in-depth analyses of kinase-dependent tyrosine
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In-depth analyses of kinase-dependent tyrosine phosphoproteomes based on metal ion
functionalized soluble nanopolymers
Anton B. Iliuk,1 Victoria A. Martin,2 Bethany M. Alicie,2 Robert L. Geahlen,2,4 and W. Andy
Tao1,2,3,4*
1Departments of Biochemistry, 2Medicinal Chemistry & Molecular Pharmacology, and
3Chemistry, and the 4Purdue University Center for Cancer Research, Purdue University, West
Lafayette, IN 47907
*Corresponding author:
watao@purdue.edu
Running title: Phosphoproteomics by PolyMAC
MCP Papers in Press. Published on June 17, 2010 as Manuscript M110.000091
Copyright 2010 by The American Society for Biochemistry and Molecular Biology, Inc.
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ABBREVIATIONS EDC 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride EGFP Enhanced green fluorescence protein FDR False discovery rate GO Gene ontology IMAC Immobilized metal ion affinity chromatography MES 2-(N-morpholino)ethanesulfonic acid PAMAM Polyamidoamine PolyMAC Polymer-based Metal-ion Affinity Capture SYK Spleen tyrosine kinase XIC Exacted ion chromatograms
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ABSTRACT
The ability to obtain in-depth understanding of signaling networks in cells is a key objective of
systems biology research. Such ability depends largely on unbiased and reproducible analysis of
phosphoproteomes. We present here a novel proteomic tool, Polymer-based Metal-ion Affinity
Capture (PolyMAC), for the highly efficient isolation of phosphopeptides to facilitate
comprehensive phosphoproteome analyses. This approach uses polyamidoamine (PAMAM)
dendrimers multi-functionalized with titanium ions and aldehyde groups to allow the chelation
and subsequent isolation of phosphopeptides in a homogeneous environment. Compared to
current strategies based on solid phase micro- and nano-particles, PolyMAC demonstrates
outstanding reproducibility, exceptional selectivity, fast chelation times, and high
phosphopeptide recovery from complex mixtures. Using the PolyMAC method combined with
antibody enrichment, we identify 794 unique sites of tyrosine phosphorylation in malignant
breast cancer cells, 514 of which are dependent on the expression of Syk, a protein-tyrosine
kinase with unusual properties of a tumor suppressor. The superior sensitivity of PolyMAC
allows us to identify novel components in a variety of major signaling networks including cell
migration and apoptosis. PolyMAC offers a powerful and widely applicable tool for
phosphoproteomics and molecular signaling.
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INTRODUCTION
Reversible phosphorylation of proteins is a major mechanism for the regulation of multiple
cellular processes (1,2). Mass spectrometry-based phosphoproteomics provides a method for the
global analysis of protein phosphorylation and molecular signaling in cells (3,4). Despite the
great progress that has been made over the past few years, the isolation of phosphopeptides and
their analysis by mass spectrometry is still a considerable challenge due to the typically low
stoichiometry of protein phosphorylation and the resulting low abundance of phosphopeptides.
An early step in any phosphoproteome analysis is the isolation of phosphopeptides, preferably
with high efficiency, selectivity, sensitivity and reproducibility. Currently there are three major
strategies for the isolation of phosphopeptides: antibody-based affinity capture, chemical
derivatization of phosphoamino acids, and metal ion-based affinity capture. Antibody-based
methods are used mainly for the selective isolation of phosphotyrosine containing proteins or
peptides (5-8). Chemical derivatization methods begin with the β-elimination of phosphates from
phosphoserine and phosphothreonine (9) or the formation of phosphoramidates by reactions with
amines (10) to selectively immobilize phosphopeptides. Metal ion-based affinity capture
techniques use immobilized metal affinity chromatography (IMAC) with Fe(III)(11,12) or
Ga(III)(13), and for the past a few years a more successful metal oxides approach (i.e., TiO2
(14,15), and ZrO2 (16,17)), for the selective binding of phosphorylated peptides. Almost all of
the current isolation methods are based on solid phase extractions, which, due to the nature of the
heterogeneous environment and nonlinear binding dynamics when dealing with extremely low
abundant phosphopeptides, can yield inconsistent results from one run to the next, even when
using the same protocol.
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We introduce here a new reagent and a novel chemical strategy termed Polymer-based Metal-ion
Affinity Capture (PolyMAC) for the isolation of phosphopeptides with exceptionally high
reproducibility, selectivity and sensitivity. This approach is based on a metal-ion functionalized
soluble nanopolymer to chelate phosphopeptides in a homogeneous aqueous environment. We
present here the preparation and the characterization of PolyMAC reagents and compare these
with existing techniques that employ IMAC or TiO2. To illustrate the utility of this approach for
the analysis of complex systems, we further demonstrate that the PolyMAC technology greatly
facilitates the characterization of the spleen tyrosine kinase (Syk)-dependent phosphoproteome
of malignant breast cancer cells.
The onset and development of breast tumors takes place through a series of complex processes
that include the suppression of oncoprotective genes and activation of oncogenes (18). Among
the tumor suppressors identified in breast cancer is Syk, a protein-tyrosine kinase whose
expression is reduced in many breast cancer cells and is completely absent in highly tumorigenic
cells (19,20). Moreover, when re-expressed in malignant breast cancer cells, Syk inhibits cell
motility, growth, invasiveness and tumor formation while promoting cell-cell adhesion (19).
While the role of Syk in signaling through antigen receptors is well characterized, little is known
about the effectors and the pathways that it regulates in breast epithelial cells. We applied,
therefore, the highly efficient PolyMAC approach to profile the Syk-dependent tyrosine
phosphoproteome in invasive breast cancer cells by analyzing sites of tyrosine-phosphorylation
present before and after the induction of its expression. Among a total of nearly 800 sites of
tyrosine-phosphorylation, we identified over 500 sites that are dependent on the expression of
Syk. These phosphorylated sites are present on enzymes participating in signaling networks that
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are involved in such processes as cell-cell interactions, cell migration and apopotosis. Overall,
PolyMAC represents a remarkable chemical tool for the in-depth study of complex signaling
networks.
