589 0.0437 0.0527 0.036 0.0162 0.0679 0.038 0.0344 0.0484 0.062 0.0632 0.0879 0.0948 0.066 0.0459 0.0706 0.0625 0.0613 0.0502 0.034 0.027 0.0264
C 0.0246 0.0358 0.0508 0.0588 0.054 0.0415 0.0436 0.0333 0.0453 0.0442 0.0408 0.0477 0.0442 0.0747 0.0642 0.0382 0.0434 0.0501 0.0784 0.0524 0.0426 0.017 0.0342 0.0121
D 0.0072 0.0338 0.0508 0.0688 0.043 0.0424 0.0418 0.0609 0.0351 0.0371 0.0424 0.049 0.0428 0.0603 0.0489 0.0718 0.0333 0.0483 0.0551 0.0614 0.0437 0.0359 0.0325 0.0168
E 0.0202 0.0136 0.0456 0.0349 0.0446 0.0236 0.0294 0.0507 0.0231 0.0321 0.0284 0.0373 0.0309 0.0464 0.0514 0.0337 0.0304 0.063 0.0627 0.0535 0.0396 0.0285 0.0413 0.0172
F 0 0.0134 0.0386 0.0452 0.0258 0.0258 0.0165 0.0272 0.0317 0.0173 0.0231 0.0328 0.0385 0.0356 0.024 0.0295 0.0186 0.0331 0.054 0.0557 0.0319 0.0357 0.0469 0.0176
G 0.0162 0.0211 0.0291 0.0275 0.0266 0.0293 0.013 0.0428 0.0247 0.0248 0.0189 0.0284 0.0246 0.0372 0.0325 0.0355 0.0234 0.0159 0.0298 0.023 0.0219 0.0267 0.0285 0.0104
H -0.0138 0.0238 0.0316 0.0372 0.017 0.0117 0.023 0.0799 0.0241 0.0336 0.0279 0.0185 0.0231 0.0439 0.0611 0.0273 0.0162 0.0299 0.0245 0.0292 0.0215 0.0263 0.0314 0.0128
I 0.0042 0.0089 0.039 0.0331 0.0202 0.0137 0.0597 0.0216 0.0272 0.0171 0.018 0.0245 0.018 0.0354 0.0327 0.0233 0.009 0.04 0.0339 0.0253 0.0136 0.0248 0.0158 0.0135
J 0.0037 0.0119 0.0162 0.0397 0.0239 0.0111 0.0468 0.0242 0.0152 0.0223 0.0104 0.0312 0.0314 0.0281 0.0264 0.0175 0.0155 0.0196 0.0232 0.0223 0.0162 0.0171 0.0138 0.0089
K 0.008 0.004 0.0523 0.0129 0.0195 0.0321 0.02 0.0257 0.0176 0.02 0.0118 0.0174 0.0157 0.0251 0.0252 0.0167 0.0167 0.0119 0.0206 0.0185 0.0111 0.012 0.01 0.0062
L 0.0094 0.0148 0.016 0.0174 0.0187 0.0029 0.0125 0.0307 0.028 0.0203 0.0126 0.0215 0.0206 0.0357 0.0373 0.0126 0.0112 0.0203 0.023 0.0332 0.0176 0.012 0.012 0
M 0.0079 0.0113 0.0167 0.0167 0.01 0.016 0.0168 0.0192 0.0281 0.0212 -0.0071 0.0148 0.0211 0.0314 0.0284 0.0116 0.0076 0.014 0.0204 0.0141 0.0114 0.0137 0.0129 0.0067
N 0.0019 0.0111 0.0181 0.0065 0.0122 0.008 0.0132 0.0094 0.0186 0.0147 0.0101 0.0114 0.0208 0.0409 0.0257 0.013 0.0144 -0.0011 0.018 0.0147 0.0157 0.0095 0.0055 -0.0012
O -0.007 -0.0006 -0.0056 -0.004 -0.0076 0.001 -0.0075 0.0075 -0.0045 -0.0026 -0.0142 -0.0096 -0.002 0.0105 0.0223 0.0139 0.0117 -0.0071 0.0017 0.0107 -0.0053 -0.003 -0.008 -0.0154
P 0.0023 0.002 0.0295 0.0381 0.0479 0.0243 0.0503 0.0307 0.0313 0.0246 0.0134 0.0236 0.0183 0.0251 0.0307 0.0259 0.0166 0.0315 0.0561 0.0335 0.0211 0.0262 0.0055 -0.0044
Mw
(KD)
180
75
48
35
28
17
10
180
75
48
35
28
17
10
HexokinaseActivity(NADPHΔ340nm)
pH 3 pH 10
Direct Measurement of Hexokinase Activity from Normal (upper panel)
and Breast Cancer Serum (lower panel) after PEP

