Sensitive Approaches for the Assay of the Global Protein Tyrosine

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Sensitive approaches for the assay of the global protein tyrosine phosphorylation in complex samples using a mutated SH2 domain Yanan Li, Yan Wang, Mingming Dong, Hanfa Zou, and Mingliang Ye Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b03812 • Publication Date (Web): 24 Jan 2017 Downloaded from http://pubs.acs.org on February 1, 2017

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Analytical Chemistry

Sensitive approaches for the assay of the global protein tyrosine phosphorylation in complex samples using a mutated SH2 domain

Yanan Li1,2, Yan Wang1,2, Mingming Dong1,2, Hanfa Zou1,2,#, Mingliang Ye1,2* 1

CAS Key Laboratory of Separation Science for Analytical Chemistry, Dalian Institute of

Chemical Physics, Chinese Academy of Sciences (CAS), Dalian, 116023, China E-mail: [email protected], Fax: +86-411-84379620; Tel: +86-411-84379620, +86-411-84379610 2

University of Chinese Academy of Sciences, Beijing, 100049, China

# Deceased April 25, 2016

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Abstract Temporal tyrosine phosphorylation (pTyr) plays a crucial role in numerous cellular functions. The characterization of the tyrosine phosphorylation states of cells is of great interest for understanding the mechanisms that underlie. In this study, we developed sensitive and cost-effective methods for the assay of the global protein tyrosine phosphorylation in complex samples by using a novel engineered pTyr binding protein, Src SH2 domain triple-point mutant (Trm-SH2). Taking the advantage of the pan-specific interaction of Trm-SH2 to pTyr, a high throughput approach was developed to determine the total protein tyrosine phosphorylation level in a sample. This method allowed the detection of 0.025 ng tyrosine phosphorylated proteins. The Trm-SH2 was further exploited to develop method to profile the global tyrosine phosphorylation state. When this approach was applied to analyze the tyrosine phosphoproteome upon stimulation, distinct patterns were observed. This approach is readily to be used in many research and clinical fields for the analysis of tyrosine phosphorylated proteins in complex samples, including classifying aberrant phosphotyrosine-dependent signaling in cancer.

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INTRODUCTION Tyrosine phosphorylation signaling system involves in numerous cellular functions, including differentiation, proliferation, motility and apoptosis1, when deregulated, it can result in disease, most notably cancer, in which tyrosine kinases are now the major class of drug targets2. Accordingly, development of assay methods for elucidating tyrosine phosphorylation allowing the comprehensive detection of activated signaling system is essential for the understanding of many physiological and pathological processes of signal transduction3. The global characterization and quantitative analysis of tyrosine phosphorylated proteins are challenging. Tyrosine phosphorylation only accounts for ∼0.05% of total protein phosphorylation, and it is a very transient and dynamical event4, those facts make its analysis difficult. Immunological methods using anti-phosphotyrosine antibodies are the dominant methods for the detection or enrichment of tyrosine phosphorylated proteins4-7. In the past decade, liquid chromatography coupled with mass spectrometry (LC-MS) has evolved into a powerful tool for proteomics analysis. LC-MS analysis of phosphotyrosyl (pTyr) peptides enriched by anti-phosphotyrosine antibodies from the protein digest of total cell lysate could identify hundreds of pTyr sites on numerous proteins7. However, many research groups do not have access to such highly specialized instruments. As alternatives to the comprehensive analysis by MS-based method, chemiluminescent, colorimetric or fluorometric methods in western blotting or enzyme-linked immunosorbent assay (ELISA) format are often used to assay the phosphorylation-dependent signaling. These methods rely heavily on the quality and accessibility of the antibodies. There are several pan-specific anti-phosphotyrosine antibodies, including PT66, anti-pTyr-4G10, PY99, P-Tyr-100, which are commercially available. However, they are often suffering from poor reproducibility, especially between different batches, and bias in recognizing surrounding sequences. Besides, antibodies are too expensive to use liberally. Since 1986, followed by the discovery of the Src Homology 2 (SH2) domain, which selec3