EXPERIMENTAL PROCEDURES
Preparation of PolyMAC-Ti reagent
Polyamidoamine dendrimer generation 4 (PAMAM G4) solution (200 μl; provided as 10%
(wt/vol) in methanol; Sigma) was dried in a microfuge tube and re-dissolved in 3 ml of 150 mM
MES (2-(N-morpholino)ethanesulfonic acid; pH 5.5) buffer and transferred into a 10-ml round-
bottom flask with a magnetic stir bar. Then 6.5 mg of 2-carboxyethyl-phosphonic acid, 6 mg of
glyceric acid, 10 mg of N-hydroxysuccinimide, and 100 mg EDC (1-ethyl-3-(3-
dimethylaminopropyl) carbodiimide hydrochloride) were added into the flask and stirred
overnight. The solution was dialyzed against water to remove any remaining excess reagents.
Next, 40 mg of sodium (meta)periodate was added to the solution and incubated for 40 min with
agitation in the dark at room temperature. The mixture was dialyzed overnight against water. The
reagent solution was transferred into a microfuge tube, 10 uL titanium oxychloride stock (Sigma)
was added and incubated for 1 hour with agitation at room temperature. The solution was finally
dialyzed overnight to remove any unbound titania and the final PolyMAC-Ti product was stored
at 4°C.
Characterization of PolyMAC-Ti reagents
Preparation of simple peptide mixtures and isotopic labeling of the angiotensin II
phosphopeptide. Human angiotensin II peptide (MW 1045 Da) and its phosphorylated form
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(MW 1125 Da) were bought directly from Sigma-Aldrich. The model protein β-lactoglobulin
was denatured and reduced in 8 M urea containing 5 mM dithiothreitol for 30 minutes at 37 °C.
The protein was further alkylated with 15 mM iodoacetamide for 1 hour in the dark at room
temperature. The sample was diluted 6-fold with 50 mM trimethylammonium carbonate and
digested overnight with proteomics grade trypsin (Sigma) at 1:100 ratio at 37 °C. The resulting
peptides were dried down using SpeedVac concentrator and resuspended in water at 100
pmol/uL concentration.
To measure accurate phosphopeptide recovery, the phosphorylated angiotensin peptide was
isotopically labeled with a “light” and ”heavy” amine-specific reagents (iTAG) developed in our
own lab (21) (see Fig S1 for the structure of isotopic tag).
Isolation of phosphopeptides from a simple peptide mixture using PolyMAC-Ti. The peptide
mixture (100 pmol each) was resuspended in 300 μl of the loading buffer A (150 mM HEPES,
pH 6.7), to which 5 nmol of the PolyMAC-Ti reagent was added. The mixture was incubated for
5 minutes (for the binding kinetics assay, 1 uL of the solution was taken out after designated
incubation time for MALDI experiments) and transferred into a spin column (Boca Scientific)
containing the Affi-Gel Hydrazide beads (Bio-Rad). The column was gently agitated for addition
10 minutes, and then centrifuged down at 2,300 × g for 30 seconds to collect the unbound
flowthrough. The gel was washed once with 200 uL loading buffer A, twice with the washing
buffer (100 mM acetic acid, 1% trifluoroacetic acid, 80% acetonitrile) , and once with water. The
phosphopeptide was eluted off the dendrimer by incubation the gel twice with 100 uL of 400
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mM ammonium hydroxide solution for 5 minutes. The eluates were collected and dried down
completely under vacuum.
Preparing DG-75 cell lysate samples. DG-75 cell culture was grown to 50% confluency in
RPMI-1410 media substituted with 10% inactivated FBS, 1% sodium pyruvate, 0.5%
streptomycin/penicillin, and 0.05% β-mercaptoethanol. Before collection, the cells were washed
with PBS, collected, and frozen at -80 °C. Cells were lysed in 1 mL of lysis solution (50 mM
Tris-HCl, pH 7.5, 150 mM NaCl, 5 mM EDTA, 1% NP-40, 1 mM sodium orthovanadate, 1x
phosphatase inhibitor cocktail (Sigma), 10 mM sodium fluoride) for 20 minutes on ice. The cell
debris was cleared at 16,100 × g for 10 minutes, and supernatant containing soluble proteins
collected. The concentration of the cell lysate was determined using the BCA assay. In each
analysis, 100 ug of the DG-75 protein lysate was denatured and reduced in 50 mM trimethyl
ammounium bicarbonate containing 0.1% RapiGest (Waters) and 5 mM dithiothreitol for 30
minutes at 37 °C. The proteins were further alkylated in 15 mM iodoacetamide for 1 hour in the
dark at room temperature, and digested with proteomics grade trypsin at 1:100 ratio overnight at
37 °C. The pH was adjusted below 3 and the sample was incubated for 40 minutes at 37 °C. The
sample was centrifuged down at 16,100 × g to remove RapiGest and to collect supernatant. The
sample was desalted with a 100-mg Sep-Pak C18 column (Waters).
Isolation of phosphopeptides from DG75 lysate using the PolyMAC-Ti reagent. The enrichment
of the complex samples using the PolyMAC-Ti reagent was similar to the above-described
PolyMAC protocol except for minor changes. Firstly, the loading buffer A was replaced with
loading buffer B (100 mM glycolic acid, 1% trifluroacetic acid, 50% acetonitrile). Secondly,
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only 100 uL of the loading buffer B was used (instead of 300 uL as previously described) to
incubated the peptide mixture with the PolyMAC. Lastly, 200 uL of the capturing buffer
(300mM HEPES, pH 7.7) was added to the mixture immediately before transferring the solution
into the spin column with Affi-Gel Hydrazide beads to a final pH of 6.3.
Phosphopeptide enrichment using IMAC and TiO2. IMAC phosphopeptide capture was done
using the PHOS-Select Iron beads (Sigma) according to the protocol modified from the
manufacturer. Briefly, the 50 uL of the PHOS-Select resin slurry was transferred into a spin
column and washed twice with water. The peptide mixture was resuspended in 200 uL IMAC
loading buffer (250 mM acetic acid, 30% acetonitrile), added to the resin, and the mixture was
incubated for 1 hour. For binding kinetics step, 1 uL of the solution was taken out designated
incubation time and spotted on a MALDI plate. The flowthough was collected by centrifugation
at 2,300 × g for 30 seconds. The resin was washed once with 200 uL of the loading buffer for 5
minutes, twice with the washing buffer (250 mM acetic acid, 0.1% TFA, 80% acetinitrile), and
one last time with water. Phosphopeptides were eluted twice with 100 uL of 400 mM of
ammonium hydroxide and dried completely under vacuum.
