Profiling EGFR Kinase Inhibitor Resistance

Profiling EGFR Kinase Inhibitor Resistance
Pathways in Non-Small Lung Cancer Cells
Ryan D. Bomgarden,1 Ryan Jacobs, 2 Jason Fong, 2 David Moravec, 2 Gregory M. Botting, 2 Neelu Puri, 2
Rosa Viner, 3 Michael Blank, 3 John C. Rogers1
1
Thermo Fisher Scientific, Rockford, IL; 2University of Illinois at Chicago, Rockford, IL;
3
ThermoFisher Scientific, San Jose, CA
Overview
Purpose: To determine the mechanism of tyrosine kinase inhibitor (TKI) resistance
through quantitative LC/MS research analysis of differential changes in protein
pathway levels.
Methods: Parental and TKI-resistant cell lines were treated with and without growth
factors (EGF or HGF) and TKI inhibitors erlotinib or SU11274. Samples from each
treatment condition were labeled with Thermo Scientific™ TMT10plex™ reagents and
analyzed/quantified by LC/MS using a Thermo Scientific™ Orbitrap™ Fusion™
Tribrid™ mass spectrometer.
Results: Changes in protein expression and/or protein phosphorylation between
parent and TKI-resistant cell lines were observed in several key signaling pathways.
Introduction
Tyrosine kinase inhibitors (TKIs) against epidermal growth factor receptor (EGFR)
have positive therapeutic effects in a subset of non-small cell lung cancer (NSCLC)
patients; however, their clinical efficacies are limited due to the development of TKI
resistance. To study the potential mechanism of TKI resistance, two NSCLC erlotinibresistant cell lines were established by exposing the cells to progressively increasing
concentrations of inhibitor. Differences in protein expression and signal transduction
pathway activation between parental and drug-resistant cells after EGF and/or erlotinib
treatment were assessed by western blotting (Figure 1) and mass spectrometry (MS).
For MS analysis, Thermo Scientific™ Tandem Mass Tag™ (TMT™) reagents were
used for sample multiplexing and measuring differences in relative protein abundance.
Various pathways implicated in drug-resistance were examined for differences in
protein expression using an Orbitrap Fusion Tribrid instrument.
Methods
Sample Preparation
NSCLC cell lines (H2170 and H358) were established with resistance to the EGFRTKI, erlotinib, exhibiting a 4-5 fold higher IC50 than the parent cell line.1 Parental and
drug-resistant NSCLC cell lines were left untreated or treated with EGF or 10 µM
erlotinib alone, or treated with both EGF and erlotinib, for 2.5 minutes. Parental and
resistant H358 cells were also treated with HGF and SU11274. After reduction and
alkylation, proteins from the up to ten different conditions were digested with trypsin,
labeled with TMT10plex reagents, and combined before LC/MS analysis (Figure 2).
Magnetic Fe-NTA resin was also used for phospho-peptide enrichment of TMT-labeled
samples. Combined peptide samples (unenriched or phospho-enriched) were also
fractionated by high pH reverse phase in a novel spin column format containing a
polystyrene divinyl benzene resin to generate eight fractions for LC/MS analysis.
Western blotting using antibodies against p-EGFR, EGFR, p-mTOR, mTOR, p-S6
kinase, S6 kinase, p-ERK, and ERK was also performed.2
Results
Characterization of H358 cel
Initial characterization of erloti
antibodies showed p-EGFR to
19-fold) in H2170 cells, but do
c-Met/EGFR downstream sign
found to be 2–4-fold upregulat
antibodies revealed little chang
and β-actin expression betwee
treatment conditions (Figure 1
FIGURE 1. Western blot anal
protein (B) levels of mTOR, E
drug resistant H2170 and H3
A
FIGURE 2. Schematic of TMT
analysis. A) Proteins from pa
lines treated with diluent, EG
reduced, alkylated, and dige
Labeled peptides from each
phospho-peptides, fractiona
and analyzed by LC/MS anal
during MS acquisition but ge
for relative quantitation.
