Quantitative profiling of protein tyrosine kinases in human cancer cell lines by multiplexed parallel reaction monitoring assays

We discovered an error after this was published as a Paper in Press. Specifically, we discovered that an m / z tolerance setting had inadvertently changed in the filtering software when some of the MS/MS spectra of phosphotyrosine peptides were generated for annotation. This error affected the number of phosphotyrosine sites confidently identified in our study. We wish to withdraw this article and submit a corrected version for review. Protein (PTKs) play key roles in Dysregulation of PTK-activated pathways, often by receptor overexpression, gene amplification, or genetic mutation, is a causal factor underlying numerous cancers. In this study, we have developed a parallel reaction monitoring (PRM)-based assay for quantitative profiling of 83 PTKs. The assay detects 308 proteotypic peptides from 54 receptor tyrosine kinases and 29 nonreceptor tyrosine kinases in a single run. Quantitative comparisons were based on the labeled reference peptide method. We implemented the assay in four cell models: 1) a comparison of proliferating versus epidermal growth factor (EGF)-stimulated A431 cells, 2) a comparison of SW480Null (mutant APC) and SW480APC (APC restored) colon tumor cell lines, and 3) a comparison of 10 colorectal cancer cell lines with different genomic abnormalities, and 4) lung cancer cell lines with either susceptibility (11-18) or acquired resistance (11-18R) to the epidermal growth factor receptor tyrosine kinase inhibitor erlotinib. We observed distinct PTK expression changes that were induced by stimuli, genomic features or drug resistance, which were consistent with previous reports. However, most of the measured expression differences were novel observations. For example, acquired resistance to erlotinib in the 11-18 cell model was associated not only with previously reported upregulation of MET, but also with upregulation of FLK2 and downregulation of LYN and PTK7. Immunoblot analyses and shotgun proteomics data were highly consistent with PRM data. Multiplexed PRM assays provide a targeted, systems-level profiling approach to evaluate cancer-related proteotypes and adaptations. Data are available through Proteome eXchange Accession PXD002706. In this study, we describe a multiplexed PRM-based assay for quantitation of 83 PTKs, which detects 308 proteotypic peptides from 54 receptor tyrosine kinases and 29 nonreceptor tyrosine kinases in a single scheduled run. We demonstrate analysis of PTK expression changes driven by stimulation, genomic abnormalities or drug resistance in tumor cell lines. PTK expression changes are selective and distinct for the models studied. The profiling of PTK expression changes in cell models provides proof of concept for application of the approach to systems-level analysis of PTK signaling and adaptation in cancer.

This article has been withdrawn by the authors.
We discovered an error after this manuscript was published as a Paper in Press. Specifically, we discovered that an m/z tolerance setting had inadvertently changed in the filtering software when some of the MS/MS spectra of phosphotyrosine peptides were generated for annotation. This error affected the number of phosphotyrosine sites confidently identified in our study. We wish to withdraw this article and submit a corrected version for review. This article has been withdrawn by the authors.
We discovered an error after this manuscript was published as a Paper in Press. Specifically, we discovered that an m/z tolerance setting had inadvertently changed in the filtering software when some of the MS/MS spectra of phosphotyrosine peptides were generated for annotation. This error affected the number of phosphotyrosine sites confidently identified in our study. We wish to withdraw this article and submit a corrected version for review. This article has been withdrawn by the authors.
We discovered an error after this manuscript was published as a Paper in Press. Specifically, we discovered that an m/z tolerance setting had inadvertently changed in the filtering software when some of the MS/MS spectra of phosphotyrosine peptides were generated for annotation. This error affected the number of phosphotyrosine sites confidently identified in our study. We wish to withdraw this article and submit a corrected version for review.
completely avoid interfering ions, which can hamper precise and specific protein quantification (12). In addition, MRM relies on a predefined and experimentally validated set of peptides and peptide fragmentations that requires considerable effort to develop (13).
High resolution and accurate mass peptide analysis now can be achieved with new generation mass spectrometers, such as the Q Exactive (ThermoFisher Scientific). These instruments combine the quadrupole precursor ion selection with the high resolution and high accuracy of an Orbitrap mass analyzer. Recent reports describe several modes of operation for targeted peptide analysis, the most powerful of which is termed parallel reaction monitoring (PRM), which generates both high resolution precursor measurements and high-resolution, full scan MS/MS data, from which transitions can be extracted postacquisition (14,15). A key feature of this approach is the highly specific extraction of signals for target peptides of interest, thus reducing interference from nominally isobaric contaminants.