Phosphopeptide enrichment with TiO2 beads (Glygen) was carried out with some modifications
according to the latest protocol (22) in batch mode. Briefly, 5 mg of the TiO2 beads were
transferred into a spin column and washed twice with water. The peptide mixture was
resuspended in 300 uL TiO2 loading buffer (1M glycolic acid, 5% TFA, 80% acetonitrile), added
to the beads, and the mixture was incubated for 1 hour (for binding kinetics step, 1 uL of the
solution was taken out after 30 minute incubation and spotted on a MALDI plate). The
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flowthough was collected by centrifugation at 2,300 × g for 30 seconds. The resin was washed
once with the loading buffer, twice with the washing buffer (1% TFA, 80% acetonitrile), and
with water again. Phosphopeptides were eluted twice with 100 uL of 400 mM of ammonium
hydroxide and dried completely under vacuum. A similar protocol was used with TiO2
nanoparticles from GL Sciences for comparison.
Isolation of tyrosine phosphopeptides from MDA-MB-231 lysate
MDA-MB-231 wild-type (Syk-negative) and TRS (Tet-response system) Syk-inducible cell
cultures were prepared, grown and stimulated as described before (23). Briefly, both cell lines
were grown to 50% confluency. Syk-inducible cells were incubated with 1 ug/mL of tetracycline
for 20 hours at 37°C to induce Syk expression. Both sets of cells were then lightly stimulated
with 20 uM sodium pervanadate for 15 min at 4°C. Cells were collected, washed, and lysed as
described above. After BCA assay, the samples were normalized to 5 mg each. The samples
were denatured, alkylated and digested as described above. The pH of the samples was adjusted
to 7.4 with 100 mM Tris-HCl buffer (pH 8.0), and 200 ul of the PT66 phosphotyrosine antibody
beads slurry (Sigma) was added to the peptide samples. The mixture was incubated overnight at
4 °C with agitation (7). The supernatant was carefully removed, and the beads were washed
twice with 500 uL of the lysis buffer with no phosphatase inhibitors (10 minutes each) and twice
with water (2 minutes each). Tyrosine phosphopeptides were eluted by incubating the beads 3
times with 100 uL of 0.1% TFA with 10 minute vigorous agitation, twice with 100 uL of 0.1%
TFA in 50% acetonitrile (10 minutes each) (24), and twice with 50 uL of 100 mM glycine, pH
2.5 (30 min each with vigorous agitation). All eluates were combined and dried completely in the
speedvac. The peptide mixture was resuspended in 300 μl of the loading buffer A (150 mM
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HEPES, pH 6.7), to which 5 nmol of the PolyMAC-Ti reagent was added. The mixture was
incubated for 5 minutes and transferred into a spin column containing the Affi-Gel Hydrazide
beads. The column was gently agitated for addition 10 minutes, and then centrifuged down at
2,300 × g for 30 seconds to collect the unbound flowthrough. The gel was washed extensively
and tyrosine phosphopeptides were eluted by incubation the gel twice with 100 uL of 400 mM
ammonium hydroxide solution for 5 minutes. The eluates were collected and dried down
completely under vacuum.
Mass spectrometry analyses
MALDI-TOF analyses. MALDI-TOF single MS analyses were performed on simple peptide
mixtures using ABI 4800 MALDI-TOF/TOF mass spectrometer. The dried peptide samples were
resuspended in 100 uL of 0.1% TFA, 0.35 uL of each sample (~10 fmol per peptide) was mixed
with equal amount of α-hydroxycinnamic acid matrix (10 mg/mL in 0.1% TFA, 80%
acetonitrile) and spotted on a MALDI plate. The laser was set at 4,000 intensity and data were
acquired at 500 pulses per spectrum.
LTQ-Orbitrap analyses. Peptide samples were re-dissolved in 8 uL of 0.1% formic acid and
injected into an Agilent nanoflow 1100 HPLC system. The reverse phase C18 was performed
using an in-house C18 capillary column packed with 5 μm C18 Magic beads resin (Michrom; 75
μm i.d. and 12 cm of bed length) on an 1100 Agilent HPLC (25). The mobile phase buffer
consisted of 0.1% HCOOH in ultra-pure water with the eluting buffer of 100% CH3CN run over
a shallow linear gradient over 60 min with a flow rate of 0.3 µl/min. The electrospray ionization
emitter tip was generated on the prepacked column with a laser puller (Model P-2000, Sutter
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Instrument Co.). The Agilent 1100 HPLC system was coupled online with a high resolution
hybrid linear ion trap orbitrap mass spectrometer (LTQ-Orbitrap XL; Thermo Fisher). The mass
spectrometer was operated in the data-dependent mode in which a full-scan MS (from m/z 300-
1700 with the resolution of 30,000 at m/z 400) was followed by 4 MS/MS scans of the most
abundant ions. Ions with charge state of +1 were excluded. The mass exclusion time was 180 s.
Data Acquisition and Analysis
The LTQ-Orbitrap raw files were searched directly against homo sapien database with no
redundant entries (67,250 entries; human International Protein Index (IPI) v.3.64) using
SEQUEST algorithm (26) or MASCOT on Proteome Discoverer (Version 1.0; Thermo Fisher).
Proteome Discoverer created DTA files from the raw data with minimum ion threshold of 15 and
absolute intensity threshold of 50. Peptide precursor mass tolerance was set at 5 ppm, and
MS/MS tolerance was set at 0.8 Da. Search criteria included a static modification of cysteine
residues of +57.0214 Da and a variable modifications of +15.9949 Da to include potential
oxidation of methionines and a modification of +79.996 on serine, threonine or tyrosine for the
identification of phosphorylation. Searches were performed with full tryptic digestion and
allowed a maximum of two missed cleavages on the peptides analyzed from the sequence
database. False discovery rates (FDR) were set for 2% for each analysis. Proteome Discoverer
generates a reverse “decoy” database from the same protein database, and any peptide passing
the initial filtering parameters that were derived from this decoy database are defined as false
positive identification. The minimum cross-correlation factor (Xcorr) filter was the re-adjusted
for each individual charge state separately in order to optimally meet the predetermined target
FDR of 2% based on the number of random false-positive matches from the reversed “decoy”
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database. Thus, each dataset had its own passing parameters. The number of unique
phosphopeptides and non-phosphopeptides identified were then manually counted and compared.
