One-Step SH2 Superbinder-Based Approach for Sensitive Analysis of

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One-step SH2 superbinder based approach for sensitive analysis of tyrosine phosphoproteome Yating Yao, Yan Wang, Shujuan Wang, Xiaoyan Liu, Zhen Liu, Yanan Li, Zheng Fang, Jiawei Mao, Yong Zheng, and Mingliang Ye J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/acs.jproteome.9b00045 • Publication Date (Web): 15 Mar 2019 Downloaded from http://pubs.acs.org on March 18, 2019

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Journal of Proteome Research

One-step SH2 superbinder based approach for sensitive analysis of tyrosine phosphoproteome Yating Yao1,2, Yan Wang1,2, Shujuan Wang3, Xiaoyan Liu1,2, Zhen Liu1,2, Yanan Li1,2, Zheng Fang1,2, Jiawei Mao1,2, Yong Zheng3*, Mingliang Ye1,2*

1CAS

Key Laboratory of Separation Sciences for Analytical Chemistry, National

Chromatographic R&A Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences (CAS), Dalian 116023, China; 2University 3State

of Chinese Academy of Sciences, Beijing 100049, China;

Key Laboratory of Proteomics, Beijing Proteome Research Center, National

Center for Protein Sciences (Beijing), Beijing Institute of Lifeomics, Beijing 102206, China * To

whom correspondence should be addressed:

（ Y. Zheng ）

Phone: +86-010-61777070. Fax ： +86-010-61777050. Email ：

[email protected] (M.L.

Ye)

Phone:

+86-411-84379610.

Fax:

+86-411-84379620.

[email protected].

1

ACS Paragon Plus Environment

E-mail:

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Abstract Tyrosine phosphorylation plays a major role in regulating cell signaling pathways governing diverse biological functions such as proliferation and differentiation. Systemically mapping phosphotyrosine (pTyr) sites is the key to understand molecular mechanisms underlining pTyr-dependent signaling. Although mass spectrometry-based technologies have been widely used for pTyr site profiling and quantification, their applications are often hindered by the poor efficiency in current multi-step enrichment procedures for inherently low abundance pTyr peptides, especially under physiological conditions. Taking advantage of the sequence-independent high affinity of SH2 superbinder towards pTyr residues, we have developed a simplified one-step pTyr peptide enrichment method that uses immobilized SH2 superbinder for unbiased and robust enrichment of endogenous pTyr peptides from biological samples. By eliminating the prerequisite global phosphopeptide enrichment step in our previously developed two-step method, we minimized sample loss and improved peptide capture efficiency. Applying this method to Jurkat cells at resting state, where the tyrosine phosphorylation level is low, both the number of identified pTyr peptides and sites are increased by three folds comparing to the two-step method. Specifically, we were able to identify 511 non-redundant pTyr peptides, corresponding to 403 high confidence pTyr sites, from Jurkat cells with high level technical reproducibility (Pearson’s correlation coefficient as high as 0.94). Further applying this method to two human breast cancer cell lines, BT474 and HCC1954, before and after EGF stimulation, we demonstrated that this approach could be a powerful tool for illustrating pTyrdependent signaling network controlling cellular behaviors such as drug resistance.

Keywords: Phosphoproteomics, SH2 superbinder, protein tyrosine phosphorylation, phosphopeptide enrichment, LC−MS/MS