30. 30
Direct Measurement of Protease Activity from Normal (upper panel)
and Breast Cancer Serum (lower panel) after PEP
Fluorescence(RFU)

31. 31
Measurement of Hexokinase Activity from Normal (upper panel)
and Breast Cancer Serum after AlbuVoidTM enrichment and PEP

32. 32
Protease Activity from Normal (upper panel) and Breast Cancer
Serum after AlbuVoidTM enrichment and PEP

33. NormalSerum
ProteaseActivity
BreastCancerSerum
ProteaseActivity
Protease Activity Profile from Normal Serum and
Breast Cancer Serum (EMD Millipore)

34. Lung Cancer Biomarker Discovery
In the slide next is the comparison between normal
(upper panel)and lung cancer (lower panel) patient
(N=20) serum for their glycolytic enzyme activity
using the PEP technology, large number of functional
features were identified.

35. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
A 0.0676 0.0439 0.0565 0.0461 0.1044 0.0505 0.0435 0.0438 0.0479 0.0492 0.0646 0.056 0.0674 0.0679 0.0634 0.0677 0.0562 0.077 0.1576 0.0645 0.0815 0.0681 0.0692 0.0545
B 0.067 0.0492 0.051 0.0806 0.2662 0.0986 0.1981 0.1272 0.041 0.0473 0.0544 0.0659 0.0643 0.0698 0.082 0.0557 0.0528 0.0513 0.0545 0.0472 0.0551 0.04 0.0478 0.0471
C 0.0464 0.0331 0.0147 0.0095 0.0364 0.065 0.1658 0.099 0.0232 0.0187 0.0208 0.0205 0.0191 0.0112 0.0175 0.0119 0.0119 0.0049 0.0184 0.008 0.026 0.0098 0.0244 0.0088
D 0.0502 0.0309 0.0149 0.0215 0.0235 0.017 0.0797 0.1009 0.0189 0.0062 0.0207 0.0176 0.0163 0.008 0.0067 0.0118 0.0124 0.0124 0.0129 0.0259 0.0169 0.0143 0.0224 0.0134
E 0.0943 0.0637 0.0382 0.045 0.0513 0.0502 0.0461 0.0485 0.0587 0.0574 0.0601 0.0531 0.0615 0.0438 0.0442 0.042 0.0509 0.0313 0.0436 0.0263 0.0784 0.0297 0.0359 0.0263
F 0.0711 0.0461 0.037 0.0404 0.0468 0.0492 0.0591 0.0374 0.0504 0.0502 0.0519 0.0545 0.0538 0.0417 0.0441 0.0371 0.0416 0.0333 0.0363 0.0233 0.0511 0.0627 0.05 0.022
G 0.0636 0.0213 0.0181 0.0191 0.0214 0.0226 0.0145 0.0142 0.0215 0.0383 0.026 0.0198 0.0133 0.0104 0.0096 0.0104 0.0174 0.0105 0.0322 0.011 0.0543 0.0349 0.0369 0.0175
H 0.0426 0.0313 0.0244 0.0161 0.013 0.0502 0.0311 0.1204 0.0186 0.0089 0.0142 0.0134 0.012 0.0082 0.0109 0.016 0.0102 0.0166 0.022 0.0103 0.027 0.1418 0.0968 0.0059
I 0.0956 0.0475 0.0373 0.0368 0.0492 0.0485 0.0864 0.0758 0.0616 0.051 0.0468 0.0457 0.0493 0.0465 0.0434 0.0372 0.0525 0.0667 0.1312 0.031 0.0968 0.1407 0.0678 0.0287
J 0.0894 0.0477 0.0294 0.0409 0.0429 0.0468 0.0408 0.0433 0.0503 0.0417 0.0453 0.0428 0.0478 0.0311 0.0377 0.0455 0.1563 0.1848 0.1113 0.0428 0.0528 0.0339 0.0616 0.0374
K 0.0781 0.0324 0.0125 0.0149 0.0198 0.0177 0.015 0.0091 0.0559 0.0195 0.0519 0.0144 0.0219 0.0049 0.0155 0.0013 0.1086 0.1636 0.3759 0.01 0.0157 0.0071 0.0351 0
L 0.0502 0.0293 0.0119 0.0113 0.0209 0.0225 0.0192 0.0103 0.0595 0.0389 0.1486 0.1055 0.0169 0.0093 0.0096 0.0629 0.1847 0.0456 0.0181 0.0189 0.005 0.0096 0.04 0.0216
M 0.1536 0.0957 0.0804 0.0788 0.0835 0.0836 0.0824 0.0841 0.1411 0.2984 0.3769 0.0956 0.0903 0.088 0.1846 0.1551 0.1067 0.0779 0.0819 0.0653 0.0772 0.073 0.0914 0.076
N 0.1485 0.0996 0.0653 0.0576 0.0672 0.0713 0.0696 0.059 0.1677 0.2246 0.2931 0.066 0.0662 0.0634 0.1125 0.0905 0.0737 0.0689 0.0621 0.0599 0.0633 0.0583 0.0716 0.0713