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tively recognize specific phosphotyrosyl motifs8-10, researchers have developed many strategies to profile the global tyrosine phosphorylated state, such as far-western blotting11-16, SH2 rosette assay11, 14-16, oligonucleotide-tagged multiplex (OTM) assay3, 14, etc. The SH2 domains based approaches are promising alternatives for the antibody-based tyrosine phosphorylated proteins analysis approaches. Unfortunately, due to the transient and dynamic protein-protein interactions occurring in a cell, the natural SH2-pTyr ligand interaction, which has a Kd in the 0.1 to 10 M range, is moderate in strength17, and the affinity is much lower than that of antibody-antigen interaction. The low binding affinity makes these methods less efficient. Besides, the human genome encodes about 121 SH2 domains in 111 proteins18, and each SH2 domain has different binding properties for phosphotyrosyl peptides with high specificity19. Thus the approaches using SH2 domains can only be used to detect specific types of tyrosine phosphorylated proteins, and cannot be used to detect the level of global protein tyrosine phosphorylation. Recently, we found that the SH2 domain-derived phosphotyrosine superbinder was a good replacement for anti-phosphotyrosine antibodies for the enrichment of pTyr peptides from complex protein digest for proteomics analysis20. The superbinder was obtained by introducing three mutations into its pTyr-binding pocket, which made the resulting mutated SH2 domains exhibited nano-to-micromolar affinities to the pTyr-containing peptides21. The markedly enhanced affinity made the superbinder almost pan-specific to pTyr residue. We have demonstrated that this superbinder enabled deeper and broader coverage of the Tyr phosphoproteome than conventional pan-specific anti-phosphotyrosine antibodies20. In that study, we identified ~20,000 distinct pTyr peptides and more than 10,000 pTyr sites from nine human cell lines22, resulted in the unprecedented deep coverage for Tyr phosphoproteome analysis. With the success of using the SH2 superbinder as the affinity reagent for Tyr phosphoproteome analysis, we expect that the phosphotyrosine superbinder could be used to detect the protein tyrosine phosphorylation in complex sample using the chemiluminescent 4


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methods in far-western blotting or ELISA format. Indeed, sensitive approaches for the assay of the global protein tyrosine phosphorylation in complex sample using the superbinder were successfully developed in this study. This new approach, which shows good specificity, high affinity, and low cost compared to antibody-based approach, can be employed to measure either total protein tyrosine phosphorylation levels of cell lysates or global changes in protein tyrosine phosphorylation levels of cell lysates.

EXPERIMENTAL SECTION Materials and Reagents. E. coli DH5α and lysozyme were purchased from Takara. Antibodies of phospho-tyrosine mouse mAb (P-Tyr-100), phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) antibody, p44/42 MAPK (Erk1/2) (137F5) rabbit mAb and phospho-EGF receptor antibody sampler kit were from cell signaling technology. GST tag polyclonal antibody and rabbit anti GAPDH monoclonal antibody were purchased from proteintechTM. Antibodies of goat anti-rabbit IgG H&L (HRP) and rabbit anti-mouse IgG H&L (HRP) were purchased from abcam. RPMI1640 medium, penicillin, streptomycin and fetal bovine serum were purchased from Gibco Invitrogen (Car1sbad, USA). Tryptone, yeast extract powder and agarose were purchased from AOBOX biotechnology. Ni-NTA (Ni-nitrilotriacetic acid) beads were from GE Healthcare. All water used in these experiments was prepared using a Mill-Q system (Millipore, Bedford, MA). All other chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA). Expression and purification of N-terminal His-GST-tagged SH2 fusion protein. Bacterial expression vectors that express wide type Src SH2 domain (Wt-SH2) and Src SH2 domain triple-point mutant (Trm-SH2, superbinder) were gifts from Shawn S. C. Li, University of Western Ontario, London. The expression of these fusion proteins were conducted by standard protein expression method. Briefly, E. coli cultures were grown in Lu5