Phosphorylation site localization from CID mass spectra was determined by Sequest Xcorr
scores and only one phosphorylation site was counted using the top scored phosphopeptide for
any phosphopeptide with potential ambiguous phosphorylation sites (∆Xcorr score of 0 in
Supplementary Tables refers to top ranked identification).
Gene Ontology and IPA pathways analysis
Cellular location, protein category, functions and processes were listed for identified proteins in
the Syk-induced sample. The data were exported and InforSense (InforSense Ltd, London, UK)
version 4.1 for Gene Ontology (GO) annotation was used as a part of Proteome Discoverer V1.0
software to group components, functions, and processes for proteins identified in the
aforementioned experiments. The resulting analyses were exported as xml files.
For pathway analyses, high confidence peptides (<2% FDR) from Syk-negative and Syk-
induced samples were manually compared and phosphotyrosine peptides identified only in Syk
induced sample were selected. The corresponding proteins were assumed to have increased
tyrosine phosphorylation on certain sites due to Syk presence. To identify canonical and
metabolic pathways and their protein partners, Ingenuity Pathway Analysis (IPA) (Ingenuity
Systems), was used for categorizing these proteins and grouping then according to processes,
localization, and networking. The IPA analyses criteria were set to include only known human
cellular proteins and their interactions. The resulting data files were exported as xml files. Top
networks identified were further inspected using NCBI and Swissprot websites, as well as the
peer-reviewed literature.
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Immunoprecipitation and Western blot experiments
MDA-MB-231 wild-type and Syk-inducible cell cultures were prepared as described above.
Cells were collected and lysed in lysis buffer with protease and phosphatase inhibitors (50 mM
Tris-HCl, pH 7.5, 150 mM NaCl, 5 mM EDTA, 1% NP-40, 1 mM sodium orthovanadate, 1x
phosphatase inhibitor cocktail (Sigma), 10 mM sodium fluoride, 1x Mini Complete protease
inhibitor cocktail (Roche)) for 20 minutes on ice. Samples were normalized based on protein
concentration and 1-2 mg of total lysate were used for immunoprecipitation (IP) experiments.
For phosphotyrosine immunoprecipitation, 2 mg of lysates were incubated with 40 uL of PT66
anti-phosphotyrosine antibody immobilized on agarose beads slurry (Sigma) for 2 hours at 4°C.
For protein immunoprecipitation, 1 mg of lysates were pre-incubated with 20 uL Protein A/G
agarose beads for 20 min at 4°C to remove non-specific binding. Then the lysates were incubated
with 4 ug of the selected antibodies (anti-MAPRE1, anti-Scrib, anti-TRIP4 or anti-dynactin) for
2 hours at 4°C. Finally, the samples were incubated with 40 uL of Protein A/G agarose beads for
2 hours at 4°C. The beads were washed, and the bound proteins were eluted off by boiling the
beads in 2x SDS loading buffer for 5 min. Lysates (50 ug each) and the IP eluents were run on a
12% SDS gel and transferred onto a PVDF membrane. The membranes were blotted against the
corresponding proteins (e.g. EB1, dynactin, Scrib, or TRIP4). Then the membranes were stripped
and reblotted against pTyr using 4G10 anti-phosphotyrosine antibody. Finally, the membranes
were stripped again and reblotted against GAPDH and Syk as controls.
RESULTS
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Strategy and design of PolyMAC reagents
We devised the PolyMAC agents based on a soluble, globular nanopolymer (i.e., dendrimer)
which was multi-functionalized with reactive groups for the site-specific chelation of
phosphopeptides, and with ‘handle’ groups that facilitate the isolation of immobilized
phosphopeptides through a highly efficient bio-conjugation (Figure 1). This design takes
advantage of not only the multifunctionalized nanoparticles, but more importantly, the soluble
nature of the molecule, allowing for the chelation of a limited sample of phosphopeptides in the
solution phase for optimum efficiency and maximum yield. In a second step, the attached
phosphopeptides are captured on a solid support (e.g., agarose or magnetic beads) through highly
efficient coupling between two bioconjugation groups on the soluble polymer and on the solid
phase, respectively. A high concentration ratio of the reactive group to the ‘handle’ group
facilitates the complete recovery of the bound phosphopeptides while eliminating the need for
extra steps to remove excess reagents.
In this study we first modified the polyamidoamine (PAMAM) dendrimer generation 4.0 with
phosphonate groups. Subsequently, titanium (IV) ions were immobilized on the surface through
the chelation to phosphonate groups. In addition, we also functionalized the dendrimer with
aldehyde as the “handle” group (Figure 1). The density of titanium ions and aldehyde groups can
be easily controlled through the synthesis procedure and the current PolyMAC-Ti reagent has 35
titanium ions and 6 aldehyde groups per molecule. PolyMAC-Ti agents bound with
phosphopeptides are isolated from the solution phase through the formation of hydraone from the
hydrazine and aldehyde reaction (<5 minutes) by coupling to the hydrazide agarose gel. Primary
amine groups on peptides may react with aldehyde groups but the reaction is reversible (27).
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Nonphosphopeptides are readily washed away, and the phosphopeptides are recovered using
ammonia aqueous solution.
PolyMAC isolates phosphopeptides from standard peptide mixtures
The ability of PolyMAC-Ti to enrich phosphopeptides was tested first by incubating the reagent
with a mixture of angiotensin II (m/z 1046) and tyrosine-phosphorylated angiotensin II (m/z
1126). For comparison, the same peptide mixture also was incubated with IMAC-Fe(III) resin
and TiO2 nanoparticles using most recent published conditions (22,28,29). Recoveries of
phosphopeptide were analyzed by matrix-assisted laser desorption ionization (MALDI) time of
flight (TOF)/TOF mass spectrometry (Figure 2; Supplementary Fig. S2 for the raw files). The
phosphopeptide was captured instantly once the PolyMAC-Ti reagent was added to the peptide
mixture as shown by the disappearance from the solution of a peak at m/z 1126 indicating fast
binding kinetics. In contrast, the time required to capture the phosphopeptide from solution was
much longer for the solid phase reagents (IMAC and TiO2 beads). In fact, IMAC was unable to
completely deplete the phosphopeptide from the solution in 30 min. Phosphopeptides were then
eluted from all three supports and the original starting amount of nonphosphopeptide (m/z 1046)
was spiked into the eluate as an internal standard to measure the yield of each technique. By
comparing the ratio of intensities of the peaks for the two peptides in the eluate to that in the
original sample, we quantified the relative recoveries of phosphopeptide for each procedure. The
average yields for recovery of phosphopeptide were close to 25% for IMAC resin, 50% for TiO2
nanoparticle and >90% for PolyMAC-Ti (Figure 2).