A
Liquid Chromatography
Samples were separated by RP-HPLC using a Thermo Scientific™ Dionex™
UltiMate™ 3000 system connected to a Thermo Scientific™ EASY-Spray™ column,
25 cm × 75 µm or a Thermo Scientific™ Acclaim™ PepMap™ C18 column over a 3 hr
5–30% gradient (A: water, 0.1% formic acid; B: acetonitrile, 0.1% formic acid) at a
300 nL/min flow rate.
Mass Spectrometry
Spectra were acquired using an Orbitrap Fusion Tribrid mass spectrometer using top
speed FT MS2 (HCD) at resolution of 60,000 at m/z 200.
Data Analysis
Spectral data files were analyzed using Thermo Scientific™ Proteome Discoverer™
software version 1.4 using the SEQUEST® HT search engine with a precursor mass
tolerance of 10 ppm and fragment mass tolerance of 0.02 Da. Carbamidomethylation
(+57.021 Da) for cysteine and TMT isobaric labeling (+229.162 Da) for lysine and
N-terminus residues were treated as static modifications with variable methionine
oxidation (+15.996 Da). Data was searched against a Swiss-Prot® human database
with a 1% FDR criteria using the Percolator algorithm.3
Proteome Discoverer software was used to calculate TMT reporter ratios with mass
tolerance ±10 ppm without applying the isotopic correction factors. A protein ratio was
expressed as a median value of the ratios for all quantifiable spectra of the peptides
pertaining to that protein.4 Hierarchical clustering and heat map analysis was
performed using Spotfire® analytics software.
2 Profiling EGFR Kinase Inhibitor Resistance Pathways in Non-Small Lung Cancer Cells
B
se inhibitor (TKI) resistance
ntial changes in protein
ated with and without growth
11274. Samples from each
™ TMT10plex™ reagents and
c™ Orbitrap™ Fusion™
phosphorylation between
veral key signaling pathways.
wth factor receptor (EGFR)
all cell lung cancer (NSCLC)
e to the development of TKI
sistance, two NSCLC erlotinibls to progressively increasing
ssion and signal transduction
cells after EGF and/or erlotinib
and mass spectrometry (MS).
g™ (TMT™) reagents were
s in relative protein abundance.
xamined for differences in
rument.
with resistance to the EGFRparent cell line.1 Parental and
eated with EGF or 10 µM
or 2.5 minutes. Parental and
U11274. After reduction and
ns were digested with trypsin,
e LC/MS analysis (Figure 2).
tide enrichment of TMT-labeled
hospho-enriched) were also
olumn format containing a
tions for LC/MS analysis.
FR, p-mTOR, mTOR, p-S6
d.2
Results
Relative Quantitation of Protein Ab
Characterization of H358 cells by Western Blot Analysis
Initial characterization of erlotinib-resistant (ER) cell lines using phospho-specific
antibodies showed p-EGFR to be constitutively auto-phosphorylated (upregulated
19-fold) in H2170 cells, but downregulated 6-fold in ER H358 cells. Interestingly,
c-Met/EGFR downstream signaling proteins p-mTOR, p-S6 kinase, and p-ERK were
found to be 2–4-fold upregulated in ER H2170 and H358 cells (Figure 1A). Targeted
antibodies revealed little change in the degree of total mTOR, EGFR, S6 kinase, ERK,
and β-actin expression between the parental and drug-resistant cell lines under all
treatment conditions (Figure 1B).
FIGURE 1. Western blot analysis for the phosphorylated isoforms (A) and total
protein (B) levels of mTOR, EGFR, ERK, S6 kinase, and β-actin in parental and
drug resistant H2170 and H358 cell lines
A
TMT10plex reagents enabled the sim
abundances in response to EGF stim
Orbitrap Fusion mass spectrometer,
peptides were able to be identified fr
90% at the peptide level employing H
High pH reversed-phase fractionatio
sample increased the number of qua
unfractionated samples. Hierarchical
relative protein abundance between
FIGURE 3. Number of quantifiable
fragmentation on the Orbitrap Fus
phospho-peptide enrichment. Res
files.