A particularly useful approach to targeted proteome analysis is to configure multiplexed assay panels for proteins and their modified forms involved in specific pathways or networks. Koomen and colleagues first described this approach with their MRM analyses of components of the Wnt signaling pathway (16) and later expanded to multiple signaling pathways (17). Multiplexed MRM assay panels have been used to quantify phosphotyrosine sites in tyrosine kinase signaling networks (18) and to monitor the protein expression status of cellular metabolic pathways (19).
This article has been withdrawn by the authors.
We discovered an error after this manuscript was published as a Paper in Press. Specifically, we discovered that an m/z tolerance setting had inadvertently changed in the filtering software when some of the MS/MS spectra of phosphotyrosine peptides were generated for annotation. This error affected the number of phosphotyrosine sites confidently identified in our study. We wish to withdraw this article and submit a corrected version for review. which detects 308 proteotypic peptides from 54 receptor tyrosine kinases and 29 nonreceptor tyrosine kinases in a single scheduled run. We demonstrate analysis of PTK expression changes driven by stimulation, genomic abnormalities or drug resistance in tumor cell lines. PTK expression changes are selective and distinct for the models studied.
The profiling of PTK expression changes in cell models provides proof of concept for application of the approach to systems-level analysis of PTK signaling and adaptation in cancer.
This article has been withdrawn by the authors.
We discovered an error after this manuscript was published as a Paper in Press. Specifically, we discovered that an m/z tolerance setting had inadvertently changed in the filtering software when some of the MS/MS spectra of phosphotyrosine peptides were generated for annotation. This error affected the number of phosphotyrosine sites confidently identified in our study. We wish to withdraw this article and submit a corrected version for review. This article has been withdrawn by the authors.
We discovered an error after this manuscript was published as a Paper in Press. Specifically, we discovered that an m/z tolerance setting had inadvertently changed in the filtering software when some of the MS/MS spectra of phosphotyrosine peptides were generated for annotation. This error affected the number of phosphotyrosine sites confidently identified in our study. We wish to withdraw this article and submit a corrected version for review. processed in parallel to minimize the effects of systematic errors. Pellets were resuspended in 100 μL 100 mM ammonium bicarbonate (AmBic) and 100 μL trifluoroethanol were added, followed by sonication (3 × 20 s). Samples were incubated at 60°C for 60 min at 1000 rpm on an Eppendorf Thermomixer and sonicated again (3 × 20 s). Protein concentration was estimated with the bicinchoninic acid assay (Pierce, Rockford, IL). Proteins (100 μg for PRM assays; 200 μg for shotgun analyses) were reduced with 40 mM tris(2carboxyethyl)phosphine ( and 100mM dithiothreitol and alkylated 50mM iodoacetamide. Samples were diluted in 50 mM AmBic, pH 8.0 and trypsinized overnight at 37 °C at a trypsin/protein ratio of 1:50, w/w). The resulting peptide mixture was lyophilized overnight and peptides were desalted as described (22).

PRM analysis
PRM analyses were performed on a Q-Exactive mass spectrometer equipped with an Easy nLC-1000 pump and autosampler system (ThermoFisher Scientific). For each analysis, 2 µL of each sample was injected onto an in-line solid-phase extraction column (100 µm x 6 cm) packed with ReproSil-Pur C18 AQ 3 μm resin (Dr. Maisch GmbH) and a frit generated with liquid silicate Kasil 1 and washed with 100% solvent A (0.1 % formic acid) at a flow rate of 2 µL/min. After a total wash volume of 7 µL, the pre-column was placed in-line with a PicoFrit capillary column (New Objective, 11 cm x 75 µm) packed with the same resin.
The peptides were separated using a linear gradient of 2% -35% solvent B (0.1% formic acid in acetonitrile) at a flow rate of 300 nL min -1 over 40 min, followed by an increase to 90% B over 4 min and held at 90% B for 6 min before returning to initial conditions of 2% B.
For peptide ionization, 1800 V was applied and a 250 °C capillary temperature was used.
All samples were analyzed using a multiplexed PRM method based on a scheduled This article has been withdrawn by the authors.