To evaluate the PolyMAC-Ti reagent using a more complex system, we measured its ability to
recover phosphorylated angiotensin II (m/z 1126) from a mixture of the phosphopeptide added at
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a ratio of 1:100 to a tryptic digest of unphosphorylated β-lactoglobulin (Supplementary Figure
S3). The phosphopeptide was hardly detectable by MS due to the high background of
nonphosphopeptides in the starting mixture. After enrichment using PolyMAC-Ti, the only
detectable peak in the spectrum was the phosphopeptide, showing the excellent selectivity of the
method. A similar recovery of phosphopeptide of greater than 90% was observed when a
nonphosphorylated peptide was included as an internal standard (data not shown).
PolyMAC isolates phosphopeptides from complex cell lysate
To demonstrate the utility of PolyMAC for biological studies, we analyzed phosphopeptides
isolated from extremely complex samples, the whole cell extracts of the human Burkitt’s
lymphoma B cell line, DG-75. A total of 100 µg of lysate was digested with trypsin and desalted
using a Sep-Pak C18 column. The digest was incubated with 5 nmol of PolyMAC-Ti reagent for
1 min, and then for an additional 5 min with 50 µl of hydrazine-agarose. A parallel analysis
using TiO2 beads (Glygen; Results with TiO2 from GL Sciences were included in the
Supplementary Material) under optimal conditions (22,30) was carried out for comparative
purposes. Glycolic acid (100 mM) was added to the binding solution to improve the selectivity
for the isolation of phosphopeptides (22,29). Phosphopeptides were eluted with ammonia, dried
under vacuum, reconstituted in 0.1% formic acid and analyzed by LC-MS/MS. The experiment
was repeated three times, each time using a different cell lysate and a different batch of TiO2 and
PolyMAC-Ti reagents. MS/MS data were analyzed using the SEQUEST software by searching
against the human protein database and reverse database to estimate the false discovery rate
(FDR). In single 90-min gradient LC-MS/MS runs, an average of 877 unique phosphopeptides
representing 1003 sites of phosphorylation from 100 µg of total lysate were identified using
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PolyMAC-Ti (Figure 3; see Supplementary Table S1-6 for the full lists). In comparison, an
average of 420 unique phosphopeptides representing 498 phosphorylation sites was identified
from the same sample using the TiO2 method. The number of identified phosphopeptides is
consistent to a recent report using Titania stagetips in which an average of 580 phosphopeptides
were identified from 150 µg of HeLa cell lysate (31). To increase the confidence in our
phosphopeptide identification, we also carried out database searches using MASCOT. Using the
same FDR cutoff, the database search with both SEQUEST and MASCOT resulted in over 85%
overlap in phosphopeptide identifications, which is consistent with previous studies on the
comparison of different database search algorithms (32,33). Although phosphopeptide
identification has remained a challenge, the results indicate that our phosphopeptide
identification is reliable. The detailed results with both MASCOT and SEQUEST search are
included in the Supplementary Material. PolyMAC-Ti has extremely high selectivity for
phosphopeptides of 96± 0.6%, compared to the selectivity of 77±7.5% for TiO2 beads.
Furthermore, the PolyMAC-Ti method exhibited lower variability than the TiO2 method in terms
of the selectivity and identity of the recovered phosphopeptides. For three parallel analyses using
TiO2 beads, 38.4% of phosphopeptides were identified by all three analyses, and ~ 50% overlap
between every two analyses, again consistent to the recent literature report (31). In contrast,
55.2% of phosphopeptides were identified by all three analyses, and ~70% overlap between
every two analyses, when using PolyMAC-Ti. To further investigate the reproducibility, we
enriched one batch of phosphopeptides and then split the sample equally in halves and analyzed
them by LC-MS/MS. We observed around 70% of overlap in phosphopeptides in the analyses,
indicating that the limit of ~70% reproducibility in identifying phosphopeptides might attribute
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to inherent factors in the LC-MS/MS analysis (data not shown) (32,33). The experiments,
therefore, demonstrated the remarkable reproducibility of PolyMAC-based enrichment method.
Analysis of the Syk-dependent tyrosine phosphoproteome in breast cancer cells
Next, we applied the PolyMAC strategy to analyze Syk-dependent signaling in breast cancer
cells. A line of highly invasive MDA-MB-231 cells, which normally lack Syk, was generated in
which the expression of an enhanced green fluorescence protein (EGFP)-tagged form of the
kinase could be induced by treatment with tetracycline (Tet) (23). Because sites of tyrosine
phosphorylation are much less abundant than sites of serine or threonine phosphorylation, we
coupled an immuno-affinity step to the PolyMAC method to enrich for phosphotyrosine
containing peptides. An overall flow chat for the analysis of Syk-dependent tyrosine
phosphorylation is illustrated in Figure S9. Although there have been reports that tyrosine
phosphopeptides can be analyzed with single stage affinity purification based on anti-pY
antibodies (7,34), including recent large scale phosphotyrosine profiling (34,35), tandem affinity
approaches combining anti-pY antibodies and metal oxide improve the selectivity and
confidence in the identification of tyrosine phosphopeptides (5,8). We carried out parallel
experiments to compare three methods using anti-tyrosine phosphopeptide PT66 alone,
PT66+TiO2 and PT66+PolyMAC purifications. From 750 µg of DG75 extract in three equal
portions (250 µg for each experiment), three methods resulted in the identification of 135, 202
and 315 pY sites, respectively (Fig. S10). Relatively low selectivity of 40% with the PT66 alone
method may contribute to the interference from non-phosphopeptide signals that suppress
tyrosine phosphopeptide signals, leading to poorer identification.
