robustness of phospho akt-mtor pathway assay...
TRANSCRIPT
Penny Jensen1; Bhavin Patel1; Leigh Foster1; Aaron Gajadhar2; Sebastien Gallien3; Jonathan R. Krieger4; Jiefei Tong5; Michael F. Moran4,5,6; Rosa Viner2; Andreas Huhmer2; Kay Opperman1; John Rogers1
1Thermo Fisher Scientific, Rockford, IL; 2Thermo Fisher Scientific, San Jose, CA; 3Thermo Fisher Scientific, PMSC, Cambridge, MA; 4SPARC Biocentre, The Hospital for Sick Children, Toronto, Canada; 5Program in Cell Biology, The Hospital for Sick
Children, Toronto, Canada; 6Department of Molecular Genetics, University of Toronto, Canada
RESULTS
ABSTRACT Purpose: The AKT/mTOR pathway plays a central role in tumor progression and drug resistance. Quantitative
measurement of alterations in the expression of pathway proteins and post-translational modifications (PTM) is necessary
for understanding cancer biology. Highly accurate monitoring of these pathway proteins has not been achieved, due to
poor reproducibility, unreliable quantitation, and lack of standardized methods and reagents. To overcome these
challenges, the novel SureQuantTM pathway panels have been applied, which utilize an optimized multiplex
immunoprecipitation to targeted mass spectrometry (mIP-tMS) workflow. SureQuant assays can quantitate multiple
proteins, PTMs and interacting partners, which creates new possibilities for a broad range of applications, including
cancer, drug development, and research into precision medicine.
Methods: The SureQuant total and phospho pathway panels contain two modules: 1) The IP-MS Sample Prep Module
includes reagents necessary to immunoenrich AKT pathway, RAS, or TP53 proteins, and perform MS sample preparation
in one day 2) The Absolute or Relative Quantitation Modules include a Pierce™ LC-MS/MS System Suitability Standard,
AQUA Ultimate Heavy and/or AQUA Ultimate Light Peptides, and verified MS instrument and data analysis methods.
Serum-starved, inhibitor-treated (LY294002/NVP-BEZ235/Rapamycin) HCT116, A549, and MCF7 cells were stimulated
with hIGF-1. SureQuant AKT pathway panels (total and phospho) were used to determine the absolute concentration of
target peptides using targeted MS analysis. The panels were benchmarked against Western blotting using three
unstimulated, hIGF-1 stimulated or inhibited cell lysates, as well as several tissue/xenograft lysates.
Results: Previously, we verified antibodies and target peptides to AKT and RAS pathways using an optimized mIP-tMS
workflow. From the standard curve, all target peptides were monitored with <20% CV, 3 orders of magnitude dynamic
range, linearity (R2) >0.97, and accuracy of 80-120% in a complex matrix. Using the SureQuantTM pathway panels,
absolute quantitation of 37 target peptides in unknown samples was achieved with <20% CV across multiple cancer cell
lines. The SureQuant pathway analysis workflow allowed absolute quantitation of target peptides from positive control
lysate with <15% individual operator %CV and <20% combined %CV using PRM analysis. Kit performance was evaluated
through analysis of abundance levels between three different cancer cell lines, A549, HCT116, and MCF7, using the
SureQuant AKT Total and Phospho assay showed preferences for certain inhibitors in specific cell lines treated with hIGF-
1. The PI3K inhibitor LY294002 functioned the best in HCT116 cells whereas the dual PI3K/Rapamycin inhibitor NVP-
BEZ235 worked predominantly in A549 cells. Analysis by mass spectrometry allowed for more accurate and informative
data with the determination of fmol levels of protein expression and capability to discriminate between isoforms of many
proteins that are unable to procure with western blot analysis. Absolute quantitation of 12 phosphorylated AKT pathway
targets was obtained from five patient derived lung tumor xenograft samples. Additionally, all 12 total and 12 phospho
AKT pathway targets were quantitated from three different tissue lysates, Lung, Large Intestine, and Breast tumor.
CONCLUSIONS
▪ PierceTM LC-MS/MS System Suitability Standard (7 x 5 mixture) achieves appropriate linearity and dynamic range to assess
system performance prior to acquisition of unknown samples.
▪ SureQuantTM Multiplex IP-MS and Absolute Quantitation Modules for AKT pathway proteins allowed simultaneous absolute
quantitation of multiple total and phospho AKT pathway proteins in treated cell lines and tumor samples with high accuracy
and precision (CV <20%).