We discovered an error after this manuscript was published as a Paper in Press. Specifically, we discovered that an m/z tolerance setting had inadvertently changed in the filtering software when some of the MS/MS spectra of phosphotyrosine peptides were generated for annotation. This error affected the number of phosphotyrosine sites confidently identified in our study. We wish to withdraw this article and submit a corrected version for review.
inclusion list containing the 314 target precursor ions representing PTK and LRP standard peptides. The full scan event was collected using a m/z 380 -1500 mass selection, an Orbitrap resolution of 17,500 (at m/z 200), target automatic gain control (AGC) value of 3 X 10 6 and a maximum injection time of 30 ms. The PRM scan events used an Orbitrap resolution of 17,500, an AGC value of 1 X 10 6 and maximum fill time of 80 ms with an isolation width of 2 m/z. Fragmentation was performed with a normalized collision energy of 27 and MS/MS scans were acquired with a starting mass of m/z 150. Scan windows were set to 4 min for each peptide in the final PRM method to ensure the measurement of 6-10 points per LC peak per transition.
All PRM data analysis and data integration was performed with Skyline software (23).
Instrument quality control assessment was done as described previously (24). Quantitative analyses were done by the labeled reference peptide (LRP) method we described previously (24) using U-13 C 6 , U-15 N 4 -arginine-labeled alkaline phosphatase (AP) peptide (AAQGDITAPGGA*R), β-galactosidase (BG) peptide (APLDNDIGVSEAT*R), and β-actin (ACTB) peptide (GYSFTTTAE*R) as the reference standard mixture. The standard mixture (25 fmol of eath standard peptide per sample) was added immediately following tryptic digestion. Five transitions for each peptide were extracted from the PRM data. The intensity rank order and chromatographic elution of the transitions were required to match those of a synthetic standard for each peptide measured. Summed peak areas from the five target peptide transitions were divided by the summed peak area for the five reference standard peptide transitions to give a peak area ratio and coefficients of variation (CVs) were calculated across replicates for each treatment.
This article has been withdrawn by the authors.
We discovered an error after this manuscript was published as a Paper in Press. Specifically, we discovered that an m/z tolerance setting had inadvertently changed in the filtering software when some of the MS/MS spectra of phosphotyrosine peptides were generated for annotation. This error affected the number of phosphotyrosine sites confidently identified in our study. We wish to withdraw this article and submit a corrected version for review. This article has been withdrawn by the authors.
We discovered an error after this manuscript was published as a Paper in Press. Specifically, we discovered that an m/z tolerance setting had inadvertently changed in the filtering software when some of the MS/MS spectra of phosphotyrosine peptides were generated for annotation. This error affected the number of phosphotyrosine sites confidently identified in our study. We wish to withdraw this article and submit a corrected version for review. PTK7 (ab55633) and ACTB (ab199622) (Abcam). Membranes were probed with fluorophore-conjugated secondary antibodies (Invitrogen) and proteins were visualized on a fluorescence scanner (LI-COR Odyssey; LIC-COR). Sample loads were normalized for total protein concentration before reducing with dithiothreitol and adding NuPAGE® lithium dodecyl sulfate sample buffer and then were boiled for 7 min at 90°C.
iTRAQ labeling and phosphotyrosine enrichment for analysis of 11-18 and 11-18R cells Peptide labeling with iTRAQ 4plex (AB Sciex) was performed as previously described (25,26). Briefly, for each analysis, 1 X 10 7 cells (equivalent to 400 μg peptide before desalting and labeling) for each cell line was labeled with one tube of iTRAQ 4plex reagent. The 11-18 cells were labeled with the iTRAQ 4-plex as follows: 114-and 116-channels, 11-18 cells; 115-and 117-channels 11-18R cells. Lyophilized samples were dissolved in 60 μL of 500 mM triethylammonium bicarbonate, pH 8.5, and the iTRAQ reagent was dissolved in 70 μL of isopropanol. The solution containing peptides and iTRAQ reagent was vortex mixed and then incubated at room temperature for 1h and concentrated to 40 μL under vacuum.
Samples labeled with four different isotopomeric iTRAQ reagents were combined and evaporated to dryness. Peptides then were dissolved in 400 μL of immunoprecipitation buffer (100 mM Tris, 100 mM NaCl, and 1% Nonidet P-40, pH 7.4) and the pH was adjusted to 7.4 before phosphotyrosine immunoprecipitation.
We discovered an error after this manuscript was published as a Paper in Press. Specifically, we discovered that an m/z tolerance setting had inadvertently changed in the filtering software when some of the MS/MS spectra of phosphotyrosine peptides were generated for annotation. This error affected the number of phosphotyrosine sites confidently identified in our study. We wish to withdraw this article and submit a corrected version for review. added and the mixture was incubated for 8 h at 4°C with gentle mixing by rotation. Antibody conjugated Protein G then was rinsed and iTRAQ 4plex labeled peptides were resuspended in the immunoprecipitation buffer, added to the conjugated Protein G and incubated overnight at 4°C with rotation. Conjugated Protein G agarose was rinsed with 400 μL of immunoprecipitation buffer and 4 × 400 μL of rinse buffer (100 mm Tris, pH 7.4), and peptides were eluted into 70 μL of 100 mm glycine pH 2. Peptides were desalted with Stage Tips (ThermoFisher Scientific).