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Syk-negative and Syk-EGFP-expressing MDA-MB-231 cells (- and + Tet) were grown to 50%
confluency. Western blotting analyses indicated a marked elevation of tyrosine phosphorylation
in cells expressing Syk-EGFP with mild pervanadate treatment (Figure 4). Cell lysates were
digested with trypsin. Phosphotyrosine-containing peptides were immunoprecipitated with anti-
phosphotyrosine antibodies linked to agarose beads, eluted under mildly acidic conditions,
subjected to PolyMAC-Ti enrichment, and analyzed by nano-flow LC-MS/MS. To evaluate the
efficiency of isolation of phosphotyrosine-containing peptides, we labeled phospho-angiotensin
II with one of two amine-specific isotope tagging reagents of different molecular mass (Figure
S1 for structures of the tagged phosphopeptides) (21). The “light” isotope tagged phospho-
angiotensin II (10 fmol) was added to the sample prior to enrichment. After enrichment, 10 fmol
of phospho-angiotensin II labeled with the “heavy” isotope tag was added prior to the LC-
MS/MS analysis. As shown in Figure 5 (Supplementary Fig. S11 for the raw files), greater than
90% of the light isotope tagged phospho-angiotensin was recovered as measured by the ratio of
the light (m/z,MH2+= 620.299) to heavy (m/z, MH2+ = 623.316) phosphopeptides. In contrast,
TiO2 was only able to recover ~10% of the light isotope-labeled phosphopeptide. This
measurement of phosphopeptide enrichment and recovery indicates that the PolyMAC method
provides an extremely efficient approach for the analysis of sites of tyrosine phosphorylation.
A much larger number of phosphotyrosine-containing peptides was identified in samples from
cells induced to express Syk-EGFP as compared to Syk-deficient cells (820 versus 377 unique
phosphopeptides), consistent with the Western blotting data. Selectivity for the recovery of
phosphopeptides using the PolyMAC-Ti reagent was excellent (only 51 nonphosphopeptides
were identified among a total of 871 peptides identified, corresponding to >94% selectivity in the
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Syk-EGFP-induced sample). Overall, we identified 514 unique sites of tyrosine phosphorylation
on 458 different proteins that were present only in samples from Syk-EGFP expressing cells
(Fig. 6A; see Supplementary Table S7-8 for the full lists). To complement identification, we
also examined the exacted ion chromatograms (XICs) to quantify the relative abundance of
phosphopeptides recovered from the two samples. After signal normalization using spiked
standard phosphopeptides, we measured the relative abundance of tyrosine phosphopeptides
from cells expressing or lacking Syk. The majority of phosphopeptides that were not identified
as being present in one sample also were not detectable in the corresponding raw MS data. Only
a small fraction of these phosphopeptides were detected with extremely low intensity and poor
MS/MS spectra. These data indicated that, although lack of identification of a phosphopeptide is
not evidence for its absence, it is sufficient to reflect a change of phosphorylation within a
signaling pathway. For example, cortactin, a cortical actin-associated protein known to play a
critical role in stabilizing the adherens junction, was recently discovered as a direct substrate of
Syk in breast cancer cells, although the phosphorylation sites were not identified (23). Our
analysis identified 3 phosphorylation sites on cortactin that were modified exclusively in the
Syk-expressing MDA-MB-231 cells (Supplementary Table S8). The further analysis of these
tyrosine phosphoproteomes will provide a unique opportunity for researchers to gain insights
into important signaling pathways in breast cancer cells.
Gene ontology (GO) analyses of proteins with enhanced tyrosine phosphorylation in Syk-
induced cells grouped by molecular function, biological process and subcellular localization
indicate roles for the kinase in a variety of cellular locations, functions and processes (Figure
S12). Possible cellular signaling pathways influenced by the expression of Syk were further
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assessed using the induced phosphorylated proteins as input. Our data revealed elevated levels of
tyrosine phosphorylation in a number of cancer-related networks, notably cancer cell movement
and cell death (Figure 7 and see Supplementary Figure S13 and Table S9 for the list of
components and other examples).
To confirm the identification and novel functions of tyrosine-phosphorylated proteins, we
selected several phosphoproteins to explore further, including a microtubule-associated protein
MAPRE1, a nuclear transcriptional coactivator TRIP4/ASC-1, a cell polarity protein SCRIB, and
a microtubule-based motor protein Dynactin. Tyrosine phosphorylated proteins were
immunoprecipitated from lysates of MDA-MB-231 cells lacking or expressing Syk-EGFP using
anti-phosphotyrosine antibodies. The resulting immune complexes were examined by Western
blotting with antibodies against MAPRE1, TRIP4, SCRIB, or Dynactin. As shown in Fig. 5B,
the phosphoproteins were enriched in anti-phosphotyrosine immune complexes prepared from
Syk-expressing cells. Similarly, the phosphorylation of each protein on tyrosine was confirmed
by the Western blotting of anti-protein immune complexes with anti-phosphotyrosine antibodies.
DISCUSSION
Signal transduction pathways modulated by protein-tyrosine kinases are often difficult to dissect
because of complexities of interacting networks, a large number of substrates and low levels of
phosphorylation. MS-based phosphoproteomics is presently the most powerful tool for profiling
dynamic phosphorylation events on a global scale. A drawback of most phosphoproteomic
approaches is a less than satisfactory reproducibility and selectivity during the phosphopeptide
enrichment step. We reasoned that inconsistencies are due largely to the heterogeneity of solid
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phase extraction-based isolation methods as represented by approaches that use IMAC or metal
oxides. Heterogeneous isolation conditions can suffer from nonlinear kinetic behavior, unequal
distribution of and/or access to functional groups, and solvation problems. This presents a
serious problem for the recovery of low-abundant phosphopeptides from complex samples. The
PolyMAC approach instead allows the isolation of low abundant phosphopeptides through
chelation of Ti(IV) in a homogeneous, aqueous solution. A high concentration of metal ions in
solution is achieved through the immobilization of the ions on soluble polymers for fast
chelation. Then, in a second step, the PolyMAC-phosphopeptide complexes are gathered from
the solution by covalent coupling to a solid support. Besides the hydrazine-aldehyde coupling
pair described here, a variety of other highly efficient pairs can be envisioned including azide-
alkyne (click chemistry), thiol-idoacetyl, thiol-maleimide, thioester-cysteine, etc. The
preparation of PolyMAC reagents and their use for phosphopeptide isolation could be easily
automated for high reproducibility and high throughput. The density of functional groups (Ti(IV)
and aldehyde) on the surface of the dendrimer can be adjusted during the synthetic steps. The
PolyMAC reagents are much more amenable to analysis using NMR, MS and other
spectroscopic methods than TiO2 or IMAC reagents.