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Introduction Dynamic protein tyrosine phosphorylation represents a fundamentally important biochemical event at the cross roads of a wide range of cellular signaling pathways. Aberrant regulation of tyrosine phosphorylation is often implicated in the formation and development of malignant tumors as well as other diseases (1-3). The more pivotal role of tyrosine phosphorylation, compared with other types of phosphorylation such as serine/threonine phosphorylation, is manifested by the fact that many tyrosine kinases (TKs), the mediator of tyrosine phosphorylation, are attractive drug targets and a number of TK inhibitors have been successfully applied for cancer therapy (4, 5). As such, a comprehensive mapping of tyrosine phosphorylation sites and their dynamic responses to stimuli in cells will provide critical information on regulatory mechanisms underlining certain physiological and pathological conditions. Mass spectrometry (MS)-based phosphoproteomics is now routinely used as a powerful tool for quantitative profiling of phosphotyrosine (pTyr)-containing peptides on a global scale (6). However, there are still several challenges that have to be faced in laboratories for this type of analysis to be successful. First of all, the frequency of occurrence of tyrosine phosphorylation is much lower than that of serine/threonine phosphorylation with an estimated occurrence less than 1% of the whole phosphoproteome in human (7). Moreover, tyrosine phosphorylation may happen on low abundance protein and/or at low stoichiometry. As a result, recovering pTyr peptides with high specificity and efficiency from samples with high non-pTyr background prior to MS analysis becomes a determinative step in pTyr site mapping. This is typically done by immunoprecipitation (IP)-based assays using commercially available pan-anti-pTyr antibodies such as pTyr-1000 and 4G10. This approach has been robustly applied to identify thousands of pTyr sites in various studies (6, 8, 9). Nevertheless, since such studies usually require large amount of samples, the preventive cost associated with acquiring sufficient antibodies often limits the widespread use of this method, especially in academic laboratories (10). Recently, a Src Homology 2 (SH2) domain-derived pTyr-binding reagent, termed 3



SH2 superbinder, has been demonstrated to be a cost-effective and functionally superior alternative for anti-pTyr antibodies (11). The SH2 superbinder is engineered by introducing three point mutations into the pTyr-binding pocket of the wild type Src SH2 domain. The resulting mutant exhibited 100-fold or greater affinity towards pTyr peptides than its nature counterpart in a context-independent manner, making it an ideal low-cost and pan-specific affinity reagent for pTyr peptides isolation (12). We have demonstrated that the SH2 superbinder, either chelated on Ni2+-NTA beads via His tag or covalently immobilized onto sepharose beads, clearly outperformed commercial anti-pTyr antibodies both in yield and in number of pTyr peptides identified (11, 13). Upon eluting with a competitive pTyr peptide (biotin-pYEEI), purified pTyr peptides can be readily analyzed by MS to resolve site information, and to some extent, their binding affinities to SH2 superbinders (14). Alternatively, the superbinder can be immobilized on a monolithic capillary column to construct a microreactor for pTyr peptide extraction from a minute amount of samples such as protein complexes obtained from a co-immunoprecipitation experiment (15). The generation of SH2 superbinders represents a significant improvement in studying pTyr-centric phosphoproteomics. Nevertheless, most SH2 superbinder-based pTyr profiling studies up-to-date are done in artificially stimulated cells. Although treatments with cell permeable tyrosine phosphatase inhibitors such as pervanadate can boost the overall pTyr level and facilitate detection, the information obtained reflects a rather spurious signaling network (16). Comprehensive and accurate illustration of pTyr-dependent cellular signaling networks under physiologically relevant conditions remains a significant challenge. It is thus important to further optimized current SH2 superbinder-based methods to improve the overall recovery of pTyr peptides, especially low copy number ones from resting or physiologically-stimulated cells. At present, widely adopted SH2 superbinder or anti-pTyr antibody-based enrichment strategies commonly include a metal affinity-based enrichment step, such as TiO2 or titanium ion immobilized metal affinity chromatography (Ti4+-IMAC), as a “polishing” step before or/and after pTyr peptide extraction (9, 11, 13, 17). This workflow is used mainly based 4


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on the presumption that TiO2 or IMAC could help to minimize the interferences of nonphosphorylated peptides. However, with previous studies showing that SH2 superbinders bind pTyr peptides with exceptional high specificity and efficiency, we argue that the IMAC-based polishing step is likely to have a negative impact on peptide recovery due to excessive sample handling associated sample loss for the analysis of low level tyrosine phosphorylation samples. To test this notion, we established and optimized a simplified one-step SH2 superbinder-based method for pTyr peptide enrichment without Ti4+-IMAC purification (Figure 1a). Efficient enrichment of a panel of eight mass spectrometryamenable pTyr peptides with sequences covering representative SH2 domain motifs indicates SH2 superbinders can be used a pan-pTyr affinity purification reagent for tyrosine phosphoproteomics analysis. Applying this method to cell lines at resting or physiologically-stimulated state, we found this method to be simple, robust and high yield. More importantly, when compared with the two-step (i.e. Ti4+-IMAC + SH2 superbinder) procedure, our method allows us to identified 3 folds more pTyr sites in 3 mg sample derived from unstimulated Jurkat cells, indicating that the one-step method is particularly advantageous for pTyr profiling in samples with low level tyrosine phosphorylation.