Phosphotyrosine peptide analysis by LC-MS/MS
Phosphotyrosine peptide separations were performed using an Easy nLC-1000 pump and autosampler system (ThermoFisher Scientific). Injections were done with a 10 μL loop and the injection volume was 5 μL. Phosphotyrosine peptides were separated using a linear gradient of 2% -50% Solvent B (0.1% formic acid in acetonitrile) over 140 min. A Q-Exactive mass spectrometer (ThermoFisher Scientific; Bremen, Germany) equipped with nanoelectrospray was used for phosphotyrosine analysis. The mass spectrometer was This article has been withdrawn by the authors.
We discovered an error after this manuscript was published as a Paper in Press. Specifically, we discovered that an m/z tolerance setting had inadvertently changed in the filtering software when some of the MS/MS spectra of phosphotyrosine peptides were generated for annotation. This error affected the number of phosphotyrosine sites confidently identified in our study. We wish to withdraw this article and submit a corrected version for review. phosphorylation of serine, threonine, and tyrosine residues. MS/MS spectra of tyrosine phosphorylated peptides were manually validated to confirm peptide identification and phosphorylation site localization. Annotated MS/MS spectra for all phosphotyrosine assignments are provided in Supplemental Dataset 1. Phosphotyrosine peptide iTRAQ ratios were normalized based on the mean relative protein quantification ratios obtained from the total protein (i.e. protein expression analysis).

RNA-seq analysis
The RNA samples were sequenced following the protocols recommended by the manufacturer (Illumina). Briefly, poly-A was purified and then fragmented into small pieces.
Using reverse transcriptase and random primers, RNA fragments were used to synthesize the first and second strand cDNAs. Following end repair, addition of an "A" base, adapter This article has been withdrawn by the authors.
We discovered an error after this manuscript was published as a Paper in Press. Specifically, we discovered that an m/z tolerance setting had inadvertently changed in the filtering software when some of the MS/MS spectra of phosphotyrosine peptides were generated for annotation. This error affected the number of phosphotyrosine sites confidently identified in our study. We wish to withdraw this article and submit a corrected version for review.

Experimental design and statistical rationale
For PRM analyses in cell culture experiments, three replicate cell cultures were analyzed for each cell culture and treatment. For 10 colorectal cancer cell lines, two replicate cell cultures were analyzed. Student's t-test was performed with the pair-wise comparisons to determine statistical significance of differences.
This article has been withdrawn by the authors.
We discovered an error after this manuscript was published as a Paper in Press. Specifically, we discovered that an m/z tolerance setting had inadvertently changed in the filtering software when some of the MS/MS spectra of phosphotyrosine peptides were generated for annotation. This error affected the number of phosphotyrosine sites confidently identified in our study. We wish to withdraw this article and submit a corrected version for review.

Development of PTK PRM assay panel
We developed a PRM assay for quantitation of 83 PTKs, which measures proteotypic peptides from 54 receptor tyrosine kinases and 29 nonreceptor tyrosine kinases in a single run. We experimentally optimized PRM transition parameters with chemically synthesized peptides. Target peptides were required to be between 7 and 25 amino acids long and were selected based on uniqueness and anticipated chemical stability. Peptides containing cysteine or methionine residues were not excluded. Although priority was given to peptides that were previously identified in the shotgun data set with high MS/MS spectral quality (21, 30, 31), additional predicted peptides were selected in silico. Each PTK protein was monitored by 3-4 proteotypic peptides. The complete panel contained 308 proteotypic peptides representing the 83 target PTKs (Table S1).
Although stable isotope dilution provides the most precise targeted quantitation method and is least subject to interferences, the cost of high purity isotope-labeled standards for all selected PTK peptides would be prohibitive. We thus employed the LRP method (24), in which a single isotope-labeled peptide serves as a normalization standard for all of the measured peptides. As we have previously reported, LRP-based quantitation provides intermediate precision between label-free analyses and stable isotope dilution-based quantitation (24). Summed peak areas for target peptide transitions were divided by the summed peak area for the LRP standard transitions to give normalized peak area, and coefficients of variation (CVs) were calculated across replicates for each treatment.
This article has been withdrawn by the authors.