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ria-Broth (LB) medium with 50 g/ml kanamycin to an OD600 of 0.6-0.8 and then grown with 200 M isopropyl β-D-1-thiogalactopyranoside (IPTG) overnight at 16°C, next harvested, and tied by centrifugation at 6,500 g for 30 min at 4°C. The cultures were re-suspended in chilled lysis buffer containing 0.05% NT-40, 1% protease inhibitor cocktail (v/v) and 1mg/ml lysozyme in phosphate-buffered saline (PBS) solution (pH7.4), and the lysate was sonicated and centrifuged. Subsequently, the supernatant was purified with Ni-NTA beads and then the size exclusion chromatography. Finally, protein concentration was determined by the bicinchoninic acid (BCA) assay, and the purity was confirmed by SDS-PAGE (Figure S1). Cell culture, stimulation and cell lysis. HeLa cells were grown in RPMI1640 medium supplemented with 10% fetal bovine serum, 1% penicillin and streptomycin in a humidified atmosphere of 5% CO2 in air at 37°C. HeLa cells about 80% confluency were starved in medium without serum overnight, then the serum-starved HeLa cells were untreated or treated with epidermal growth factor (EGF) at a final concentration of 100 ng/ml for 1, 3, 5, 10, 20 min, respectively. Subsequently, the cells were washed with ice-cold PBS and extracted with chilled lysis buffer (Strong RIPA, 2% protease inhibitor cocktail (v/v), 1 mM Na3VO4, 1 mM NaF, 1 mM Na4P2O7, 1 mM β-glycerophosphate disodium), and then the lysate was sonicated and centrifuged at 25,000 g for 30 min at 4°C. Finally, the protein concentration of the supernatant was determined by BCA assay, and the total cellular proteins were frozen in -80°C。 Dephosphorylation of phosphoproteins HeLa cell lysates (750 g in 0.75 mg/ml) obtained from the cells stimulated with EGF for 20 min were incubated with 200 units of alkaline phosphatase (ALP) from bovine intestinal mucosa in 1*ALP buffer (20 mM Tris-Cl, 150 mM NaCl, 1 mM MgCl2, 0.1 mM ZnCl2, pH 8.0) at 37°C. An aliquot (100 L) was taken from the reaction solution at 0, 3, 5, 10, 20, 30, 60 min and the enzymatic reaction activity was stopped by adding the Na3VO4 to the final 6


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concentration of 8 mM and incubating at 37°C for 5 min. The resulting protein mixture for each time point was stored at -80 °C for further analysis. Dot-blot analysis for standard tyrosine phosphorylated proteins. Dot-blot analysis of standard tyrosine phosphorylated proteins was performed as described15-16, 23-24. 1-4 l of serial dilutions of phosphotyrosine-BSA, BSA, β-casein or the mixtures of all of them were spotted on nitrocellulose membranes (0.45m, BIO-RAD, USA) and allowed to dry for 1-2 h at room temperature (RT), followed by blocking with blocking buffer of 5% skimmed milk in TBST (0.1% Tween-20, 150 mM NaCl, 20 mM Tris-HCl, pH 8.0) for 2 h at RT. The membranes were then incubated with 2 g/ml Trm-SH2, 2 g/ml Wt-SH2, and P-Tyr-100 diluted 1:1000 in blocking buffer overnight at 4°C, respectively. After washing three times, for Trm-SH2 and Wt-SH2 based approaches, the membranes were then incubated at RT for 1 h with GST tag polyclonal antibody diluted 1:15000 in blocking buffer, for P-Tyr-100 based approach, the membrane was incubated at RT for 2 h with relevant HRP-labelled secondary antibody diluted 1:10000 in blocking buffer. Finally, for P-Tyr-100 based approach, after washing, HRP substrate (Western BrightTM Peroxide, Advansta, USA) was added before the detection by BioImaging systems (Fusion FX5-XT). Meanwhile, for the Trm-SH2 and Wt-SH2 based approaches, the membranes were incubated at RT for 1h with relevant HRP-labelled tertiary antibody diluted 1:10000 in blocking buffer before the addition of the HRP substrate. Trm-SH2 based ELISA detection of tyrosine phosphorylated proteins. Trm-SH2 based ELISA detection of tyrosine phosphorylated proteins was performed as described25-26. Standard proteins (e.g. phosphotyrosine-BSA) or HeLa cell lysates, were immobilized onto a polystyrene 96-well microplate (Corning® 96 well white flat bottom polystyrene high bind microplate) at the designated amount in 100 l of carbonate buffer, pH 9.6, at 37°C for 2 h. Then the solution was removed and the wells were washed three times with 200 l of TBST (0.05% Tween-20, 150 mM NaCl, 20 mM Tris-HCl, pH 7.4), 7