In addition, the dendrimer can be functionalized with different metal ions such as Fe(III) or
Ga(III). Our data sets suggest that PolyMAC-Ti may exhibit selectivity for singly
phosphorylated peptides, an observation documented by others using TiO2 or ZrO2 for the
isolation of phosphopeptides (36). As a solution, a strategy that uses IMAC and TiO2 separation
strategies in sequence has been proposed for the separation of multi-phosphorylated and then
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monophosphorylated peptides (36). An analogous strategy could be adopted easily by the
sequential use of PolyMAC-Fe and PolyMAC-Ti.
We demonstrate in this study that the PolyMAC method offers a rapid process for the recovery
of phosphopeptides. The entire isolation procedure including chelation of phosphopeptides,
recovery of PolyMAC-phosphopeptide complexes on agarose beads, washing, and elution
requires less than 30 min while still retaining exceptionally high selectivity, reproducibility and
recovery yields that are superior to the commonly used TiO2 method. PolyMAC is generally
applicable for the isolation of phosphopeptides modified on serine, threonine or tyrosine. By
combining PolyMAC with an anti-phosphotyrosine enrichment step, we have developed a
strategy for the in-depth analysis of tyrosine phosphoproteomes.
To test this strategy, we probed the phosphotyrosine proteome of invasive breast cancer cells
expressing the Syk protein-tyrosine kinase and identified 794 sites of tyrosine phosphorylation,
514 of which were largely dependent on the expression of the kinase. GO annotation of the
phosphoproteome identified prominent subclasses of substrates involved in important processes
that regulate cell growth, motility and survival. The large number of tyrosine phosphorylated
cytoskeletal (17%) and plasma membrane (10%) proteins is consistent with the described roles
for Syk in modulating cancer cell motility, invasion and cytoskeletal rearrangements (23). The
identification of substrates important in the regulation of apoptosis also is consistent with the
reported role for Syk in the enhancement of cell survival (37). In addition, 27% of the
phosphotyrosine-containing proteins were annotated to the nucleus, consistent with previous
studies suggesting important roles for the kinase in the nucleus (23,38,39). The most abundant
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category for the molecular function of substrates was protein binding (44%), which is consistent
with the known role of Syk and tyrosine-phosphorylation in regulating protein-protein
interactions.
The identification of sites of tyrosine phosphorylation on important components of signaling
networks offers exciting new experimental targets in cancer research. Protein-tyrosine kinases
play vital roles in human cancer, most often as the products of dominant, transforming genes. In
contrast, Syk appears to play an unusual role in a subset of cancers by restricting their metastatic
behavior, but the full repertoire of substrates for Syk, especially in epithelial cells, is not known.
A previous study identified a number of proteins potentially phosphorylated on tyrosine in Syk-
expressing breast cancer cells, but sites of phosphorylation were not determined (40).
Interestingly, the majority of the phosphorylation sites identified in this study were characterized
by a high abundance of acidic amino acid residues surrounding the phosphorylated tyrosine,
which is consistent with the known substrate specificity of Syk (Fig. S14-15). While it is not yet
known which of the substrates are most important for mediating Syk’s effects on epithelial cell
function, it is intriguing that known and verified substrates such as MAPRE1 and TRIP4 are
involved in activities (i.e., regulation of microtubule structure and activation of NF-κB) that are
known to be modified as a function of Syk’s expression. Further work will be needed to
characterize fully the role of these and other substrates in the Syk-dependent regulation of
epithelial cell metabolism.
ACKNOWLEDGMENT
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This project has been funded in part by an NSF CAREER award (WAT), a 3M general fund
(WAT), and by National Institutes of Health grants GM088317 (WAT), CA115465-03 (RLG and
WAT) and CA037372 (RLG). We also thank the Tang group in the Indiana University-
Bloomington for providing the software ProteomicsToolBox to generate tandem mass spectra
galleries.
SUPPLEMENTARY MATERIALS
Supplementary data set containing 12 figures and 9 tables are available on the internet through
the MCP site. The tandem mass spectrum gallery are available free of charge at
https://proteomecommons.org/tranche/data-
downloader.jsp?fileName=bsHq3AjE6NwgpqA2UrjU3hmUr%2Biw6R%2FfKMxHcKlf2g15dK
zWoKnAuX%2F%2FN%2Byya3zhtJkwi4qhRAe6LookPYr88RbFWpYAAAAAAAg42Q%3D
%3D.
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FIGURE CAPTION Figure 1 Schematic representation of the PolyMAC method. The PolyMAC strategy
consists of two reaction steps: a homogeneous chelation between phosphopeptides
and the PolyMAC-Ti reagent in the solution phase and a heterogeneous reaction
between the PolyMAC reagent and the solid-phase support.
Figure 2 Side-by-side comparisons of phosphopeptide recoveries using IMAC, TiO2 and
PolyMAC-Ti methods. A peptide mixture consisting of angiotensin II (m/z 1046)
and phospho-angiotensin II (m/z 1126) was analyzed by MALDI-TOF MS prior
to enrichment (column 1) and after incubation with each reagent for the indicated
times (column 2); The analysis of the peptides present in the flow-through and in
the eluate are shown in columns 3 and 4, respectively; quantification of
phosphopeptide recovery is shown in column 5 (the same amount of m/z 1046
peptide was added as an internal standard). Red bar indicates the standard
deviation of recovery yield on multiple repeats.
Figure 3 Analyses of phosphopeptides from digests of DG75 B cell lysates using
PolyMAC-Ti and TiO2. A). Average and standard deviation in the number of
phosphopeptides identified using PolyMAC-Ti or TiO2 in three separate analyses.
