to develop and implement a clia-ready protein …...sandip chavan1, kiah bowers1, lancia darville1,...
TRANSCRIPT
Sandip Chavan1, Kiah Bowers1, Lancia Darville1, Bin Fang1, Sam Massoni2, Theresa Boyle1, Eric B. Haura1 and John M. Koomen1
To Develop and Implement a CLIA-ready Protein Biomarker Assay
Platform using LC-MRM Quantification
Treatment options for non-small cell lung cancer (NSCLC), especially
adenocarcinoma, include a number of targeted therapies against kinases as well
as therapeutic antibodies that target immune checkpoint proteins (ICPs) (1).
The large number of treatment options has led to the need of detailed molecular
classification of tumors for personalized treatment (2).
Current clinical methods, such as fluorescence in-situ hybridization (FISH) and
immunohistochemistry (IHC), have well documented limitations (3).
To complement these strategies, we develop liquid chromatography-multiple
reaction monitoring mass spectrometry (LC-MRM) assays for quantitative
analysis of receptor tyrosine kinases (RTKs) and immune checkpoint proteins to
assist selection of FDA-approved companion diagnostics.
A secondary goal is the clinical translation of assays for cancer signaling proteins
to further understand tumor biology.
1. Moffitt Cancer Center, Tampa, FL 2. New England Peptide, Inc., Gardner, MA
BACKGROUND & OBJECTIVES
TARGET PROTEINS AND THEIR CORRESPONDING PEPTIDES
STRATEGY FOR LC-MRM PANEL DEVELOPMENT
MULTI-POINT RESPONSE CURVE FOR SELECTED PEPTIDES IMPLEMENTATION OF IMMUNO-MRM ASSAY IN LUNG CANCER CELL LINES
Protein Peptide Sequence Protein Peptide Sequence
AKT1 SLLSGLLKMK03
GQPFDVGPR
AKT2
EGISDGATMK NYLQSLPSK
EGISDGATMKMTOR
VLGLLGALDPYK
SLLAGLLK LFDAPEAPLPSR
AKT3
TDGSFIGYK
PTEN
IYNLCAER
DEVAHTLTESR GVTIPSQR
EGITDAATMK IYSSNSGPTR
CADH1TAYFSLDTR
PD-1LAAFPEDR
NDVAPTLMSVPR VTQLPNGR
CADH2
GPFPQELVRPD-1L2
ATLLEEQLPLGK
LSDPANWLK TPEGLYQVTSVLR
LNGDFAQLNLKPD-1L1
LQDAGVYR
EGFR
IPLENLQIIR LFNVTSTLR
NLQEILHGAVRMET
TEFTTALQR
GSTAENAEYLR VADFGLAR
ERBB2GIWIPDGENVK
RETVFLSPTSLR
ELVSEFSR LLEGEGLPFR
ERBB3LTFQLEPNPHTK
CTLA4AQLNLATR
GDSAYHSQR SPLTTGVYVK
MP2K1VSHKPSGLVMAR ALK SNQEVLEFVTSGGR
IPEQILGK NTRK1 WEEEGLGGVPEQK
AXL APLQGTLLGYR NTRK2 SNEIPSTDVTDK
ROS1 IQDQLQLFR NTRK3 VVSLEEPELR
Clinically relevant Proteins
Peptide Selection
Peptide Evaluation
Assay Development
Assay Characterization
Implementation
Stable, proteotypic, easy to
synthesize, devoid of
modifications/mutations, etc.