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and blocked with 200 l blocking buffer of 5% skimmed milk in TBS for 1 h at 37°C. Subsequently, each well was incubated with 100 l 2 g/ml Trm-SH2 or P-Tyr-100 diluted 1:1000 in blocking buffer at 37°C for 2 h. While for negative controls, 100 l blocking buffer was added. After washing as above, for Trm-SH2 and Wt-SH2 based approaches, the wells were then incubated at RT for 1 h with GST tag polyclone antibody diluted 1:15000 in blocking buffer, for P-Tyr-100 based approach, the wells were incubated at RT for 2 h with relevant HRP-labelled secondary antibody diluted 1:10000 in blocking buffer. Finally, after washing, for P-Tyr-100 based approach, 100l of the HRP substrate (SuperSignal® ELISA Pico Chemiluminescent Substrate, Thermo, USA) was added, and the plate was agitated for 1 min before detecting the signal (BioTek® Synergy H1/H1M microplate reader). For the Trm-SH2 based approach and the negative control, the wells were incubated at 37°C for 1 h with 100 l of relevant HRP-labelled tertiary antibody diluted 1:10000 in blocking buffer before the addition of the HRP substrate. Trm-SH2 based Western blotting detection of tyrosine phosphorylated proteins. Trm-SH2 based Western blotting detection of tyrosine phosphorylation proteins was performed as described15, 27. HeLa cell lysate containing 30 g protein was run alongside on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), transferred onto a PVDF membrane (0.45 m, Millipore, USA). Then the membrane was blocked with blocking buffer of 5% skimmed milk in TBST (0.1% Tween-20, 150 mM NaCl, 20 mM Tris-HCl, pH 8.0) for 2 h at RT. Subsequently, for the primary antibodies binding (2 g/ml Trm-SH2, 2 g/ml Wt-SH2, P-Tyr-100 diluted 1:1000 in blocking buffer), the reactions were carried out overnight at 4°C, at the same time, for the negative control, only blocking buffer was added. Next, followed by washing 3 times, the membranes were incubated for 1 h at RT with GST tag polyclone antibody diluted 1:15000 in blocking buffer or relevant HRP-labelled secondary antibody diluted 1:10000 in blocking buffer. Finally, for P-Tyr-100 based approach, after washing, HRP substrate (Western BrightTM Peroxide, Advansta, USA) 8


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was added and the bands were detected by BioImaging systems (Fusion FX5-XT). For the Trm-SH2, Wt-SH2 based approaches and the negative control, the membranes were incubated at RT for 1 h with relevant HRP-labelled tertiary antibody diluted 1:10000 in blocking buffer before the addition of the HRP substrate.