▪ Analysis of abundance levels between three different cancer cell lines using the SureQuantTM AKT Total and Phospho kits
revealed preferences for certain inhibitors in specific cell lines, with PI3K inhibitor LY294002 demonstrating highest efficacy
in HCT116 cells, whereas the dual PI3K/Rapamycin inhibitor NVP-BEZ235 was most effective in A549 cells. Orthogonal
evaluation between PRM assays and Western Blot analysis of three cancer cell lines treated with hIGF-1 and various
inhibitors, showed similar trends in the protein expression changes, albeit the level of precision and dynamic range
achieved with the SureQuant kit is difficult or impossible to achieve with Western Blot analysis.
▪ SureQuantTM AKT pathway kits are amenable to diverse sample sources and allowed identification of target proteins from
cell lysate, tissues and patient derived xenograft tissue samples.
▪ SureQuantTM AKT pathway kits are amenable to diverse sample sources and allowed identification of target proteins from
cell lysate, tissues and patient derived xenograft tissue samples.
REFERENCES1. Logue JS, Morrison DK. Complexity in the signaling network: insights from the use of targeted inhibitors in cancer therapy. Genes Dev. 2012 Apr 1; 26(7):641-50.
2. Mendoza MC, Er EE, Blenis J. The Ras-ERK and PI3K-mTOR pathways: cross-talk and compensation. Trends Biochem Sci. 2011 Jun;36(6):320-8
3. Carr SA, Abbatiello SE, Ackermann BL et al. Targeted Peptide Measurements in Biology and Medicine: Best Practices for Mass Spectrometry-based Assay
Development Using a Fit-for-Purpose Approach. Mol Cell Proteomics. 2014 Mar; 13(3):907-17.
4. Ackermann BL . Understanding the role of immunoaffinity-based mass spectrometry methods for clinical applications. Clin Chem. 2012 Dec; 58(12):1620
TRADEMARKS ANDS LEGAL INFORMATION
© 2019 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries.
This information is not intended to encourage use of these products in any manner that might infringe the intellectual property rights of
others.
Applications of Mass Spectrometry Targeted Assays for Quantitative Analysis of Cancer Signaling Proteins
MATERIALS AND METHODS
Figure 1. Thermo Scientific™ SureQuant™ Multiplex IP to Targeted MS Modules & Kits
• 10 reactions (samples)
• Biotinylated antibodies mix
• Positive control lysate
• Streptavidin magnetic beads
• Wash buffers (A and B)
• Trypsin
• Sample prep buffer
• Reduction/alkylation reagents
• 10% TFA
• Low-binding tubes
• 10 reactions (samples)
• AQUA heavy peptide mix
• AQUA light peptide mix
• 6 protein digest matrix
• 7 x 5 system suitability standard
• Peptide Diluent
• Instrument method with Skyline doc
• Low-binding tubes
Relative quantitation module
• 10 reactions (samples)
• AQUA heavy peptide mix
• 7 x 5 system suitability standard
• Peptide Diluent
• Instrument method with Skyline doc
• Low-binding tubes
AKT pathway:
total targets
AKT pathway:
phospho targets
AKT1/AKT2/
AKT3
PTEN
IRS1
IGF1R
GSK3a
GSK3b
PRAS40
(AKT1S1)
mTOR
P70S6K
TSC2
AKT1/AKT2
pSer473/pSer474
PTEN pSer380
IRS1 pSer312
IGF1R
pTyr1162/1163
GSK3a pSer21
GSK3b pSer9
PRAS40 pThr246
mTOR pSer2448
P70S6K pThr389
TSC2 pSer939
IP/MS sample prep module Absolute quantitation module
Cell Lines and Tissue Lysate: A549, HCT116 and MCF7 cells were grown in Ham’s F-12K media, McCoy’s 5A Media
and DMEM Media, respectively, with 10% FBS/1xPenStrep to ~70-80% confluency. Cells were serum starved with 0.1%
charcoal stripped FBS for 24 hours before stimulation with 100 ng/mL of IGF for 15 minutes. Breast, Lung, and Large
Intestine Tumor and Normal Adjacent Tissue were purchased from BioIVT. Cells and tissue samples were lysed with IP
Lysis buffer (Thermo Fisher Scientific PN#87788) supplemented with 1X HALT Protease and Phosphatase inhibitor
cocktail (Thermo Fisher Scientific PN#78440).