100000
90000
B
80000
70000
60000
50000
40000
30000
20000
10000
0
FIGURE 2. Schematic of TMT10plex reagent sample preparation and LC/MS
analysis. A) Proteins from parental and H358 erlotinib-resistant NSCLC cell
lines treated with diluent, EGF, Erlotinib, or both EFG/Erlotinib were extracted,
reduced, alkylated, and digested before being labeled with TMT10plex reagents.
Labeled peptides from each treatment condition are combined, enriched for
phospho-peptides, fractionated by high pH reversed-phase chromatography,
and analyzed by LC/MS analysis. B) TMT10plex-labeled peptides co-elute
during MS acquisition but generate 10 unique reporter ion masses during MSn
for relative quantitation.
A
Load (L)
Protein Groups (ID)
Protein Groups (Quan)
Total peptides (ID)
Total peptides (Quan)
3770
3553
24897
22796
FIGURE 4. Clustering analysis bas
protein expression for 5,485 prote
parental versus erlotinib-resistant
is normalized to untreated H358 p
H358 parental
EGF/Erlotinb
EGF
Scientific™ Dionex™
fic™ EASY-Spray™ column,
Map™ C18 column over a 3 hr
rile, 0.1% formic acid) at a
mass spectrometer using top
0.
P
e
B
fic™ Proteome Discoverer™
ngine with a precursor mass
02 Da. Carbamidomethylation
229.162 Da) for lysine and
s with variable methionine
Swiss-Prot® human database
MT reporter ratios with mass
ion factors. A protein ratio was
iable spectra of the peptides
eat map analysis was
Thermo Scientific Poster Note • PN-64138-ASMS-EN-0614S 3
HGF/SU1
Relative Quantitation of Protein Abundance Using TMT10plex Reagents
Pathway Analysis of H358
) cell lines using phospho-specific
auto-phosphorylated (upregulated
ld in ER H358 cells. Interestingly,
mTOR, p-S6 kinase, and p-ERK were
and H358 cells (Figure 1A). Targeted
of total mTOR, EGFR, S6 kinase, ERK,
nd drug-resistant cell lines under all
TMT10plex reagents enabled the simultaneous comparison of relative protein
abundances in response to EGF stimulation, exposure to erlotinib, or both. Using the
Orbitrap Fusion mass spectrometer, almost 7,800 protein groups and 90,000 unique
peptides were able to be identified from samples, with a quantitation rate exceeding
90% at the peptide level employing HCD MS2 quantitation/fragmentation (Figure 3).
High pH reversed-phase fractionation of unenriched and phospho-peptide enriched
sample increased the number of quantified proteins almost 3-fold compared to
unfractionated samples. Hierarchical cluster analysis revealed numerous changes in
relative protein abundance between parental and resistant cell lines (Figure 4).
Protein expression profiling
SU11274-resistant (SR) cells
downregulated in ER cells b
study also showed ~2 fold lo
Epidermal growth factor rece
(SHC1, 5C) were both phosp
partial inhibition to erlotinib t
containing protein 1 (SND1,
(GNA1, 5E) both were phosp
osphorylated isoforms (A) and total
kinase, and β-actin in parental and
FIGURE 3. Number of quantifiable proteins and peptides using MS2 HCD
fragmentation on the Orbitrap Fusion MS after high pH fractionation and
phospho-peptide enrichment. Results are the MudPIT search of two replicate
files.