Amenable to robust detection, generate characteristic
MS/MS fragmentation patterns and high-performing
on LC-MRM
Quality of peptide signal, optimization
of the signal and selection of top
performing transitions
Analysis of a response curve
and intra batch linearity to
determine LLOD and LLOQ
Quantitation in cell
lines, lung tissues
and FFPE tissues
Selection of candidate
biomarkers using
known biology
Peptide Selection for
Targeted Protein
Published
assays
Peptide
Library
Discovery
data
In house (purified
proteins, cell
line, tissues)
Public data
(NIST, Human
Proteome, SRM
Atlas, CPTAC assay
portal)
y = 5E-05x + 0.044R² = 0.999
y = 6E-05x - 0.026R² = 1
y = 4E-05x + 0.001R² = 1
0
5
10
15
20
25
30
35
0 200000 400000 600000
Peak
Are
a R
ati
o
(SIS
/Lig
ht)
Amount (amol)
Quantiva
QE Plus
Altis
0.001
0.01
0.1
1
10
1 100 10000 1000000
Peak
Are
a R
ati
o
(SIS
/Lig
ht)
Amount (amol)
Quantiva
QE Plus
Altis
0.001
0.01
0.1
1
10
1 100 10000 1000000
Peak
Are
a R
ati
o
(SIS
/Lig
ht)
Amount (amol)
Quantiva
QE Plus
Altis
y = 4E-05x + 0.103R² = 0.998
y = 5E-05x + 0.071R² = 0.999
y = 3E-05x + 0.032R² = 0.999
0
5
10
15
20
25
30
0 200000 400000 600000
Peak
Are
a R
ati
o
(SIS
/Lig
ht)
Amount (amol)
Quantiva
QE Plus
Altis
Figure 2: Reverse calibration curve for all the peptides are performed with LC-MRM (TSQ
Quantiva™ and TSQ Altis™) and LC-PRM (Q Exactive™ Plus) . An example of working curve
and log plots with peak area ratios on the y-axis and the corresponding concentration on
the x-axis for PD-1 peptides- A) LAAFPEDR and B) VTQLPNGR.
A)
B)
10000
100000
1000000
10000000
10000000
1E+09
1 100 10000 1000000
Peak
Are
a
Amount (amol)
SIS
Light
10000
100000
1000000
10000000
10000000
1E+09
1 100 10000 1000000
Peak
Are
a
Amount (amol)
SIS
Light
Figure 3: Reverse calibration curve for all the peptides are performed with LC-MRM. A
working curve and log plots with peak area on the y-axis and the corresponding
concentration on the x-axis for PD-1-LAAFPEDR peptide A) TSQ Quantiva™ and B) TSQ
Altis™.
REPEATABILITY ASSESSMENT OF PEPTIDES IN LC-MRM
Medium (2 fmol)
(%CV=4)
High (20 fmol)
(%CV=3)
Low (250 amol)
(%CV=17)
Medium (2 fmol) High (20 fmol)Low (250 amol)
Protein Peptide sequence Low Medium High Protein Peptide sequence Low Medium High
AKT1 SLLSGLLK 13.2 4.7 2.2 MK03 GQPFDVGPR 19.2 6.7 3.6
AKT2 EGISDGATMK 16.7 8.1 3.1 CTLA4 AQLNLATR 20.4 7.8 2.4
AKT2 SLLAGLLK 14.6 7.2 1.9 CTLA4 SPLTTGVYVK 18.3 6.9 2.8
AKT3 TDGSFIGYK 17.6 8.0 3.4 CTLA4 SPLTTGVYVK 18.3 6.9 2.8
AKT3 DEVAHTLTESR 20.5 8.6 5.0 MK03 NYLQSLPSK 12.2 4.8 4.5
AKT3 EGISDGATMK 12.5 6.9 3.1 MTOR VLGLLGALDPYK 56.9 33.2 44.1
CADH1 TAYFSLDTR 12.3 4.4 5.2 MTOR LFDAPEAPLPSR 10.2 3.3 2.5
CADH1 NDVAPTLMSVPR 12.8 2.3 4.4 PTEN IYNLCAER 15.8 7.6 3.9
CADH2 GPFPQELVR 12.4 1.9 4.7 PTEN GVTIPSQR 19.2 6.7 4.0
CADH2 LSDPANWLK 11.9 3.4 4.0 PTEN IYSSNSGPTR 17.4 7.5 5.9
CADH2 LNGDFAQLNLK 14.1 5.4 2.8 PD-1 LAAFPEDR 17.1 8.4 3.6
ALK SNQEVLEFVTSGGR 35.5 10.6 18.3 PD-1 VTQLPNGR 17.2 9.1 4.0
AXL APLQGTLLGYR 10.5 3.6 3.7 PD-1L2 ATLLEEQLPLGK 14.9 5.4 2.7
ROS1 IQDQLQLFR 12.1 4.3 3.4 PD-1L2 TPEGLYQVTSVLR 60.5 17.4 22.6
MET TEFTTALQR 20.6 6.5 4.3 PD-1L1 LQDAGVYR 17.2 9.1 4.0
MET VADFGLAR 17.6 5.8 3.6 PD-1L1 LFNVTSTLR 9.9 5.1 2.2
EGFR IPLENLQIIR 18.8 9.0 6.9 ERBB3 LTFQLEPNPHTK 15.7 4.2 5.4