O 0.1118 0.0489 0.0621 0.0309 0.0389 0.0435 0.0401 0.0337 0.1299 0.4718 0.2018 0.0447 0.0325 0.033 0.042 0.0456 0.0325 0.0232 0.0357 0.0336 0.0474 0.039 0.0342 0.0313
P 0.7759 0.7237 0.4237 0.0034 0.0619 0.0736 0.0479 0.053 0.0906 0.6007 0.4574 0.0548 0.0461 0.0462 0.0419 0.0825 0.1451 0.0675 0.0603 0.0526 0.0484 0.0373 0.0296 0.0166
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
A 0.0485 0.0451 0.7698 0.0765 0.0833 0.0797 0.0662 0.075 0.1667 0.0737 0.0664 0.0408 0.053 0.0479 0.067 0.0515 0.0466 0.0472 0.0481 0.0535 0.0579 0.0329 0.0298 0.0271
B 0.0532 0.0518 0.0464 0.0424 0.049 0.0469 0.0729 0.0615 0.1137 0.047 0.0439 0.0473 0.0534 0.0428 0.0501 0.0357 0.0512 0.0433 0.0667 0.0396 0.0442 0.0422 0.0442 0.0317
C 0.0498 0.0365 0.0284 0.0576 0.0307 0.0428 0.0214 0.0222 0.0265 0.0205 0.0195 0.0241 0.026 0.0309 0.0238 0.0193 0.0164 0.0209 0.1131 0.0948 0.1115 0.0337 0.0185 0.0301
D 0.0468 0.0393 0.0277 0.0282 0.031 0.0271 0.0306 0.0241 0.0208 0.0214 0.0243 0.0211 0.0262 0.0232 0.0255 0.016 0.0227 0.0192 0.1837 0.2614 0.3684 0.0477 0.0257 0.0338
E 0.0726 0.0605 0.056 0.0398 0.054 0.0531 0.0477 0.0472 0.0699 0.056 0.0515 0.0526 0.0669 0.0478 0.0513 0.0424 0.0525 0.0442 0.1481 0.2524 0.3534 0.0799 0.0592 0.0381
F 0.0602 0.0486 0.0446 0.0533 0.0558 0.0497 0.049 0.0613 0.0521 0.0499 0.0487 0.0455 0.0466 0.0482 0.0507 0.0439 0.0513 0.0416 0.2083 0.3571 0.3005 0.0685 0.0493 0.0344
G 0.0533 0.0419 0.0333 0.0429 0.0324 0.0378 0.0287 0.033 0.0296 0.0349 0.0268 0.0322 0.0301 0.0235 0.0255 0.0162 0.0257 0.0244 0.0465 0.0387 0.0218 0.0119 0.0313 0.0169
H 0.0484 0.0407 0.0318 0.0298 0.0285 0.0309 0.0336 0.0336 0.027 0.0282 0.0273 0.0293 0.0311 0.0293 0.0279 0.0192 0.015 0.0151 0.0216 0.0167 0.027 0.0155 0.0262 0.023
I 0.0764 0.053 0.0526 0.0569 0.0472 0.0543 0.0552 0.0581 0.0552 0.0479 0.053 0.0573 0.0632 0.0567 0.0573 0.0452 0.0525 0.0423 0.0463 0.0394 0.039 0.0312 0.0544 0.0459
J 0.0824 0.0424 0.0513 0.0558 0.0499 0.0517 0.0554 0.0507 0.0504 0.052 0.055 0.055 0.0528 0.0524 0.0463 0.0424 0.0461 0.0418 0.0414 0.0281 0.044 0.0282 0.053 0.0483
K 0.0605 0.0323 0.0301 0.0315 0.0312 0.0293 0.0347 0.0406 0.0247 0.0204 0.0227 0.0217 0.0229 0.0213 0.0166 0.0148 0.0203 0.0136 0.0213 0.0103 0.0179 0.0193 0.0249 0.0202
L 0.0603 0.0383 0.0256 0.0283 0.0343 0.0337 0.0319 0.027 0.0286 0.0242 0.0286 0.0171 0.0234 0.0214 0.0156 0.0148 0.013 0.0156 0.0318 0.0171 0.0175 0.0101 0.019 0.027
M 0.1282 0.087 0.0658 0.0764 0.0733 0.0909 0.088 0.093 0.0828 0.0829 0.0854 0.0882 0.0952 0.1137 0.0868 0.0902 0.093 0.0867 0.1017 0.0863 0.0847 0.0762 0.0912 0.0954
N 0.1178 0.0784 0.0654 0.0768 0.0882 0.0872 0.0818 0.0702 0.0689 0.0718 0.0727 0.0684 0.0733 0.0913 0.0766 0.0733 0.0732 0.0749 0.0843 0.0718 0.0886 0.0662 0.0988 0.1108
O 0.0528 0.122 0.0471 0.0387 0.0367 0.0376 0.0316 0.0332 0.0361 0.0353 0.0332 0.0341 0.0399 0.4747 0.0356 0.0334 0.0392 0.0448 0.0431 0.0522 0.038 0.0578 0.0542 0.0349
P 0.1175 0.0666 0.0599 0.0754 0.0658 0.0742 0.0959 0.0603 0.0519 0.0567 0.0615 0.0677 0.052 0.257 0.0663 0.0532 0.0723 0.0741 0.098 0.0934 0.4635 0.0767 0.0566 0.0615