Multiplex Immunoprecipitation to MS Sample Preparation and MS Quantitation: The SureQuantTM IP and MS
Sample Preparation Modules for AKT Pathway (PN# A40081, A40086, A40091), were used to immunoenrich relevant
protein targets. The SureQuantTM Absolute Quantitation Modules for AKT Pathway (PN# A40083, A40093) was used to
generate calibration curves and determine concentrations of target peptides from unknown samples.
Liquid Chromatography and Mass Spectrometry: PierceTM LC-MS/MS System Suitability Standard (7 x 5 Mixture)
(PN# A40010) was used to assess dynamic range and sensitivity (LLOQ) of the nanoLC-MS system prior to running
calibration curves or unknown samples. IP-enriched and trypsin digested samples were then desalted on-line using the
Thermo Scientific™ Acclaim™ PepMap 100 C18 Trap Column (PN#164564) followed by seperation using a Thermo
Scientific™ EASY-Spray C18 column (PN#ES800). For discovery MS and targeted PRM-MS analysis, the samples were
analyzed using the Thermo Scientific™ Dionex™ UltiMate™ 3000 RSLCnano System and Thermo Scientific™ Q
Exactive™ HF Hybrid Quadrupole-Orbitrap Mass Spectrometer. Verified instrument acquisition methods were used, as
well as inclusion lists relevant to each Absolute Quantitation Module.
MS Data Analysis: Discovery MS data were analyzed with Thermo Scientific™ Proteome Discoverer™ to assess percent
sequence coverage, unique peptides, areas/intensities of identified peptides, and PTMs. For targeted MS data analysis,
Skyline software (University of Washington) were used to measure limit of quantitation (LOQ) from the calibration curve
and target analyte concentration from unknown samples.
hIGF-1 Stimulation
Figure 2. Experimental Workflow for Multiplex IP-MS Assays Figure 3. AKT Pathway
Protein Elution
1 h
Traditional MS Sample Prep Workflow for IP
SureQuantTM Target Assay MS Sample Prep Workflow for IP
Protein
Elution
0.2h 1h 0.5h 0.5h 18.5h
1 h
Dry Samples Alkylation
Reduction Protein DigestionDry and reconstitute
~21 hours
total
~4 hours total 1h 0.1h
Protein DigestionReduction
Alkylation Dry and reconstitute
2h
Figure 4. MS Sample Prep Workflow Optimization for Immuno-enriched (IP) Samples
INTRODUCTIONMultiplex Immunoprecipitation to Mass spectrometry (IP-MS) kits from Thermo Fisher Scientific are developed for simultaneous
enrichment and quantitation of total abundance and phosphorylation levels of multiple proteins from the AKT/mTOR Signaling
Pathway. The immunoenriched, digested samples are spiked with heavy peptide internal standards, which can then be
processed using discovery MS (DDA) and targeted MS (PRM) methods for analysis.