RESULTS AND DISCUSSION Profiling the global tyrosine phosphorylation state by using Trm-SH2. There are more than 100 SH2 domains in the human genome, and different SH2 domains bind to different classes of pTyr sites18-19. Due to their different specificities, the profiling of the global tyrosine phosphorylation state using different SH2 domains often resulted in different patterns. In other words, many pTyr sites in a cell lysate cannot be detected by using only one SH2 domain. To profile the global tyrosine phosphorylation state in a cell lysate requires the reagent to recognize all the pTyr sites. Trm-SH2 is almost pan-specific to pTyr sites as it has exhibited submicromolar Kd values for the GGpYGG peptide where the Gly residues give minimum contribution to binding due to no side-chain21. We found recently that the superbinder Trm-SH2 was able to enrich almost all type of pTyr peptides from protein digest, which resulted in the deepest coverage for tyrosine phosphoproteome analysis20. Therefore, the Trm-SH2 could be a promising reagent to profile the global tyrosine phosphorylation state of cells. The scheme to profile the tyrosine phosphorylation state using Trm-SH2 was given in Figure 1a. Cell lysates are first run on SDS-PAGE and then transferred onto a membrane. Subsequently, the membrane is incubated with Trm-SH2, which binds specific to pTyr sites on the proteins. Because there is a GST tag on the Trm-SH2, the membrane is further incubated with GST antibody to recognize the bound Trm-SH2 followed by incubating with HRP labelled tertiary antibody for detection. Distinct patterns should be observed by chemiluminescent detection when HRP substrate is added.

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Dot-blot experiments were first performed to investigate the performance of this assay method. Three protein standards, Tyr phosphoprotein phosphotyrosine-BSA (pY-BSA), non-phosphoprotein BSA and Ser/Thr phosphoprotein β-casein, were used as the test samples. These proteins with different amounts were separately spotted onto NC membranes and detected with Trm-SH2. Though the exposure time was extended to 12 s for over-exposure to maximize the detection sensitivity, this method could only detect Tyr phosphoprotein pY-BSA but not the non-phosphoprotein BSA or the Ser/Thr phosphoprotein β-casein (Figure 2a). Above result indicated that this assay method is specific to tyrosine phosphorylated protein. It should be mentioned that the Trm-SH2 was derived from Src SH2, which has the specificity to bind pY-E-x-[L/I]-K motif20-21. However, the pY-BSA was prepared by conjugating the O-phospho-L-tyrosine to BSA, which clearly does not have such motif. The binding of Trm-SH2 to pY-BSA also indicated that it recognizes the pTyr sites in a pan-specific fashion, which is important to detect the global protein tyrosine phosphorylation. We then investigated if this assay method is able to detect pTyr protein in protein mixture. The mixtures of above three proteins with different amounts were spotted onto NC membranes and detected with Trm-SH2. The exposure time was kept short (2 s) to keep the linear relationship between the amounts of pTyr protein and the signal intensity of the spots. It was found the intensity of the spots were only correlated with the amount of pY-BSA but independent on other two proteins (Figure 2b), which further indicated the superior specificity of Trm-SH2 to pTyr protein. Then, we compared the sensitivity of using three different reagents, Trm-SH2, WT-SH2 and P-Tyr-100, to detect pY-BSA for dot-blot experiments. Serial dilutions of pY-BSA ranging from 1 ng to 100 ng were spotted on NC membranes and detected with Trm-SH2, Wt-SH2 and P-Tyr-100, respectively. With the same exposure time (50 s), Trm-SH2 based approach had the highest sensitivity (Figure 2c), which could detect the signal as little as 5 ng of pY-BSA. It is not surprise that the Wt-SH2