EGFR NLQEILHGAVR 25.6 4.2 3.2 ERBB3 GDSAYHSQR 55.0 66.1 11.9
EGFR GSTAENAEYLR 11.6 3.5 4.4 MP2K1 VSHKPSGLVMAR 19.8 6.8 3.4
ERBB2 GIWIPDGENVK 11.6 3.5 4.4 MP2K1 IPEQILGK 20.4 7.9 2.6
ERBB2 ELVSEFSR 16.0 5.4 3.8 NTRK1 WEEEGLGGVPEQK 50.0 8.8 7.9
RET VFLSPTSLR 8.8 2.8 5.2 NTRK2 SNEIPSTDVTDK 19.1 8.0 5.1
RET LLEGEGLPFR 15.4 5.1 1.3 NTRK3 VVSLEEPELR 11.7 4.5 5.3
Figure 4: LLOQ and Linear region from response curve are used to determine low, medium and high
concertation of the SIS to assess the repeatability. Bar plot represents contribution of each of the five
transitions selected to the total MS signal (ex. EGFR-IPLENLQIIR*). Transition ratio was reproducible across
three biological replicates processed and analyzed on three different days.
Figure 5: Peak Area for Repeatability of low, medium and high Concentrations for EGFR
Peptide in a 3 x 3 Experiment (IPLENLQIIR*).
Table 2: CV(%) for target peptides from three biological replicates processed and
analyzed across three different days (>20 % in red).
Panel of Lung Cancer Cell lines
Pro
tein
Am
ou
nt
(fm
ol/
μg
)
Panel of Lung Cancer Cell lines
Pro
tein
Am
ou
nt
(fm
ol/μ
g)EGFR
AKT1
CONCLUSIONS AND FUTURE DIRECTIONS
REFERENCES
ACKNOWLEDGEMENTS
Figure 7: Heat map for the expression of 25 different proteins across 15 lung cancer cell
lines. Red color denotes high expression while blue denotes low expression and grey is not
detected.
Figure 6: The expression of EGFR and AKT1 across lung cancer cell lines.
LC-MRM assays have been developed for 46 peptides corresponding to 24
target proteins.
The reverse calibration curve for target peptides is reproducible across different
platforms (LC-MRM and LC-PRM).
The performance of two different instruments (Quantiva with Nanospray 1
source and Altis with EasySpray source) was compared. Chromatographic
peaks were sharper on Altis with better reproducibility. Through peak areas
were lower on Altis than Quantiva, overall performance in terms of LLOQ was
either similar or slightly better.
Inter and intra-assay variability is within the acceptable range (<20% CV) for
most peptides.
The biological implementation of assay is being performed in lung cancer cell
lines and FFPE patient samples.
Assay will be made available on CPTAC Assay Portal upon completion
(assays.cancer.gov).
1) Zappa et al. Transl Lung Cancer Res. (2016) 5(3):288-300
2) Soo et al. Lung cancer. (2018) 115, 12-20.
3) Inamura et al. Cancers. (2018) 14;10(3).
4) Morales-Betanzos et al. Mol Cell Proteomics. (2017) 16(10):1705-1717
5) Conlon et al. Mol Cell Proteomics. (2013) 12(10):2714-23
6) Hoffman et al. J Proteome Res. (2018) 17(1):63-75
This project builds on previous funding for the CPTAC Immuno-MRM Project
(Leidos 14X270) and is supported by Moffitt’s Lung Cancer Center of
Excellence, Moffitt Cancer Center, and NCI CCSG P30-CA076292.
Services were provided by Moffitt Cancer Center Proteomics and
Metabolomics Core, Tissue Core and Analytic Microscopy Core.
Proteomics & Metabolomics
Figure 1: Experimental design for LC-MRM assay development. Peptides for target proteins were
selected either from published assays and/or by digesting and LC-MS/MS analysis of purified
proteins/overexpression lysates .
Table 1: Clinically relevant proteins and corresponding peptides.