y = 0.935x + 7.4654R² = 0.9971
6
7
8
9
10
11
12
-1 0 1 2 3
1: GISNEGQNASIK
y = 0.9327x + 7.5556R² = 0.9969
6
7
8
9
10
11
12
-1 0 1 2 3
2: IGDYAGIK
y = 0.9832x + 7.4441R² = 0.9963
6
7
8
9
10
11
12
-1 0 1 2 3
3: TASEFDSAIAQDK
y = 1.0553x + 7.1621R² = 0.9971
6
7
8
9
10
11
12
-1 0 1 2 3
4: ELGQSGVDTYLQTK
y = 0.8584x + 7.026R² = 0.9851
6
7
8
9
10
11
12
-1 0 1 2 3
5: SFANQPLEVVYSK
y = 1.0769x + 7.1064R² = 0.993
6
7
8
9
10
11
12
-1 0 1 2 3
6: LTILEELR
y = 1.2028x + 5.6947R² = 0.9923
4
5
6
7
8
9
10
-1 0 1 2 3
7: ELASGLSFPVGFK
RT: 10.00 - 35.00 SM: 7B
10 12 14 16 18 20 22 24 26 28 30 32 34
Time (min)
0
10
20
30
40
50
60
70
80
90
100
Rela
tive A
bundance
17.27
620.8320.30
433.2625.00
783.41
31.65
691.4029.17
509.33
22.39
703.35
27.29
754.91
20.68
433.2620.03
461.7722.81
703.8425.43
783.9133.79
845.5316.98
556.80
30.63
561.8110.07
371.3112.40
371.10
13.47
371.10
NL:
1.94E8
Base Peak
F: ms MS
DDA_7x5mi
x_200fmolO
C_R1
7x5 Mixture: Peptide Groups Calibration CurvesPeptide
GroupPeptide Sequence
Precursor m/z
(Isotope label)
Conc on
Column (fmol)
1
GISNEGQNA[+4]S[+4]I[+7]K[+8] 620.8324++ (4 heavy) 200
GISNEGQNA[+4]SI[+7]K[+8] 618.8289++ (3 heavy) 20
GISNEGQNASI[+7]K[+8] 616.8253++ (2 heavy) 2
GISNEGQNASIK[+8] 613.3168++ (1 heavy) 0.5
GISNEGQNASIK 609.3097++ 0.0125
Figure 5. PierceTM LC-MS/MS System Suitability Standard (7 x 5 Mixture)
LLOQ/Linearity
Calibration curve generated for each peptide set of 5 isotopologues of the same peptide sequence showed >3 orders of
magnitude dynamic range and linearity (R2) ≥ 0.9800
Figure 6. Determination of Quantitation Limits and Linearity of AQUA Light Peptides for
SureQuantTM Pathway Kits
Calibration
Curve PointsFmol on column
CC1 200.00
CC2 20.00
CC3 2.00
CC4 0.50
CC5 0.13
CC6 0.03
0.95
0.96
0.97
0.98
0.99
1
1.01
Lin
ea
rity
(R
2)
AKT Pathway Target Peptides Linearity (R2)
y = 1.0076x - 1.2741
R² = 0.9993-3
-2
-1
0
1
2
-2 -1 0 1 2 3
AKT1: NDGTFIGYK
0
0.2
0.4
0.6
LL
OQ
(fm
ol)
AKT Pathway Target Peptides Limit of quantitation (LLOQ)
All 30 AKT pathway target peptides
were monitored with linear quantitation
(R2 ≥ 0.9800) and 2-3 orders of
magnitude (LLOQ ≤ 0.5 fmol on column)
Figure 7. Precision of AKT/mTOR Signaling Pathway Proteins Using SureQuantTM Targeted MS Kits
-10%
10%
30%
%C
V
-10%
10%
30%
%C
V
AKT SureQuantTM Pathway Mass Spec Assay kits allowed absolute quantitation of target peptides from positive control
lysate with <15% individual CV and <20% combined CV using PRM analysis.
0%
5%
10%
15%
20%
25%
30% Op1_CV
Op2_CV
Op3_CV
Combined CV
PRM quant specs:
• Individual CV <15%
• Combined CV <20%
All peptides for 11
targets passed CV
specs
Robustness of Phospho AKT-mTOR Pathway Assay Kit
%C
V
Figure 9. Absolute or Relative Quantitation of AKT/mTOR Signaling Pathway Proteins Using
SureQuantTM kits
AKT/mTOR pathway proteins were enriched through multiplex immunoprecipitation using the SureQuant™ AKT Pathway or AKT
Phospho Pathway Mass Spec Assay kit. Analyses were performed on a Thermo Scientific™ Q Exactive™ HF Orbitrap™ mass
spectrometer using directed discovery (DDA) and targeted MS (PRM) acquisition methods. DDA and PRM data were analyzed