We discovered an error after this manuscript was published as a Paper in Press. Specifically, we discovered that an m/z tolerance setting had inadvertently changed in the filtering software when some of the MS/MS spectra of phosphotyrosine peptides were generated for annotation. This error affected the number of phosphotyrosine sites confidently identified in our study. We wish to withdraw this article and submit a corrected version for review.
Although the PRM method monitored 308 target peptides, additional criteria were applied post-analysis to ensure correct identities of the signals attributed to each target peptide.
Accordingly, using Skyline, we verified that the 5 most intense transitions attributed to the target peptide co-eluted and displayed identical chromatographic retention with the synthetic peptide standards and that the order of y-ion fragment intensities matched the order for the synthetic peptide standard. An example of application these criteria to evaluate peptide PRM data are provided in Figure S1.
We applied this PRM assay panel to profile 83 PTK proteins in four cell model studies: 1) a comparison of proliferating versus epidermal growth factor (EGF)-stimulated A431 cells, 2) a comparison of SW480Null (mutant APC) and SW480APC (APC restored) colon tumor cell lines, 3) a comparison of 10 colorectal cancer cell lines with different genomic abnormalities, and 4) a comparison of lung cancer cell lines with either susceptibility or acquired resistance to the epidermal growth factor receptor tyrosine kinase inhibitor erlotinib. We compared PRM-based measurements to spectral count-based estimates and immunoblotting analyses. For the A431 cell model, the SW480 cell model and the 10 CRC cell lines, we used the previously published shotgun datasets (21,30,31). For the lung cancer cell lines study, we performed shotgun analyses for this work, as described In Supplemental Methods. Three replicate cultures of each cell line were collected for analysis. All lysates in each study were prepared together for analysis. Each sample was spiked with all three LRP reference peptide standards and the CV for each was calculated across all analyses in each of the four studies ( Table 1). In each study, the alkaline phosphatase peptide yielded the lowest CV (mean 6.76% across all four studies) and was used for normalization. This article has been withdrawn by the authors.
We discovered an error after this manuscript was published as a Paper in Press. Specifically, we discovered that an m/z tolerance setting had inadvertently changed in the filtering software when some of the MS/MS spectra of phosphotyrosine peptides were generated for annotation. This error affected the number of phosphotyrosine sites confidently identified in our study. We wish to withdraw this article and submit a corrected version for review.

Study 1: EGF stimulation in A431 cells
Recently, we reported protein expression changes produced by EGF stimulation and inhibition in A431 cells, as measured by LC-MS/MS label free shotgun proteomics (31). We employed the same model to study EGF-induced PTK expression changes. Twenty seven PTKs were detected in this study, eight of which showed significant differences (p < 0.05) ( Figure 1A). Although a decrease in EGFR protein upon ligand stimulation is welldocumented (32), other changes we observed appear to be selective and novel, including downregulation of IGF1R, PTK7, AXL and, most notably EPHA1 (0.40 fold, p < 0.0004) and upregulation of SRC, FAK2 and RET.  which was most significantly downregulated by EGF treatment. Thus, the data demonstrate that for a subset of differentially expressed proteins, label free shotgun proteomics data and PRM data are broadly concordant.
We also compared PRM with immunoblotting for selected PTK proteins ( Figure 1B). After treatment with EGF, EGFR phosphorylation at Tyr-998 dramatically increased, which reflects EGFR signaling activation. EGFR, EPHA2, PTK7, and LYN proteins, for which antibodies were commercially available, were chosen for confirmation. The immunoblot results were consistent with shotgun and PRM data. This article has been withdrawn by the authors.
We discovered an error after this manuscript was published as a Paper in Press. Specifically, we discovered that an m/z tolerance setting had inadvertently changed in the filtering software when some of the MS/MS spectra of phosphotyrosine peptides were generated for annotation. This error affected the number of phosphotyrosine sites confidently identified in our study. We wish to withdraw this article and submit a corrected version for review.  (Table S3).
A subset of the PTKs found to be differentially expressed by PRM assay also were assessed by immunoblotting ( Figure 2B). The five proteins, EGFR, EPHA2, SYK, ZAP70, and PTK7, were analyzed in three SW480Null and three SW480APC cultures and the immunoblot results were consistent with shotgun and PRM data.

Study 3: PTK expression related to different genomic abnormalities in 10 CRC cell lines
We recently described shotgun proteomic and integrated proteogenomic analyses of 10 CRC cell lines (21). These cell lines display mutations frequently associated with CRC, including KRAS, APC, TP53, PLK3CA, BRAF, and CTNNB1 ( Table S4). Six of the cell lines display microsatellite instability (MSI) and epigenetic silencing or mutation of the DNA This article has been withdrawn by the authors.