FIGURE 5. Differential exp
by TMT reporter ion quant
Phospho-peptide signals w
lot Analysis
100000
90000
B
A
Extracted from: R:\Ryan\Fusion TMT phospho\TMT10 AACR Puri\TMT10_L_MS2_25.raw #44393 RT: 1
FTMS, [email protected], z=+3, Mono m/z=710.04175 Da, MH+=2128.11069 Da, Match Tol.=0.6 Da
b₄₊
732.38123
2.5
80000
2.0
70000
b₃₊
619.29742
Intensity [counts] (10^6)
60000
50000
40000
b₅₊
831.45001
1.5
850
1.0
30000
y₅₊
609.38483
y₅²₊
305.19568
b₆²₊-H₂O, b₂₊-H₂O, y₄₊
472.32498
y₆₊
722.46869
y₃₊
359.24060
0.5
20000
10000
0.0
500
R:\Ryan\Fusion TMT phospho\TMT10 AACR Puri\TMT10_L_MS2_25.raw
#44393, RT=134.84 min
FTMS, HCD, Precursor: z=+3, Mono m/z=710.04175 Da, MH+=2128.11069 Da, Integration=Most
90
High pH
fractions (P)
4493
3592
17017
13987
80
Total
(Unique)
8689
8328
92099
84287
EGF/Erlotinb
EGF
Diluent
EGF
6024
54178
50
40
30
20
0
126
Diluent
H358 erlotinb-resistant
HGF/SU11274
63462
60
10
FIGURE 4. Clustering analysis based on TMT quantitation showing changes in
protein expression for 5,485 proteins (2 peptides per protein) between H358
parental versus erlotinib-resistant cells for different treatment conditions. Data
is normalized to untreated H358 parental cells.
H358 parental
70
Intensity [counts] (10^3)
3770
3553
24897
22796
High pH
fractions (L)
7865
7528
83626
80091
Intensity (103)
Protein Groups (ID)
Protein Groups (Quan)
Total peptides (ID)
Total peptides (Quan)
Phopsphoenriched (P)
1399
1220
2734
2252
127_N
EGF
127_C
EFG
erloti
H358 pa
B
R:\Ryan\Fusion TMT phospho\TMT10 AACR Puri\TMT10_P_MS2_17.raw #39290, RT=129.44 min
FTMS, HCD, Precursor: z=+3, Mono m/z=925.44189 Da, MH+=2774.31113 Da, Integration=Most
11
10
8995
9
8
Intensity [counts] (10^3)
sample preparation and LC/MS
8 erlotinib-resistant NSCLC cell
both EFG/Erlotinib were extracted,
ng labeled with TMT10plex reagents.
ition are combined, enriched for
reversed-phase chromatography,
plex-labeled peptides co-elute
ue reporter ion masses during MSn
Load (L)
Intensity (103)
0
7
6
5
4
3
EGF/Erlotinb
2915
217
2
1
0
126
Diluent
127_
EFG
erloti
H358 pa
C
R:\Ryan\Fusion TMT phospho\TMT10 AACR Puri\TMT10_P_MS2_17.raw #49485, RT=157.54 min
FTMS, HCD, Precursor: z=+3, Mono m/z=812.07092 Da, MH+=2434.19822 Da, Integration=Most
18
16
14
Intensity [counts] (10^3)
Intensity (103)
2-fold
No change
0.5 fold
127_N
EGF
12772
12
10
8
6
4
2
0
1724
1051
126
Diluent
127_N
EGF
127_
EFG
erloti
H358 pa
D
R:\Ryan\Fusion TMT phospho\TMT10 AACR Puri\TMT10_P_MS2_7_140322115834.raw #28258
FTMS, HCD, Precursor: z=+2, Mono m/z=777.81842 Da, MH+=1554.62956 Da, Integration=Most
250
Intensity [counts] (10^3)
Intensity (103)
200
150
100
50
9528
0
126
Diluent
10913
127_N
EGF
126
127
EFG
erloti
H358 pa
E
R:\Ryan\Fusion TMT phospho\TMT10 AACR Puri\TMT10_P_MS2_20_140323110057.raw #1992
FTMS, HCD, Precursor: z=+4, Mono m/z=517.51672 Da, MH+=2067.04506 Da, Integration=Mos
60
Intensity (103)
Intensity [counts] (10^3)
50
40
30
20
10
0
10828
126
Diluent
8438
127_N
EGF
1040
127_
EFG
erloti
H358 pa
4 Profiling EGFR Kinase Inhibitor Resistance Pathways in Non-Small Lung Cancer Cells
g TMT10plex Reagents
Pathway Analysis of H358 Cells
arison of relative protein
e to erlotinib, or both. Using the
tein groups and 90,000 unique
h a quantitation rate exceeding
ation/fragmentation (Figure 3).
and phospho-peptide enriched
lmost 3-fold compared to
revealed numerous changes in
stant cell lines (Figure 4).
