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0 100 200 300 400 500 600 700
2D_25
2D_50
2D_100
2D_200
DS
Cleanup
1D
2D
Total Number of Peaks
Met
hods
Opt
imiz
atio
na
b
cd
6 8 10 12 14 16 18 20
6
8
10
12
14
16
18
20
Ln
(I)
Ln(I)
FT 200 500 1000
Color: • • • •Features: 25 18 43 43% CV: 0.2 0.3 0.3 0.5R2: 0.99 0.98 0.96 1.0
Peak Reproducibility from 30% Fractions
143 Features129 < 10 kDa7 > 10 kDa < 20 kDa6 > 20 kDa
30% AcN30% Organic (143 Features)
FT 200 500 1000#Features 25 18 43 43CV 0.2 0.3 0.3 0.5R2 0.99 0.98 0.96 1.0
6 8 10 12 14 16 18 20
6
8
10
12
14
16
18
20
Ln
(I)
Ln(I)
FT 200 500 1000
Color: • • • •Features: 52 56 62 67% CV: 0.2 0.2 0.2 0.3R2: 0.98 0.99 0.98 0.99
237 Features205 < 10 kDa28 > 10 kDa < 20 kDa4 > 20 kDa
Peak Reproducibility from 45% Fractions
45% Organic (237 Features)
FT 200 500 1000#Features 52 56 62 67CV 0.2 0.2 0.2 0.3R2 0.98 0.99 0.98 0.99
6 8 10 12 14 16 18 20
6
8
10
12
14
16
18
20
Ln
(I)
Ln(I)
FT 200 500 1000
Color: • • • •Features: 49 73 68 63% CV: 0.4 0.2 0.3 0.3?R2: 0.99 0.99 0.97 0.91
253 Features199 < 10 kDa46 > 10 kDa < 20 kDa8 > 20 kDa
Peak Reproducibility from 80% Fractions80% Organic (258 Features)
FT 200 500 1000#Features 49 73 68 63CV 0.4 0.2 0.3 0.3R2 0.99 0.99 0.97 0.91
Met
ho
d
Supplemental Figure 1.
Pooled Serum
1) IgY-R7 Depletion Column
2) SPE w/Peptide Mactrotraps (C18/ SCX)
3) C4-RP HPLC (LC-MALDI-Spotter)
4) LC-MALDI-TOF-MS (isolate key fractions)
5) 1D-PAGE (In-gel tryptic digestion)
6) nLC-MS2
Validation by Western Blot
Increased in Ras
15626, m/z15605, m/z
6568, m/z
15605, m/z
6568, m/z
Serpina1a
1500
1000
500
0
Control Ras
Rela
tive
Inte
nsity
LysC
Rela
tive
Inte
nsity
Sub-pooled serum
IgY-R7 Depletion
C4-RP HPLC
Peptide Macrotrap
LC-MALDI Analysis
1D-PAGE on Fractions containing Peaks of Interest
In-gel Tryptic Digestion
LC-MS(MS)2 Analysis
Validation with Western Blot
Verif ication with EIA on Human Serum
Feature Increased in Kras vs. Control
6,560 6,620
m/z
Increased in Ras
15626, m/z15605, m/z
6568, m/z
15605, m/z
6568, m/z
Serpina1a
1500
1000
500
0
Control Ras
Re
lati
ve
Inte
nsi
ty
LysC
Re
lati
ve
Inte
nsi
ty
2,000
1,500
500
0
Inte
nsi
ty
C. Ras
6,568, m/z
100
50
0
Rel
. In
ten
sity
300 1,200
m/z
MS2 Sequence
Protein ID
a.b.
c.
Supplemental Figure 2.
Extracellular Membrane Cytoplasm Nuclear
Significant Hit
Receptor Ligand
Transcripton Factor
Generic Protein
Binding Protein
Transporter
Receptor
Generic Enzyme
Protease
Kinase
Legend
SupplementalFigure 3.
Recommended