Transcript Document

Isolating and Identifying Protein Interactions
within EGFR Complexes
1,
3
White ,
2
Guha ,
2
Varmus ,
1
Cristea
Laura Lee
Udayan
Harold E.
Ileana M.
1Princeton University, Princeton, NJ; 2Sloan Kettering NY, NY; 3The Pingry School, Martinsville, NJ
OVERVIEW
Introduction: Protein interactions are responsible for the majority of
cellular processes. In this study, epidermal growth factor receptor (EGFR)
and associated proteins were isolated to understand their involvement in
lung cancer.
Aim: To characterize signaling pathways involved
Fig. 4
EGFR Sequences
1
2
Wild type
3
Mutated
Deletion
L858R
in lung cancer.
E746 – A750
Method: Isolation of protein complexes on magnetic beads, followed by
protein quantification and identification using metabolic labeling and
mass spectrometry.
Metabolic Labeling during cell culture
Wild type EGFR mutated EGFR deletion EGFR
Results: We isolated wild type and mutant forms of EGFR and identified
Light media
Heavy media
Heavy media
its interacting proteins to gain insights into their function.
No modification
+6 Da R
+4 Da R
+4 Da K
+4 Da K
INTRODUCTION
• EGFR binds to epidermal growth
factor and transforming growth factor
α [1]. It is a member of the ErbB family
of receptors and, when activated, it
signals mitogen-activated protein
kinase signal transduction.
Isolate EGFR
• Overexpression of EGFR is
frequently seen in non-small cell lung
cancer (NSCLC), the leading cause of
fatal cancer in men and women.
EGFR
1
EGFR
2
Fig 1. EGFR image labeled with L858R mutation in the
activation loop and E746-A750 deletion in exon
19. (J. Guillermo Paez, 2004).
Ab (anti-EGFR)
EGFR Magnetic bead
3
Wild type, mutant
(L858R), and deletion
(E746-A750) forms of
EGFR were constructed to
study different interacting
proteins.
After changing the
genetic sequence of EGFR,
the cells were
metabolically labeled.
The process of
metabolic labeling
incorporates stable
isotope-containing amino
acids into newly
synthesized proteins.
Growth medium is
created in which all
“light” arginines and
lysines were replaced with
“heavy” amino acids.
The purpose behind
metabolic labeling is that
each population (wild
type, mutation, deletion
forms of EGFR, and
interacting proteins) are
distinguishable through
MS analysis.
II. EGFR Quantification
Fig. 8
I. Identification of wild type EGFR
Mass spectra represents the distribution of components (molecules or atoms) by
mass-to-charge ratio in a sample acquired by a mass spectrometer
Fig. 6
kDa
EGFR peptides labeled
250
150
100
MS-Fit searches
heavy isotope
labeled R and K.
Isotopic labeling
evident in
database search.
Monoisotopic
peaks labeled in
spectra and control
peptides
eliminated.
75
50
R and K
highlighted
modification’s
detailed in next
figure.
Database search
performed with all
labeled peptides.
37
Search revealed
EGFR peptides as
most prevalent in
the sample.
25
15
•Protein
Coverage: 24.7%
Sample analyzed
by mass
spectrometry.
500
0
1000
m/z
4500
C:\Documents and Settings\Ileana Cristea\Desktop\Laura White\LabeledSpectra\LW_MO5_6_1_woControl.massml (11:31 08/11/08)
Description:
Fig 8. MS-Fit results. Highest hit, EGFR protein. The modifications are
indicated by the database search engine.
EGFR peptides are
marked in the
shown spectrum.
EGFR peptide LTQLGTFEDHFLSLQR
EGFR peptide GLWIPEGEK
R Modification
K Modification
Isolate using magnetic beads
coated with anti-EGFR Ab
Elute co-purified
proteins
Identify proteins using mass
spectrometry
Search for Biological Significance
• Once isolated, the proteins were
eluted and resolved by SDS-PAGE.
• Protein bands were cut from the gel
and digested with trypsin.
• The peptides were analyzed by mass
spectrometry.
• Mass spectra were labeled and the
data was used to search databases to
confirm EGFR and identify the interacting
partners.
Fig 3. Approach for studying protein interactions [3]
1036.57
Cryogenic Cell Lysis
• EGFR and associated proteins were
isolated on magnetic beads conjugated
with anti-EGFR antibodies (Fig. 3).