Protein expression profiling studies of H358 erlotinib-resistant (ER) and H2170
SU11274-resistant (SR) cells using SILAC™ have shown that β-catenin was
downregulated in ER cells but upregulated SR cells.2 TMT10plex quantitation in this
study also showed ~2 fold lower expression of β-catenin for H358 ER cells (Figure 5A).
Epidermal growth factor receptor (EGFR/Erb2, 5B) and SHC-transforming protein 1
(SHC1, 5C) were both phosphorylated after EGF and HGF treatment but showed
partial inhibition to erlotinib treatment in ER cells. Staphylococcal nuclease domaincontaining protein 1 (SND1, 5D) and glucosamine 6-phosphate N-acetyltransferase
(GNA1, 5E) both were phosphorylated in ER cells compared to parental cells.
eptides using MS2 HCD
gh pH fractionation and
dPIT search of two replicate
FIGURE 5. Differential expression of key proteins in H358 cell lines determined
by TMT reporter ion quantitation using the Orbitrap Fusion mass spectrometer.
Phospho-peptide signals were normalized to relative protein abundance.
Extracted from: R:\Ryan\Fusion TMT phospho\TMT10 AACR Puri\TMT10_L_MS2_25.raw #44393 RT: 134.84
FTMS, [email protected], z=+3, Mono m/z=710.04175 Da, MH+=2128.11069 Da, Match Tol.=0.6 Da
β-catenin
MEEIVEGCTGALHILAR
b₄₊
732.38123
2.5
β-catenin
2.0
Intensity [counts] (10^6)
b₃₊
619.29742
b₅₊
831.45001
1.5
Parental ER
y₈₊
850.52722
1.0
y₅₊
609.38483
y₅²₊
305.19568
b₆²₊-H₂O, b₂₊-H₂O, y₄₊
472.32498
y₉₊
951.57556
y₁₁₊
1168.62781
y₆₊
722.46869
b₇₊
1017.51465
y₃₊
359.24060
0.5
y₁₂₊
1297.67126
y₁₀₊
1111.60645
0.0
500
1000
R:\Ryan\Fusion TMT phospho\TMT10 AACR Puri\TMT10_L_MS2_25.raw
#44393, RT=134.84 min
FTMS, HCD, Precursor: z=+3, Mono m/z=710.04175 Da, MH+=2128.11069 Da, Integration=Most Confident Centroid,
Integration tolerance=20 ppm
m/z
1500
2000
90
Intensity [counts] (10^3)
70792
63462
60
60249
70455
64917
62394
54178
50
40
37805
38988
129_C
130_N
Conclusion
32045
30
20
10
0
126
Diluent
8 erlotinb-resistant
127_N
EGF
127_C
EFG +
erlotinib
128_N
HGF +
erlotinib
128_C
129_N
HGF + Quan Channels Diluent
SU11274
H358 parental
B
EGF
11
10
8995
9
7736
8
EFG +
erlotinib
H358 ER
R:\Ryan\Fusion TMT phospho\TMT10 AACR Puri\TMT10_P_MS2_17.raw #39290, RT=129.44 min
FTMS, HCD, Precursor: z=+3, Mono m/z=925.44189 Da, MH+=2774.31113 Da, Integration=Most Confident Centroid, Integration tolerance=20 ppm
Intensity [counts] (10^3)
ntitation showing changes in
per protein) between H358
nt treatment conditions. Data
7570
130_C
Diluent
131
HGF

TMT labeling enables the sim
multiple experimental condit
mass of the Orbitrap instrum

High pH reversed-phase frac
samples resulted in over 7,8
quantified from ten different

Protein expression ratios fro
SILAC studies and western

Cluster analysis revealed dif
proteins.