We discovered an error after this manuscript was published as a Paper in Press. Specifically, we discovered that an m/z tolerance setting had inadvertently changed in the filtering software when some of the MS/MS spectra of phosphotyrosine peptides were generated for annotation. This error affected the number of phosphotyrosine sites confidently identified in our study. We wish to withdraw this article and submit a corrected version for review. mismatch repair genes MLH1, MSH2 and MSH6. We analyzed PTKs in three replicate cultures from each cell line and then compared PTK status as a function of genomic characteristics. The results of the PTK measurements are presented in Table S5. We then compared PTK abundance in CRC cell lines based on differences in KRAS, TP53, PIK3CA, BRAF and CTNNB2 mutations and in MSI status.
KRAS mutations impact EGFR signaling (34) and clinical responses to EGFR inhibitors (35, 36). Five of the CRC lines (DLD1, HCT116, HCT15, LOVO, and LS174T) contain both a wild type KRAS allele and a codon 12/13 mutant, whereas SW480 contains two mutant (G12V) alleles. EGFR abundance displayed substantial heterogeneity between KRAS mutant CRC cells. As shown in Figure 3A and Figure S2, EGFR was expressed at uniformly low levels in KRAS wild type cells, whereas the KRAS mutant cell lines SW480 and LOVO expressed EGFR at highest abundance. However, the KRAS mutant cell lines LS174T and HCT15 expressed EGFR at levels similar to KRAS wild type cells. Immunoblot analysis confirmed these differences in EGFR expression ( Figure 3B).
Six of the CRC cell lines (CACO2, COLO205, HT29, SW480, DLD1, and HCT15) have TP53 mutations, whereas four (HCT116, LOVO, LS174T, and RKO) do not. Comparison of PTK profiles for these cells revealed substantial variation in expression of the non-receptor PTK LYN. Figure 3C and Figure S3 shows that LYN which was expressed at uniformly low levels in TP53 mutated cell lines, but at high levels in the TP53wt cell lines LOVO and RKO. Immunoblot analysis of LYN were consistent with the PRM measurements ( Figure   3D).

This article has been withdrawn by the authors.
We discovered an error after this manuscript was published as a Paper in Press. Specifically, we discovered that an m/z tolerance setting had inadvertently changed in the filtering software when some of the MS/MS spectra of phosphotyrosine peptides were generated for annotation. This error affected the number of phosphotyrosine sites confidently identified in our study. We wish to withdraw this article and submit a corrected version for review. microsatellite stable (MSS) (CACO2, COLO205, HT29, and SW480) and MSI (DLD1, HCT116, HCT15, LOVO, LS174T, and RKO) cells. Three PTKs, EPHA4, IGF1R, and SYK displayed significant lower average expression (p < 0.05) in MSI than in MSS cells ( Figure   S4). However, protein abundances of these PTKs were highly variable within MSI and MSS cells. Six CRC cell lines (HT29, DLD1, HCT116, HCT15, LS174T, and RKO) have PIK3CA mutations. We found that PIK3CA mutant cells express on average higher ERBB3 and lower EPHA4, IGF1R, and NTRK3 than in PIK3CA wild type cells (Figure S5), although expression patterns of these three PTKs differed dramatically between PIK3CA mutant and wild type cells. Three of the CRC cell lines (COLO205, HT29, and RKO) have BRAF V600E mutations and displayed elevated ABL1, EPHB4, FAK1, and SRC ( Figure   S6). Six of the CRC cell lines (HT29, SW480, DLD1, HCT15, LOVO, and RKO) had CTNNB1 mutations and displayed decreased FGFR2 and elevated EPHB2, FGFR4, and HCK compared to CTNNB1 wild type CRC cells (Figure S7), although protein abundance of these three PTK was highly variable between cell lines. We discovered an error after this manuscript was published as a Paper in Press. Specifically, we discovered that an m/z tolerance setting had inadvertently changed in the filtering software when some of the MS/MS spectra of phosphotyrosine peptides were generated for annotation. This error affected the number of phosphotyrosine sites confidently identified in our study. We wish to withdraw this article and submit a corrected version for review. differences (p < 0.05). EGFR, FLK2, and MET were increased in 11-18R cells, whereas EPHA1, FAK1, FGFR2, IGF1R, LYN, PGFRA, PTK7, SRC, VGFR2, and YES were decreased ( Figure 4A). PRM analyses also were consistent with measurements for several of the PTKs that were detected in shotgun proteomic analyses (see Supplemental Methods and Table S6). These results also were confirmed by immunoblot analysis (Figure 4B).