Fig 5. Protein Identification using Mass
Spectrometry. Example shown highlights
the use of a MALDI Q-ToF mass
spectrometer for protein analysis. Parts
of the figure adapted from Steen and
Mann, 2004 [4].
• Mass spectrometry identifies the chemical composition of a compound or
sample on the basis of the mass-to-charge ratio (m/z).
• A MALDI quadrupole time-of-flight (Q-ToF) mass spectrometer was used to
analyze peptides from proteins isolated with EGFR. The peptides are mixed with
a matrix that absorbs at the wavelength of the laser and placed on a target. When
dry, it creates a heterogenous distribution of matrix and peptide crystals which
are then ionized.
• The ions travel through the quadrupole and enter the time-of-flight mass
analyzer.
1028.56
Freeze Cells
m/z
1920.0
0
1026.0
m/z
1040.0
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Description:
Fig 9. Examples of identified EGFR peptides. Labels show the isotopically modified R- and K-containing
peptides.
EGFR peptides had the
highest result based on
the high decision score,
the high number of
assigned peptides, and
an overall error reading
consistent with the ToF
calibration.
METHODS
Cell Culture
0
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Description:
(image from Weisenthal Cancer
Group, 2006 [2]).
Fig. 3
•High decision score:
8.0
1905.0
lung. Top image is the control
image of the tumor cell. Bottom
image is the tumor treated with
gefitinib. This treatment led to
the death of most tumor cells
I. Approach for Isolating Protein Complexes
Intensity
• In this study we examined the L858R
mutation in the activation loop and E746-A750
deletion sequence in exon 19 (Fig. 1).
Fig 2. Images of adenocarcinoma of the
Protein
Identification
1915.01
Isolated proteins
•X-Proteo Database
Search revealed EGFR
as top result
1905.00
Fig. 5
4 Da
Fig. 7
Intensity
III. Protein Identification by Mass Spectrometry
• These patients have shown good response to
gefitinib (Iressa™), a drug that inhibits the
receptor tyrosine kinase.
• NSCLC mutations are most frequent in
Japanese patients, patients that are nonsmokers, or those that have adenocarcinoma.
Fig 4. Strategy for the isolation of
mutated EGFR and associated
proteins via metabolic labeling
and magnetic bead isolation
4 Da
70
1032.58
1400
4 Da
1911.02
6 Da
Elution from Magnetic Beads
Fig. 9
Fig 6. Sample cut from SDS-PAGE, digested with trypsin and analyzed by
mass spectrometry; EGFR peptides labeled on the mass spectrum.
Fig. 2
• Some patients with NSCLC have mutations
in the EGF receptor that can be either short, inframe deletions, or substitutions around the EGF
tyrosine kinase domain.
•MS-Fit database
search revealed
EGFR as first hit.
•MOWSE score:
2.33e+10
100 kDa sample
was cut from the
SDS-PAGE into
1mm pieces to
digest the protein.
100 kDa Mass Spectra
Intensity
Combine Cell Lysates
Fig. 1
RESULTS
II. Quantitative Studies of
EGFR Interactions
•
Labeled EGFR peptides identified through MS-Fit search.
•
Triple peaks verify EGFR metabolic labeling and the successful isolation
of all the three forms of EGFR.
•
Wild type, mutation, and deletion EGFR peptides are evident in sample.
•
First peptides show the “light” isotopic peptide from wild type EGFR.
Second and third peptides show “heavy” R and K amino acids within the
mutated EGFR peptides.
Only wild type EGFR
peptides are identified.
No modifications due to
metabolic labeling were
included as search
parameters when using
the XProteo search
engine.
This approach proved successful for the isolation and quantification
of the three forms of EFGR.
We identified several interacting partners, such as GRB2, and are in
the process of determining their relative levels of association with
the wild type, L858R mutated, and E746-A750 deletion forms of
EGFR.
Knowledge of proteins that form complexes with the wild type and
mutant EGFR may lead to new therapeutic targets for NSCLC.
In order to relatively
quantify the protein
interactions with
mutant EGFR, another
database was used.
Acknowledgements
Luke De
Bonnie Kaiser
Anna Arnaudo
Chase Palisch
The Pingry School Science Department
Val Carabetta
The Members of the Cristea Lab
References
Fig 7. Database search of known masses from labeled mass spectra.
EGFR is highest match of peptides in sample.
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