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hardly detected the pY-BSA as it lacks the preferred motif for Wt-SH2 (Figure 2c), resulted in the lowest sensitivity. In the above dot-blot experiments, only one pTyr protein was used. To further validate the specificity of Trm-SH2, we used a much more complex sample, the protein digest of HeLa cells, in which many pTyr peptides coexisted with huge amount of interference peptides. This sample was subjected to affinity purification using immobilized Trm-SH2 followed with LC-MS/MS analysis20. For comparison, the analysis using P-Tyr-100 was performed in parallel. As seen in Table S1, the enrichment specificities for Trm-SH2 and P-Tyr-100 were similar, both close to 80%. Considering extremely low-abundance of pTyr, specificity of 80% was impressive. Moreover, Trm-SH2 identified more pTyr peptides, indicating higher enrichment efficiency. Thus, this approach should be applicable to real complex sample like total cell lysate. The Trm-SH2 was used to profile the global tyrosine phosphorylation state in HeLa cells in a far-western blotting format. HeLa cell lysates were obtained from the cells untreated or treated with EGF for 20 min, and then the same amounts of proteins were separated by the SDS-PAGE, transferred onto a membrane, and blotted independently using Trm-SH2, Wt-SH2, P-Tyr-100 or directly GST polyclonal antibody as the control. It can be seen from Figure 3, a significant increase in signal was observed for all three approaches, which was consistent with the fact that, tyrosine phosphorylation level increases significantly after EGF stimulation27-29. Above results indicate that these methods were all response to the tyrosine phosphorylation level. However, the patterns were quite different. It is obviously that the detection with Trm-SH2 showed much more bands, which was also in agreement with the phosphoproteomics data, indicating higher coverage and sensitivity could be obtained. Hence, the features of cost-effectiveness, high affinity and pan-specific binding make this Trm-SH2 based approach a promising method to profile the global tyrosine phosphorylation state of a complex sample. Determining total protein tyrosine phosphorylation level by using Trm-SH2. 11



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There are some well-established approaches, e.g. the BCA protein assay, to determine the total protein amount in a complex sample. It is equally important to measure the total protein phosphorylation level in individual samples. However, such method is seldom reported due to lacking of pan-specific reagents recognizing the phosphorylation sites. Recently, Tao et al presented an interesting approach to measure the total protein phosphorylation level using a reagent termed pIMAGO that is multi-functionalized with titanium ions for its superior selectivity towards phosphorylated sites25-26, 30. Tyrosine phosphorylation plays an important role for regulating biological processes. However, Tao’s method was unable to differentiate the phosphorylation occurred on Ser, Thr or Tyr and therefore cannot be used to determine the total tyrosine phosphorylation level. As Trm-SH2 is almost pan-specific to pTyr sites21, it could be a promising reagent to measure the total protein tyrosine phosphorylation level in complex sample. For this purpose, we designed an ELISA approach (Figure 1b). The samples are immobilized onto a polystyrene 96-well microplate. After blocking, Trm-SH2 is added followed with the addition of GST antibody and then HRP labelled tertiary antibody for detection. Between each step, the plate is washed with a mild detergent solution to remove any proteins or antibodies that are non-specifically bound. After the final wash step, the plate is detected by adding an enzymatic substrate to produce a chemiluminiscence signal, which allows the determination of pTyr protein amount in the sample. We firstly investigated the quantitative performance of this method using pY-BSA as the test sample. pY-BSA with serial dilutions ranging from 0.005 to 2.5 ng and from 0.01 ng to 5 ng was immobilized onto the plate for measurement using Trm-SH2 and P-Tyr-100, respectively. As shown in Figure 4a and 4b, the limit of detection (LOD) defined by a signal-to-noise ratio 3 for Trm-SH2 based approach was determined to be 0.025 ng (Figure 4a), which was nearly 4 fold lower than that of P-Tyr-100 based approach (0.1 ng). The data points above the LOD had excellent linearity (r2>0.99) for both methods. To investigate the up limit of the linear working range, more pY-BSA was immobilized onto the plate for the 12