Study 4: PTK alterations related to erlotinib resistance in lung tumor cells
Our analyses also detected increased MET abundance in 11-18R. Although MET amplification has not been reported as a characteristic of this particular cell line (20), several studies have demonstrated that in lung adenocarcinoma-derived cells, EGFR inhibition can be overcome by signaling through hepatocyte growth factor (HGF) and MET (37,38). Moreover, MET amplification is associated with acquired resistance to anti-EGFR treatment in patients (39).
To better assess the role of PTK abundance changes versus PTK activation, we performed a phosphotyrosine profiling analysis of the 11-18 and 11-18R cells with a 4-plex iTRAQ reagent ( Figure S8). This analysis identified 226 tyrosine phosphorylation sites on 133 proteins, with 64 phosphorylation sites exhibiting greater than 1.5-fold differences in tyrosine phosphorylation between 11-18 and 11-18R cells ( Table S7) Table 2. Erlotinib resistance in 11-18R cells resulted in multiple phosphorylation changes, with EPHB4, ERBB3, MET, and FAK1 phosphorylation sites increasing and IGF1R phosphorylation sites decreasing. Phosphorylation of MET is consistent with activation of HGF/MET as an adaptation associated with resistance to EGFR TK inhibitors.

This article has been withdrawn by the authors.
We discovered an error after this manuscript was published as a Paper in Press. Specifically, we discovered that an m/z tolerance setting had inadvertently changed in the filtering software when some of the MS/MS spectra of phosphotyrosine peptides were generated for annotation. This error affected the number of phosphotyrosine sites confidently identified in our study. We wish to withdraw this article and submit a corrected version for review. This article has been withdrawn by the authors.
We discovered an error after this manuscript was published as a Paper in Press. Specifically, we discovered that an m/z tolerance setting had inadvertently changed in the filtering software when some of the MS/MS spectra of phosphotyrosine peptides were generated for annotation. This error affected the number of phosphotyrosine sites confidently identified in our study. We wish to withdraw this article and submit a corrected version for review. alterations in PTK expression in multiple cellular model systems and the PRM data are highly consistent with data from western blot and shotgun LC-MS/MS analyses. Although we employed the labeled reference peptide method as a low-cost alternative to stable isotope dilution, other strategies could be employed, such as the intermediate-cost alternative of using low purity stable isotope labeled peptide standards for all analytes (40).
We observed distinct PTK expression changes in all of the cell model systems we studied.
Moreover, our analyses of colon tumor cell lines illustrated the unexpected associations of many cancer-associated mutations with PTK expression, as KRAS, BRAF, TP53, PIK3CA and CTTNB mutations all exerted distinct effects in different cells. Our analyses detected previously reported PTK expression changes induced by EGFR ligand stimulation, APC mutation or acquired resistance to erlotinib, but also identified unanticipated adaptations.
Our PTK analyses failed to detect many PTKs for which evidence of expression was found at the mRNA level. Our analysis approach could be further enhanced by targeted fractionation to selectively enrich PTK peptides of interest (41).
Although our studies were limited to cell culture models, PTK assay panels could be extended to studies of tissues. Signaling networks can be analyzed through measurements of phosphosites, but recent studies indicate that phosphorylation status in tissues is highly sensitive to tissue ischemia (42,43), whereas protein abundance remains unaffected. PTK profiling at the protein expression level thus may provide a robust alternative to study adaptation of signaling networks in human tumors.
This article has been withdrawn by the authors.
We discovered an error after this manuscript was published as a Paper in Press. Specifically, we discovered that an m/z tolerance setting had inadvertently changed in the filtering software when some of the MS/MS spectra of phosphotyrosine peptides were generated for annotation. This error affected the number of phosphotyrosine sites confidently identified in our study. We wish to withdraw this article and submit a corrected version for review. This article has been withdrawn by the authors.
We discovered an error after this manuscript was published as a Paper in Press. Specifically, we discovered that an m/z tolerance setting had inadvertently changed in the filtering software when some of the MS/MS spectra of phosphotyrosine peptides were generated for annotation. This error affected the number of phosphotyrosine sites confidently identified in our study. We wish to withdraw this article and submit a corrected version for review. This article has been withdrawn by the authors.
We discovered an error after this manuscript was published as a Paper in Press. Specifically, we discovered that an m/z tolerance setting had inadvertently changed in the filtering software when some of the MS/MS spectra of phosphotyrosine peptides were generated for annotation. This error affected the number of phosphotyrosine sites confidently identified in our study. We wish to withdraw this article and submit a corrected version for review.