in Proteome Discoverer and Skyline software, respectively. PRM analysis using the calibration curve allowed absolute
quantitation of each target peptide from positive control lysate. Western Blot data showed differential expression for
phosphorylated AKT pathway proteins with hIGF-1 stimulation and inhibitor treatments
Mar
ker
Unt
reat
edhI
GF-
1LY
2940
02/h
IGF-
1Ra
pam
ycin
/hIG
F-1
LY2+
Rap/
hIG
F-1
BEZ2
35/h
IGF-
1
MCF7 lysate HCT116 lysate
pAKT
Mar
ker
Unt
reat
edhI
GF-
1LY
2940
02/h
IGF-
1Ra
pam
ycin
/hIG
F-1
LY2+
Rap/
hIG
F-1
BEZ2
35/h
IGF-
1
72 -55 -
95 -
A549 lysate
72 -55 -
95 -
72 -55 -
95 -
Mar
ker
Unt
reat
edhI
GF-
1LY
2940
02/h
IGF-
1Ra
pam
ycin
/hIG
F-1
LY2+
Rap/
hIG
F-1
BEZ2
35/h
IGF-
1
72 -95 -130 -
pIGF1R 72 -95 -130 -
72 -95 -130 -
pPTEN 72 -55 -
72 -55 -
72 -55 -
pGSK3A
pGSK3B
pPRAS40
pmTOR
pRPS6KB1
72 -55 -
72 -55 -
72 -55 -
55 -
36-
55 -
36-
55 -
36-
cyclophilinB
55 -
36-
55 -
36-
55 -
36-
250 - 250 - 250 -
72 -55 -
72 -55 -
72 -55 -
28 -
17 -
28 -
17 -
28 -
17 -
0.00
10.00
20.00
30.00
13901 11931 11391 11373 11133 Positive
Ab
solu
te C
on
c/IP
(fm
ol) pAKT1
Peptide #2
Peptide #1
0.00
1.00
2.00
3.00
13901 11931 11391 11373 11133 Positive
Ab
solu
te C
on
c/IP
(fm
ol) pAKT2Peptide #2
Peptide #1
0.00
1.00
2.00
3.00
4.00
13901 11931 11391 11373 11133 Positive
Ab
solu
te C
on
c/IP
(fm
ol)
pAKT3Peptide #2Peptide #1
0.00
50.00
100.00
150.00
200.00
13901 11931 11391 11373 11133 Positive
Ab
solu
te C
on
c/ P
I (f
mo
l)
pAKT1S1Peptide #2
Peptide #1
0.00
10.00
20.00
30.00
40.00
50.00
13901 11931 11391 11373 11133 Positive
Ab
solu
te C
on
c/IP
(fm
ol) pGSK3A
Peptide #2Peptide #1
0.00
10.00
20.00
30.00
40.00
13901 11931 11391 11373 11133 Positive
Ab
solu
te C
on
c/IP
(fm
ol)
pGSK3BPeptide #1Peptide #2Peptide #3
0.00
10.00
20.00
30.00
40.00
13901 11931 11391 11373 11133 Positive
Ab
solu
te C
on
ce/I
P (
fmo
l)
pIGF1RPeptide #3Peptide #2Peptide #1
0.00
1.00
2.00
3.00
13901 11931 11391 11373 11133 Positive
Ab
solu
te C
on
c/IP
(fm
ol)
pIRS1Peptide #2
Peptide #1
0.00
20.00
40.00
60.00
13901 11931 11391 11373 11133 Positive
Ab
solu
te C
on
c/IP
(fm
ol) pMTOR
Peptide #1
Peptide #4
Peptide #3
0.00
5.00
10.00
15.00
20.00
13901 11931 11391 11373 11133 Positive
Ab
solu
te C
on
c/IP
(fm
ol)
pPTENPeptide #3Peptide #2Peptide #1
0.00
20.00
40.00
60.00
13901 11931 11391 11373 11133 Positive
Ab
solu
te C
on
c/IP
(fm
ol)
pRPS6KB1Peptide #2Peptide #1
0.00
5.00
10.00
15.00
20.00
25.00
13901 11931 11391 11373 11133 Positive
Ab
solu
te C
on
c/IP
(fm
ol)
pTSC2Peptide #3Peptide #2Peptide #1
Figure 12. Phospho AKT Pathway Proteins PRM Data from Lung Cancer Patient Xenograft Samples.
Accurate quantitation was obtained from five patient derived lung tumor xenograft samples using the SureQuantTM AKT Phospho
Pathway kit. Patient derived xenograft samples were lysed in 2% SDS before multiplex IP enrichment followed by Targeted MS (PRM)
analysis using the Thermo Scientific™ Q Exactive™ HF-X and EASY-nLC 1200 system. A 50cm EASY-Spray column (ES803) in
direct-injection setup was used for all LC-MS data acquisition and Skyline Software was used to generate standard curves (data not
shown) and calculate absolute concentrations of unknown samples.