H358 SR
EGFR Y1173
GSHQISLDNPDyQQDFFPK
7
6
5117
5
4
3
EGF/Erlotinb
3114
2986
2915
2171
2
1621
2237
1
0
126
Diluent
127_C
EFG +
erlotinib
128_N
HGF +
erlotinib
128_C
129_N
HGF + Quan Channels Diluent
SU11274
129_C
130_N
EGF
EFG +
erlotinib
H358 ER
R:\Ryan\Fusion TMT phospho\TMT10 AACR Puri\TMT10_P_MS2_17.raw #49485, RT=157.54 min
FTMS, HCD, Precursor: z=+3, Mono m/z=812.07092 Da, MH+=2434.19822 Da, Integration=Most Confident Centroid, Integration tolerance=20 ppm
16
Intensity [counts] (10^3)
Intensity (103)
14
12772
12
8
6065
4
126
2220
1724
1051
Diluent
127_N
EGF
127_C
EFG +
erlotinib
128_N
HGF +
erlotinib
128_C
2895
HGF + Quan Channels Diluent
SU11274
129_C
130_N
EGF
EFG +
erlotinib
H358 ER
R:\Ryan\Fusion TMT phospho\TMT10 AACR Puri\TMT10_P_MS2_7_140322115834.raw #28258, RT=91.81 min
FTMS, HCD, Precursor: z=+2, Mono m/z=777.81842 Da, MH+=1554.62956 Da, Integration=Most Confident Centroid, Integration tolerance=20 ppm
200
Intensity [counts] (10^3)
130_C
Diluent
131
HGF
H358 SR
176466
106248
100
55972
50
0
9528
10913
12695
126
127_N
127_C
Diluent
EGF
EFG +
erlotinib
21868
16730
128_N
128_C
HGF +
erlotinib
129_N
HGF + Quan Channels Diluent
SU11274
H358 parental
129_C
130_N
EGF
EFG +
erlotinib
H358 ER
R:\Ryan\Fusion TMT phospho\TMT10 AACR Puri\TMT10_P_MS2_20_140323110057.raw #19922, RT=81.23 min
FTMS, HCD, Precursor: z=+4, Mono m/z=517.51672 Da, MH+=2067.04506 Da, Integration=Most Confident Centroid, Integration tolerance=20 ppm
60
50
44133
130_C
Diluent
131
HGF
H358 SR
GNA1 S244
LDSsACLHAVGDK
For research use only. Not for
40
30
27180
21270
20
10
0
2. Jacobs, R.; Fong, J.; Morave
Blank, M.; Puri, N. 2014. Pro
Association for Cancer Rese
41602
43571
41511
1. Puri, N. and Salgia, R. Syne
inhibition, in non-small cell lu
4. Viner, R.; Scigelova, M.; Zell
2012. Thermo Fisher Scienti
149467
150
References
3. Kall, L.; Canterbury, J.; Wes
4:923–925.
SND1 S909
ADDADEFGYsR
250
E
5246
1728
129_N
H358 parental
D
H358 SR
7220
6
0
131
HGF
9590
10
2
130_C
Diluent
SHC1 Y427
ELFDDPSyVNVQNLDK
18
Intensity (103)
2-fold
No change
0.5 fold
127_N
EGF
H358 parental
C
Intensity [counts] (10^3)
EGF
70
Intensity (103)
Total
(Unique)
8689
8328
92099
84287
Intensity (103)
High pH
fractions (P)
4493
3592
17017
13987
80
Intensity (103)
pH
s (L)
5
8
26
91
A
FIGURE 6. Elements of the ErbB
quantified by LC/MS are highligh
generated by Thermo Scientific™
10828
126
Diluent
14142
8438
127_N
EGF
10403
127_C
EFG +
erlotinib
H358 parental
7003
128_N
HGF +
erlotinib
128_C
129_N
HGF + Quan Channels Diluent
SU11274
129_C
130_N
EGF
EFG +
erlotinib
H358 ER
130_C
Diluent
131
HGF
H358 SR
Tandem Mass Tag and TMT are registered tr
trademark of the University of Washington. S
(Sib) Foundation, Switzerland. SILAC is a tra
KEGG is a trademark of Kyoto University, Ka
trademarks are the property of Thermo Fishe
This information is not intended to encourage
property rights of others.
