to develop and implement a clia-ready protein …...sandip chavan1, kiah bowers1, lancia darville1,...

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.