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measurement. To prevent the saturation of signal, short integration time and small value of gain were used. It was found that the up limits were about 100 ng for both approaches (Figure S2). Thus the linear working range for the Trm-SH2 based approach was from about 0.05 ng to 100 ng, spanning over four orders of magnitude. We then investigated if this method could be used to quantify the total protein tyrosine phosphorylation in complex sample. HeLa cells were treated with EGF for 1 min and the obtained cell lysate was used as the sample. Serial dilutions of proteins ranging from 25 ng to 500 ng were added to different wells and detected with Trm-SH2, resulting in outstanding linearity (r2>0.99) (Figure 4c). Then HeLa cell lysate obtained from the cells treated with EGF for 20 min was treated with alkaline phosphatase, each assay time point containing 375 ng protein was also immobilized onto a microplate and detected with Trm-SH2. It was found that the signal decreased rapidly (Figure 4d) , indicating the signal truly depended on phosphorylation. To mimic the complex samples with different tyrosine phosphorylation levels, different amounts of pY-BSA were spiked into the EGF untreated HeLa cell lysates (200 ng). As shown in Figure 5a, the signal increased linearly (r2>0.99) with the amount of pY-BSA spiked in, indicating this method is able to quantify the total protein tyrosine phosphorylation in complex sample. Thus this approach could be a rapid approach to determine the total protein tyrosine phosphorylation level in complex sample. Temporal analysis of the protein tyrosine phosphorylation in HeLa cells after stimulation with EGF. Protein tyrosine phosphorylation is a highly dynamical post-translational modification29. Take the cells stimulated with epidermal growth factor (EGF) as an example. EGF signaling begins with activation of the EGF receptor by tyrosine phosphorylation and extends through a cascade of downstream kinases to mediate the phosphorylation of a large number of substrate proteins28. Several MS studies have focused on the global tyrosine phosphorylated “early events” after growth-factor stimulation27-28,

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. These approaches employed the

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immunoprecipitation by anti-phosphotyrosine antibodies, and large numbers of tyrosine phosphorylation sites were identified and quantified as function of stimulus, time, and subcellular location, providing valuable dynamical information of tyrosine phosphorylated proteins and closely associated binders. While it needs large amounts of proteins, and more importantly, it is often limited to analyze the dynamical processes with less than 3 time points due to the high cost and low throughput of the approach. However, the approaches developed in this study are fast, cost-effective and of high throughput. The assay method to determine total protein tyrosine phosphorylation level is performed in a polystyrene 96-well microplate, which could easily determine numerous samples from different time points. The assay method to profile the global tyrosine phosphorylation state is performed in SDS-PAGE, which could easily run multiple samples in parallel. These two assay methods were applied for temporal analysis of the protein tyrosine phosphorylation in HeLa cells after stimulation with EGF. We first measured the dynamical change in total protein tyrosine phosphorylation level using Trm-SH2. HeLa cells were untreated or treated with EGF for 1, 3, 5, 10 and 20 min, respectively, generating a six-point dynamical profile. The activation of EGFR signal pathway was verified by the antibodies of p44/42 MAPK (Erk1/2) (137F5), phospho-p44/42 MAPK (Erk1/2) (Figure S3a) and phospho-EGF receptor antibody sampler kit (Figure S3b)27-28, 32. The obtained cell lysates corresponding to 200 ng of proteins were added to different wells and detected with Trm-SH2. As illustrated in Figure 5b, a significant increase in signal was observed when the cells were treated with EGF for 1 min, and then the signal decayed afterwards. This means the total protein tyrosine phosphorylation level peaks at 1 min. Clearly, the dynamical change in total protein tyrosine phosphorylation level could be easily determined by this method. The chemiluminescence intensity also depends on the assay conditions, e.g. the amount of sample loaded, the incubation time, etc., which makes the comparison between assays difficult. Using the calibration curve (Figure 5a) generated 14


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by spiking pY-BSA into 200 ng EGF untreated HeLa cell lysate under the same assay conditions, the chemiluminescence intensities could be normalized to the amount of pY-BSA (Figure 5a). Therefore, the total pTyr amount in the HeLa cell lysates could be expressed as the equivalent amount of pY-BSA. For example, the total pTyr amounts in the lysate treated with EGF for 1 min and 20 min were equivalent to 2.56 ng and 1.67 ng pY-BSA, respectively. After normalization, the total pTyr amounts determined under different conditions should be comparable. We then determined the global tyrosine phosphorylation state profile by far-western blotting using Trm-SH2. EGF-treated HeLa cell lysates corresponding to 30 g of proteins were run on SDS-PAGE, transferred onto a membrane, and blotted using Trm-SH2. The results were shown in Figure 5c. Patterns changed significantly after the stimulation. Consistent with total protein tyrosine phosphorylation level obtained by ELISA, the intensity of the staining and the number of bands peaked at 1 min. The total dynamical changes of tyrosine phosphorylation were also in agreement with previous studies27-28, 32. Because the sites on different proteins were phosphorylated for different stages with stimulation, the patterns were distinct. If the samples are separated by two-dimensional electrophoresis (2-DE) and then detected by Trm-SH2, higher resolution patterns could be obtained, which could be used to infer the signal states of the samples or classify different cancer samples.