This article has been withdrawn by the authors.
We discovered an error after this manuscript was published as a Paper in Press. Specifically, we discovered that an m/z tolerance setting had inadvertently changed in the filtering software when some of the MS/MS spectra of phosphotyrosine peptides were generated for annotation. This error affected the number of phosphotyrosine sites confidently identified in our study. We wish to withdraw this article and submit a corrected version for review. This article has been withdrawn by the authors.
We discovered an error after this manuscript was published as a Paper in Press. Specifically, we discovered that an m/z tolerance setting had inadvertently changed in the filtering software when some of the MS/MS spectra of phosphotyrosine peptides were generated for annotation. This error affected the number of phosphotyrosine sites confidently identified in our study. We wish to withdraw this article and submit a corrected version for review.
This article has been withdrawn by the authors.
We discovered an error after this manuscript was published as a Paper in Press. Specifically, we discovered that an m/z tolerance setting had inadvertently changed in the filtering software when some of the MS/MS spectra of phosphotyrosine peptides were generated for annotation. This error affected the number of phosphotyrosine sites confidently identified in our study. We wish to withdraw this article and submit a corrected version for review. This article has been withdrawn by the authors.
We discovered an error after this manuscript was published as a Paper in Press. Specifically, we discovered that an m/z tolerance setting had inadvertently changed in the filtering software when some of the MS/MS spectra of phosphotyrosine peptides were generated for annotation. This error affected the number of phosphotyrosine sites confidently identified in our study. We wish to withdraw this article and submit a corrected version for review.
This article has been withdrawn by the authors.
We discovered an error after this manuscript was published as a Paper in Press. Specifically, we discovered that an m/z tolerance setting had inadvertently changed in the filtering software when some of the MS/MS spectra of phosphotyrosine peptides were generated for annotation. This error affected the number of phosphotyrosine sites confidently identified in our study. We wish to withdraw this article and submit a corrected version for review.
This article has been withdrawn by the authors.
We discovered an error after this manuscript was published as a Paper in Press. Specifically, we discovered that an m/z tolerance setting had inadvertently changed in the filtering software when some of the MS/MS spectra of phosphotyrosine peptides were generated for annotation. This error affected the number of phosphotyrosine sites confidently identified in our study. We wish to withdraw this article and submit a corrected version for review.   This article has been withdrawn by the authors.
We discovered an error after this manuscript was published as a Paper in Press. Specifically, we discovered that an m/z tolerance setting had inadvertently changed in the filtering software when some of the MS/MS spectra of phosphotyrosine peptides were generated for annotation. This error affected the number of phosphotyrosine sites confidently identified in our study. We wish to withdraw this article and submit a corrected version for review. This article has been withdrawn by the authors.
We discovered an error after this manuscript was published as a Paper in Press. Specifically, we discovered that an m/z tolerance setting had inadvertently changed in the filtering software when some of the MS/MS spectra of phosphotyrosine peptides were generated for annotation. This error affected the number of phosphotyrosine sites confidently identified in our study. We wish to withdraw this article and submit a corrected version for review.
We discovered an error after this manuscript was published as a Paper in Press. Specifically, we discovered that an m/z tolerance setting had inadvertently changed in the filtering software when some of the MS/MS spectra of phosphotyrosine peptides were generated for annotation. This error affected the number of phosphotyrosine sites confidently identified in our study. We wish to withdraw this article and submit a corrected version for review. This article has been withdrawn by the authors.
We discovered an error after this manuscript was published as a Paper in Press. Specifically, we discovered that an m/z tolerance setting had inadvertently changed in the filtering software when some of the MS/MS spectra of phosphotyrosine peptides were generated for annotation. This error affected the number of phosphotyrosine sites confidently identified in our study. We wish to withdraw this article and submit a corrected version for review. This article has been withdrawn by the authors.

D C
We discovered an error after this manuscript was published as a Paper in Press. Specifically, we discovered that an m/z tolerance setting had inadvertently changed in the filtering software when some of the MS/MS spectra of phosphotyrosine peptides were generated for annotation. This error affected the number of phosphotyrosine sites confidently identified in our study. We wish to withdraw this article and submit a corrected version for review. This article has been withdrawn by the authors.
We discovered an error after this manuscript was published as a Paper in Press. Specifically, we discovered that an m/z tolerance setting had inadvertently changed in the filtering software when some of the MS/MS spectra of phosphotyrosine peptides were generated for annotation. This error affected the number of phosphotyrosine sites confidently identified in our study. We wish to withdraw this article and submit a corrected version for review.