36. Sample ID Protein Full Protein Name Expected Size (kD) Size on 2-D Gel Expected pI pI on 2-D Gel
1 PLG Plasminogen 83 100 6.2 5.5, 8.5
2 Xxbac Uncharacterized protein
3 HRNR Hornein 21 25 7.1 7.2
4 TGM3 Transglutaminase3 50, 27 25 7.2
5 ENO1 Enolase 1 47 25 7.37, 6.95 7.2
6 FASN Fatty acid synthase 22 25 7.5 6.5
7 FABP5 Fatty acid binding protein5 15 25 6.3 7.2
8 CAT Catalase 60 200 6.9 4
9 PKM Pyruvate kinase 66 80 7.9 8.5
10 BLMH Bleomycin hydrolase 53 45 4.8 8
11 GAPDH
Glyceraldehyde-3-phosphate
dehydrogenase 36 40 8.6 8.2
12 ARG1 Arginase 1 35 42 5.9 7.5
13 CFH Complement factor H 155 200 5.5
14 C3 Complement component 3 75 50 5.7 9
15 SERPINA3 Serpin peptidase inhibitor, clade A 47 200 5.4 5.5
16 APOH Apolipoprotein H 50 100 8.4 8
17 LTF Lactotransferrin 80 50 8.7 9
18 A2M Alpha-2-macroglobulin 180 50 10 0
19 HPX Hemopexin 59 150 5.4 to 6.4 5
Proteins Identified with Mass Spectrometry (Lung Cancer)

37. Biology Verification: Enhanced Hexokinase Activity with Purified GAPDH
GAPDH (Units) added to the System
NADPReduction(OD340nm)
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0 0.125 0.25 0.5 1
With Substrate
No Substrate

38. Flow Chart of PEP Technology for Cancer Biomarker Discovery
PEP Technology Applied to Normal and
Cancer Patient Serum
Enzyme Activity Profile Analyzed with
Principle Component Analysis
Fractions with Distinct Enzyme Activity between the
Two Groups Analyzed by Mass Spectrometry
Enzymes Identified Are Further Validated
with Specific Antibodies
A Group of Antibodies Identified for Direct ELISA-based
Enzyme Activity Analysis as Biomarker or Diagnostic Kit
Discovery
Stage
Development
Stage

39. Protein Kinase Analysis from a Cancer Cell Line with 5 Kinase Inhibitors.
Upper panel: without inhibitor; lower panel: with inhibitor.
Promega’s ADP-Glo Protein Kinase kit was used, casein lysate was the general substrate.

40. Diabetics Biomarkers: MAO (upper panel) vs. LEAN (lower panel)

41. Diabetics Biomarkers: MNO (upper panel) vs. LEAN (lower panel)

42. Conclusions
 A 2-D Gel method was modified to retain enzyme activity or protein
function after separation.
 A Protein Elution Plate was developed to effectively transfer and
recover functional proteins from 1-D or 2-D gels.
 This PEP technology can be used for systematic enzyme assays from
a proteome.
 A large number of active hexokinase and protease fractions were
identified from human serum after 2-D gel protein separation and PEP
for protein elution.
 Potential functional biomarkers were identified from lung and breast
cancer patient serum with PEP and mass spectrometry.
 Further biological and clinical validations are needed for the
biomarker candidates identified.
42

43. 43
Acknowledgement
Mike Davies Array Bridge Inc.
Guofeng Fu Array Bridge Inc.
David Wang University of Iowa School of medicine
Matthew Kuruc Biotech Support Group
Swapan Roy Biotech Support Group
Zhenyu Sun, MD. Nantong University School of Medicine, China
Xiaofeng Chen, MD. Fudan University School of Medicine, China
Gan Wang, Ph.D. Wayne State University School of Medicine
Liang Li, MD. Zibo Central Hospital, China
Chuanguang Xiao, MD. Zibo Central Hospital, China