in-depth analyses of kinase-dependent tyrosine

1

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:

[email protected]

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.

by guest on April 13, 2019

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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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in-depth analyses of kinase-dependent tyrosine

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