Thermo Scientific Poster Note • PN-64138-ASMS-EN-0614S 5
b-resistant (ER) and H2170
shown that β-catenin was
s.2 TMT10plex quantitation in this
tenin for H358 ER cells (Figure 5A).
and SHC-transforming protein 1
nd HGF treatment but showed
taphylococcal nuclease domain6-phosphate N-acetyltransferase
compared to parental cells.
FIGURE 6. Elements of the ErbB signaling pathway. Proteins identified and
quantified by LC/MS are highlighted in green. KEGG™ pathway maps were
generated by Thermo Scientific™ ProteinCenter™ software version 3.9.
ns in H358 cell lines determined
trap Fusion mass spectrometer.
lative protein abundance.
β-catenin
LAR
Parental ER
2000
70455
62394
37805
38988
129_C
130_N
Conclusion
32045
129_N
Diluent
EGF
EFG +
erlotinib
H358 ER
7570
130_C
Diluent
131
HGF

TMT labeling enables the simultaneous quantification of protein expression from
multiple experimental conditions when coupled with the high resolution/accurate
mass of the Orbitrap instruments.

High pH reversed-phase fractionation of unenriched and phospho-peptide enriched
samples resulted in over 7,800 protein groups identified and over 5,500 proteins
quantified from ten different experimental conditions.

Protein expression ratios from TMT ion reporters correlated well with previous
SILAC studies and western blot analysis.

Cluster analysis revealed differences in regulation of key ErbB pathway signaling
proteins.
H358 SR
EGFR Y1173
GSHQISLDNPDyQQDFFPK
5117
3114
2986
2237
129_N
Diluent
129_C
EGF
130_N
EFG +
erlotinib
H358 ER
130_C
Diluent
131
HGF
H358 SR
SHC1 Y427
ELFDDPSyVNVQNLDK
9590
6065
5246
2895
1728
129_N
Diluent
129_C
EGF
130_N
EFG +
erlotinib
H358 ER
176466
130_C
Diluent
131
HGF
H358 SR
106248
129_N
129_C
EGF
H358 ER
44133
41511
130_N
EFG +
erlotinib
130_C
Diluent
2. Jacobs, R.; Fong, J.; Moravec, D.; Botting, G.; Bomgarden, R.; Rogers, J.; Viner, R.;
Blank, M.; Puri, N. 2014. Proceedings of the 106th Annual Meeting of the American
Association for Cancer Research. Apr 6-10, Washington, D.C.
4. Viner, R.; Scigelova, M.; Zeller, M.; Oppermann, M.; Moehring, T.; Zabrouskov, V.
2012. Thermo Fisher Scientific Application Note 566.
149467
Diluent
1. Puri, N. and Salgia, R. Synergism of EGFR and c-Met pathways, cross-talk and
inhibition, in non-small cell lung cancer. 2008. J. Carcinogenesis. 7:9.
3. Kall, L.; Canterbury, J.; Weston, J.; Noble, W.S.; MacCoss, M. 2007. Nat. Methods.
4:923–925.
SND1 S909
ADDADEFGYsR
55972
References
41602
131
HGF
H358 SR
GNA1 S244
LDSsACLHAVGDK
43571
For research use only. Not for use in diagnostic procedures.
27180
21270
129_N
Diluent
129_C
EGF
H358 ER
130_N
EFG +
erlotinib
130_C
Diluent
131
HGF
H358 SR
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