B). Average and standard deviation in the percentage of the total identified
peptides that were phosphopeptides in three trials. C and D). Venn diagram
illustrating the overlap in the sets of phosphopeptides identified using PolyMAC-
Ti and TiO2;
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Figure 4 Tetracycline-induced Syk expression in MDA-MB-231 breast cancer cells. A)
Western Blotting indicates elevated tyrosine phosphorylation in response to Syk
expression; B) Syk was expressed in transfected MDA-MB-231 cells with Tet
treatment, indicated by co-expression of EGFP fused on its C terminus.
Figure 5 Comparison of the recovery yield of phosphopeptides using PolyMAC-Ti versus
TiO2. One phosphopeptide was isotopically labeled to generate identical peptides
of two different masses: m/z 620.3 (light, doubly-charged), m/z 623.3 (heavy,
doubly-charged). The light version was included in the sample for enrichment and
the heavy version was spiked into the final eluate for measurement by LC-MS
using an LTQ-Orbitrap spectrometer. Extracted ion chromatograms of labeled
phosphopeptides (top panels). Average intensity of doubly-charged, labeled
phosphopetide ions (bottom panels).
Figure 6 Elevated phosphorylation by Syk induction. A) Number of identified tyrosine
phosphorylation sites in MDA-MB-231 cells with induced (+) and not induced (-)
Syk expression. B) Proteins present in lysates or in the indicated immune
complexes (IP) prepared from cells induced (+) or not induced (-) to express Syk
were separated by SDS-PAGE and analyzed by Western blotting (WB) with the
indicated antibodies.
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Figure 7 Graphic representation of two major pathways perturbed by the induced
expression of Syk. Phosphoproteins identified by MS are represented in color.
Pathway components not identified by MS are in grey. The list of component
names and some examples of other pathways can be found in Supplementary
Figures S13 and Table S9.
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Figure 1
Ti
Ti
TiP
Ti
Ti
Ti
OH
OH
OH
OH
OH
OH
C H3NHP
OO
O O
C H3
NHP
OO
O OCH3
H
O
Solid-phase beadsPolyMACPeptides by guest on A
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Figure 2
1126
1046
P
1046
1126
P
1046
1126P
1126
1046
P
1046
1046
112
6
P
1126
1046
P
Mass (m/z)
10
46
Mass (m/z)
10
46
Mass (m/z)
P
1126
1126
P
Mass (m/z)
10
46
112
6
P
Mass (m/z)
IMAC
TiO2
PolyMAC
0 hoursAfter 1 min
incubationFlowthrough Elution Recovery
~55%
~90%
1046
1126
1122.5
P
~30%
1046
1126
P
P
1126
1046 by guest on A
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0
200
400
600
800
1000
PolyMAC# p
ho
sph
osi
tes
iden
tifi
ed 1003 ± 62
498 ± 39
TiO2
0
20
40
60
80
100
PolyMAC
% e
nri
chm
ent
sele
ctiv
ity 96 ± 0.6 %
77 ± 7.5 %
TiO2
A)
Figure 3A-B
B)
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PolyMAC I
PolyMAC IIIPolyMAC II
16.4%
18.2% 20.0%
55.2%
15.1% 13.3%
11.5%
37.5%
30.1% 36.0%
38.4%
14.0% 10.1%
17.5%
TiO I2
TiO II2 TiO III2
Figure 3C-D
C)
D)
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Tet - +
anti-pY
anti-Syk
anti-GAPDH
- Tet + Tet
Syk-EGFP
Figure 4
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Original ratio
m/z
10
20
30
40
50
60
70
80
90
100
Rela
tive A
bu
nd
an
ce
620.30085z=2
620.80191z=2
623.31890z=2
623.82028z=2
621.30307z=2
624.32154z=2621.80435
z=2
m/z
10
20
30
40
50
60
70
80
90
100
Rela
tive A
bu
nd
an
ce
620.29927z=2
620.80061z=2 623.31807
z=2
623.81976z=2
621.30231z=2
624.32132z=2621.80444
z=2
PolyMAC recovery
NL:
1.99E6
Base Peak
m/z=
620.29590-
620.30210
Genesis
NL:
9.95E5
Base Peak
m/z=
623.31488-
623.32112
Genesis
0 5 10 15 20 25 30 35 40 45 50 55Time (min)
0102030405060708090
1000
102030405060708090
100
Rela
tive A
bu
nd
an
ce
RT: 26.17Area: 54240887
RT: 26.17Area: 27330625
0 5 10 15 20 25 30 35 40 45 50 55Time (min)
0102030405060708090
1000
102030405060708090
100
Rela
tive A
bu
nd
an
ce
RT: 24.39Area: 1927739
RT: 24.39Area: 1138721
NL:
5.59E4
Base Peak
m/z=
620.29590-
620.30210
Genesis
NL:
3.13E4
Base Peak
m/z=
623.31488-
623.32112
Genesis
0 5 10 15 20 25 30 35 40 45 50 55Time (min)
0102030405060708090
1000
102030405060708090
100
Rela
tive A
bu
nd
an
ce
RT: 24.47Area: 322851
RT: 24.40Area: 2311732
NL:
1.01E4
Base Peak
m/z=
620.29590-
620.30210
Genesis
NL:
7.43E4
Base Peak
m/z=
623.31488-
623.32112
Genesis
m/z
10
20
30
40
50
60
70
80
90
100
Rela
tive A
bu
nd
an
ce
623.31770z=2
623.81953z=2
624.32090z=2
620.80065z=2
620.29909z=2
621.30193z=2
TiO2 recovery
Figure 5
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Figure 6
A) B)
30 250 514
+Syk
-Syk
64 kD
4G10 IP
ASC1 WB
37 kDMAPRE1 WB
99 kDSyk WB
180 kDScrib WB
64 kD
64 kDASC1 WB
pY WBASC1 IP
-Syk
lysate
+Syk
lysate
-Syk
eluent
+Syk
eluent
MAPRE1 WB
pY WB
MAPRE1 IP
37 kD
37 kD
Dynactin WB
pY WB
Dynactin IP64 kD
64 kDDynactin WB
64 kD
Scrib WB
pY WB
Scrib IP180 kD
180 kD
GAPDH WB 49 kD
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Cancer, cellular movement Cancer, cell death
Figure 7
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