Calculated concentration (fmol) values were used to summarize data where an increase in phosphorylation was designated
by at least a 40% increase compared to untreated. Inhibition was designated if below 40% hIGF-1 treated value. All pathway
phosphorylated proteins showed increase in abundance with hIGF-1 treatment across all three cell lines, whereas inhibitors
functioned differently among three cell lines:
• The PI3K inhibitor (LY294002) was more effective than the mTOR inhibitor (Rapamycin) with effectiveness HCT116
> MCF7 > A549
• Combined LY294002 + Rapamycin treatment was most effective in HCT116 cells with effectiveness HCT116 > A549
> MCF7
• Dual inhibitor (NVP-BEZ235) worked best in A549 cells compared to LY294002 and Rapamycin together with
effectiveness A549 > HCT116 > MCF7
Figure 10. Summary of Quantitative Changes in Phospho AKT Pathway following Targeted
Inhibition
hIGF-1 Stimulation
LY294002
PI3K inhibitorRapamycin
mTOR inhibitor
NVP-BEZ235
mTOR and
PI3K inhibitor
13.3
2.20.6
13.5
17.5
31.6
1.4 0.6
4.87.8
0.3 1.4
18.9
5.0
0.5
29.5
33.4
54.4
0.1 1.13.5
9.5
0.62.0
0
10
20
30
40
50
60
fmo
l tar
get
Lung NormalLung Tumor
1.0
0.5
1.7
1.3
3.0
1.0
0.0 0.2
5.7
1.4
0.20.40.3 0.3 0.3 0.2
3.7
1.4
0.00.3
2.1
5.6
0.2 0.2
0
1
2
3
4
5
6
7
fmo
l tar
get
Lung NormalLung Tumor
Figure 8. Quantitation of Total and Phospho AKT Pathway Proteins using Different Positive
Control Lysate Amounts
Total PhosphoLung Tissue
Breast
Large Intestine
8.5
2.1 0.4
6.1 5.9 4.70.8 1.3
3.5 1.6 0.1 0.8
37.1
18.4
0.4
55.1
84.0
72.3
0.6 2.6
18.3
11.6
0.45.1
0
10
20
30
40
50
60
70
80
90
fmo
l tar
get
Breast Normal
Breast Tumor
0.4 0.40.9 0.6 0.4 0.3 0.0 0.1
2.2
0.40.1 0.3
0.7 0.6 0.6 0.31.1 1.1
0.00.4
10.6
4.9
0.2 0.5
0
2
4
6
8
10
12
fmo
l tar
get
Breast NormalBreast Tumor
7.3
1.0 0.3
16.5
20.8 20.2
1.2 0.2
7.9
0.9 0.2 0.7
16.2
2.80.8
19.9
32.2
43.3
0.7 0.6
5.3
0.7 0.2 1.3
0
10
20
30
40
50
60
fmo
l tar
get
Lg Intestine Normal
Lg Intestine Tumor
0.5 0.20.8 0.6
2.0
0.70.0 0.1
12.0
0.2 0.1 0.30.6 0.4 0.70.2
1.9
1.0
0.0 0.2
4.0
0.4 0.2 0.3
0
2
4
6
8
10
12
14
16
fmo
l tar
get
Lg Intestine Normal
Lg Intestine Tumor
SureQuantTM AKT Total and Phospho Pathway kits allowed accurate quantitation from lung, breast, and large intestine
normal and tumor tissue lysates. Skyline Software was used to generate standard curves and calculate absolute
concentrations of unknown samples.
Figure 11. Quantitation of Total and Phospho AKT Pathway Proteins in Lung, Breast, and Large
Intestine Normal and Tumor Tissue Samples.
0
20
40
60
80
100
120
50 µg 100 µg 200 µg 500 µg 1000 µg
fmo
l tar
get
lysate amount
Phospho AKT hIGF-1 MCF7 lysate pAKT1pAKT2pAKT3pAKT1S1pGSK3ApGSK3BpIGF1RpIRS1pmTORpPTENpKS6KB1
0
50
100
150
200
250
50 µg 100 µg 200 µg 500 µg 1000 µg
fmo
l tar
get
lysate amount
Total AKT hIGF-1 A549 lysateAKT1AKT2AKT3AKT1S1GSK3AGSK3BIGF1RIRS1mTORPTENKS6KB1
Total and phospho AKT pathway PRM analysis allowed absolute quantitation of all target peptides from 50 µg to 1000 µg of
positive control lysate. Overall linear correlation between lysate amount and target quantitation was observed.