Post on 12-Jan-2016
Sample peptides:
-Class 1:1,2,3…
-Class 2:1,2,3…
-Class 3:1,2,3…
SCX/RP-HPLC
Collect 100 fractions on MALDI plates
MALDI-TOF MS
for each sample
LC fraction -- m/.z --abundance
MASS-Conductor ®
SSA ®
Machine learning
feature discovery and classification
Biomarker panels
MSMS protein IDProspective validation
with quantitative mass spec (MRM)
Figure 1
0
200
400
600
800
1000
1200
1400
1600
1800
1 4 7 10 13 16 19 22 25 28 31 34 37 40 43 46 49 52 55 58 61
0
200
400
600
800
1000
1200
1400
1600
1800
1 153 305 457 609 761 913 1065 1217 1369 1521 1673 1825 1977 2129 2281 2433
AR
0
100
200
300
400
500
600
700
800
900
1000
0 20 40 60 80 100 120
S
0
100
200
300
400
500
600
700
800
900
1000
0 20 40 60 80 100 120
V
0
100
200
300
400
500
600
700
800
900
1000
0 20 40 60 80 100 120
S
0
100
200
300
400
500
600
700
800
900
1000
0 20 40 60 80 100 120
AR
0
100
200
300
400
500
600
700
800
900
1000
0 20 40 60 80 100 120
V
0
100
200
300
400
500
600
700
800
900
1000
0 20 40 60 80 100 120
Class A
Class B
Class C
A
B
HPLC Fractions
LC
-MA
LD
I in
ten
sity
HPLC fraction 13 (m/z 1200 ~ 1500)
LC
-MA
LD
I in
ten
sity
Figure 2
Pool individual urine samples
Split into 4identical samples
1
2
3
4
Spin Freeze @ -20ºC Thaw Process
SpinFreeze @ -20ºC Thaw Process
Spin Freeze @ -20ºC Thaw Process
SpinFreeze @ -20ºC Thaw Process
RT 4 hrs
Figure 3A
122
t > 3
72
t < -3
S/F/T vs. F/T/S RT/S/F/T vs. RT/F/T/S
14825 196
-3 < t < 3t < -3
14762
-3 < t < 3 t > 3
83
0MW
/100
002
42
30
1# X
100
RandomizeFeature set
20 X
P (Two-Tailed) = 0.013
Figure 3B
Transit
B
Pool 5 individual urine samples
Split into 6 identical samples
1
2
3
4
5
6
Day 1
Process
Day 2
ProcessR.T.
Day 3 Day 4 Day 5
ProcessR.T.
ProcessR.T.
ProcessR.T.
ReceiveFEDEX
Process
5 X
Figure 1
Day 2 vs. Day 1 Day 3 vs. Day 1 Day 4 vs. Day 1 Day 5 vs. Day 1 FEDEX vs. Day 1
Den
sity
0.0 0.2 0.4 0.6 0.8 1.0 0.0 0.2 0.4 0.6 0.8 1.0 0.0 0.2 0.4 0.6 0.8 1.0 0.0 0.2 0.4 0.6 0.8 1.0 0.0 0.2 0.4 0.6 0.8 1.0
Tukey HSD P value between sub groups
0
2
4
6
8
10
12
Figure 3
B
15516728
t > 3
Day 2 vs. 1
-3 < t < 3
t < -3
Day 3 vs. 1
-3 < t < 3
33
t > 3t < -3
553
Day 4 vs. 1
-3 < t < 3
38
t > 3t < -3
16325 1469
Day 5 vs. 1
-3 < t < 3
149
t < -3
15298
t > 3
761
FEDEX vs. day 1
-3 < t < 3
173
t < -3
15982
t > 3
12 16565 339
MW
/100
01
3
Figure 4
B
Distribution of CV of each feature Distribution of DCV of each feature
1.0
0.5
2.0
1.5
0.0
Day 1 Day 2 Day 3 Day 4 Day 5 FEDEX Day 2- Day 1
Day 3- Day 1
Day 4- Day 1
Day 5- Day 1
FEDEX- Day 1
0.0
-1.0
2.0
1.0
-2.0
Figure 5
B
1. THP 982.59 VLNLGPITR2. THP 1047.48 SGSVIDQSRV3. THP 1211.66 DQSRVLNLGPI
4. THP 1225.69 SRVLNLGPITR5. THP 1324.76 IDQSRVLNLGPI6. THP 1423.83 VIDQSRVLNLGPI 7. THP 1468.82 DQSRVLNLGPITR8. THP 1510.87 SVIDQSRVLNLGPI9. THP 1567.91 GSVIDQSRVLNLGPI10. THP 1581.91 IDQSRVLNLGPITR11. THP 1654.91 SGSVIDQSRVLNLGPI12. THP 1680.98 VIDQSRVLNLGPITR13. THP 1755.96 SGSVIDQSRVLNLGPIT14. THP 1768.01 SVIDQSRVLNLGPITR15. THP 1912.07 SGSVIDQSRVLNLGPITR16. THP 2040.16 SGSVIDQSRVLNLGPITRK
Figure 7
A
Sig
nal
/100
000
20
4
THP 1680.98 THP 1912.07THP 1423.83
Figure 7
B
1 2 3 4 5 6 7 8 9 10 11 12 130.00E+00
2.00E-01
4.00E-01
6.00E-01
8.00E-01
1.00E+00
1.20E+00 PR
Enbrel
CR CR
Anakinra
CRPR CR
Enbrel Anakinra A
B
C
Figure X
S/F/T vs. F/T/S RT/S/F/T vs. RT/F/T/S
Den
sity
0.0 0.2 0.4 0.6 0.8 1.0 0.0 0.2 0.4 0.6 0.8 1.0
Tukey HSD P value between sub groups
0
2
4
6
8
10
12
Total # of feature: 15030
Urine peptide biomarkers: the discovery process
Sample peptides:
-Class 1:1,2,3…
-Class 2:1,2,3…
-Class 3:1,2,3…
SCX/RP-HPLC
Collect 100 fractions on MALDI plates
MALDI-TOF MS
for each sample
LC fraction -- m/.z --abundance
MASS-Conductor ®
Machine learning
feature discovery and classification
Biomarker panels
MSMS protein IDProspective validation
with quantitative mass spec (MRM)
Allograft Acute Rejection Urine Biomarker Discovery
Peak finding
Peak alignment
Peak indexing
Supervised Data mining
Feature selection
Training
Testing
LCMS Data reduction
Unsupervised Data mining
2D - Clustering
QuantitativeLCMS
Validation
1 2 3 4
Permutation based FDR analysis of the biomarker signature
Proposed Underlying Mechanisms of Urine Naturally Occurring Peptide Biomarkers