CONCLUSION In summary, we developed a novel approach using the engineered pTyr recognizing Src SH2 superbinder domain (Trm-SH2) for the global characterization of tyrosine phosphorylated proteins in complex samples. This approach showed good specificity, high affinity, and low cost compared with the antibody-based approach. It can be employed to measure either the total protein tyrosine phosphorylation levels in complex samples or the tyrosine phosphorylation profiles of different cell states. This approach is widely applicable to the 15



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analysis of tyrosine phosphorylated proteins in many research and clinical fields, including classifying aberrant phosphotyrosine-dependent signaling in cancer.

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] (M.L. Ye);

ACKNOWLEDGEMENTS This work was supported, in part, by funds from the China State Key Basic Research Program Grants (2016YFA0501402, 2013CB911202), the National Natural Science Foundation of China (21235006, 21321064, 21535008, 81430072, 81361128015). MY is a recipient of the National Science Fund of China for Distinguished Young Scholars (21525524).

SUPPORTING INFORMATION AVAILABLE Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.

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Figure captions Figure 1. Schematics for (a) profiling the global tyrosine phosphorylation state and (b) determining total protein tyrosine phosphorylation level by using Trm-SH2. Figure 2. Dot-blot experiments for tyrosine phosphorylated proteins. (a,b) Serial dilutions of Tyr phosphoprotein pY-BSA, Ser/Thr phosphoprotein β-casein, non-phosphoprotein BSA or the mixtures of all of them were spotted on NC membranes and detected with Trm-SH2. (c) The membranes were blotted with Trm-SH2, Wt-SH2 and P-Tyr-100 and detected with the same exposure time. Figure 3. Profiling the global tyrosine phosphorylation state by using Trm-SH2. HeLa cell lysates untreated or treated with EGF for 20 min were separated by SDS-PAGE, transferred onto membranes, and detected independently using GST antibody (control), Trm-SH2, Wt-SH2 or P-Tyr-100 (the exposure time was 5 s, 5 s, 5 s, 60 s, respectively). Figure 4. Quantitative performance of the approach to determine the total protein tyrosine phosphorylation level by using Trm-SH2. (a) The limit of detection of Trm-SH2 based approach for the detection of pY-BSA (shown as Mean ± SD, n=3; integration time: 1s; gain: 135). (b) The limit of detection of P-Tyr-100 based approach for the detection of pY-BSA (shown as Mean ± SD, n=3; integration time: 1s; gain: 135). (c) Quantitation of chemiluminescence signal from Trm-SH2 based approach of EGF-treated HeLa cell lysates (shown as Mean ± SD, n=3; integration time: 1s; gain: 135). (d) EGF stimulated HeLa cell lysates were treated with alkaline phosphatase for the indicated times and each assay time point was immobilized onto a microplate and detected with Trm-SH2 (shown as Mean ±SD, n=3; integration time: 1s; gain: 135). Figure 5. Temporal analysis of tyrosine phosphorylated protein changes in HeLa cell lysates. (a) Calibration curve of pY-BSA generated by spiking it into HeLa cell lysates (shown as Mean ± SD, n=3, integration time: 1s; gain: 129). (b) Determining the total protein tyro18


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sine phosphorylation levels of EGF treated HeLa cell lysates using Trm-SH2 (shown as Mean ± SD, n=3, integration time: 1s; gain: 129). (c) The global tyrosine phosphorylation state profile of EGF-treated HeLa cell lysates by far-western blotting using Trm-SH2 (the exposure time was 18s).

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Figure 1.

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Figure 2.

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Figure 3.

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Figure 4.

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Figure 5.

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Sensitive Approaches for the Assay of the Global Protein Tyrosine

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