Material and Methods Materials and chemicals Fused-silica capillaries with 150 μm inner diameter were obtained from PolymicroTechnologies (Phoenix, AZ, USA). Acetonitrile (ACN, HPLC-grade) and methanol (MeOH, HPLC-grade) were purchased from Merck (Darmstadt, Germany). CNBr-activated Sepharose 4B was purchased from GE (Princeton, NJ, USA). Formic acid (FA) was obtained from Fluka (Buches, Germany). Trifluoroacetic acid (TFA), Tris(2-carboxyethyl)phosphine

(TCEP),

iodoracetamide

(IAA),

3-(N-

morpholino)propansulfonic acid (MOPS), sodium phosphate dibasic (Na2HPO4), sodium chloride (NaCl), 2,5-dihydroxybenzoic acid (DHB), triethylamonium 5



bicarbonate (TEAB) and trypsin (bovine, TPCK-treated) were obtained from Sigma(St. Louis, MO, USA). Other chemicals and reagents were either of analytical grade or of better grade. All of the water in the experiments was purified by a Milli-Q system from Millipore Co. (Bedford, MA). Tyrosine phosphorylated peptides were synthesized by ChinaPeptides Co. Ltd. (Shanghai, China). Cell culture and digest preparation Jurkat cells were grown in RPMI 1640 medium, supplemented with 10% fetal bovine serum, 100 μg/mL of streptomycin and 100 U/mL of penicillin in a humidified atmosphere containing 5% CO2 at 37 °C. The cells were harvested until 80% confluence by centrifugation and washed three times with PBS, and a portion of the cells were treated with 1 mM freshly prepared sodium pervanadate for 15 min at 37 °C(18). After that, the cell pellets were gently homogenized in an ice-cold lysis buffer containing 50 mM Tris-HCl (pH 7.4), 8 M urea, phosphatase inhibitor (1 mM Na3VO4, 1 mM NaF, 1 mM C3H7Na2O6P, and 10 mM Na4O7P2), 2% protease inhibitor cocktail (v/v) and 1% Triton X-100 (v/v) in ice bath, sonicated at 400 W for 10 min, and centrifuged at 12 000 g for 30 min. The supernatant was precipitated in ice-cold acetone/ethanol/acetic acid (50/50/0.1, v/v/v). Then the obtained proteins were dissolved in buffer containing 8 M urea and 100 mM TEAB (pH 8.0), and the final protein concentration was determined using the bicinchoninic acid (BCA) assay (Thermo Scientific Pierce). The re-dissolved proteins were reduced by 5 mM TCEP and alkylated by 10 mM IAA, then diluted to 1 M Urea by 100 mM TEAB buffer (pH 8.2). Protein lysate was digested with trypsin overnight at 37 °C with a ratio of enzyme-to-protein at 1:20 (w/w). After stopping the digestion with 1% TFA, the peptide mixture was kept at −80 °C or subjected to C18 solid phase extraction (Sep-Pak, Waters) for desalting, and subsequently vacuum-centrifuged to dryness. The obtained peptide mixture was kept at −20 °C for further usage. The human serum sample used in this work was collected from 10 volunteers from the Second Affiliated Hospital of Dalian Medical University (Dalian, China). And the 6


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study complied with the tenets of the Declaration of Helsinki and the Ethics Committee of the hospital. For the digestion of serum protein, human serum was diluted with buffer containing 8 M urea and 100 mM TEAB (pH 8.0), reduced with TCEP and then alkylated with IAA. After dilution to 1 M urea with 100 mM TEAB buffer (pH 8.2), proteins were digested with trypsin at a ratio of 1:20 (enzyme to protein, w/w) overnight at 37 °C. Digestion was stopped with TFA to a final concentration of 1%, and were desalted on C18 solid-phase extraction (Sep-Pak, Waters). Peptides were dried in the SpeedVac and kept at −20 °C for further usage. Phosphopeptide enrichment Phosphopeptide enrichment was performed with Ti4+-IMAC beads as described previously(19). Briefly, Jurkat cell digests were mixed with identical volume of loading buffer (80% ACN/6% TFA), and then the mixture were loaded on SPE column packed with Ti4+-IMAC beads. After that, the column was washed with 50% ACN/200 mM NaCl/6% TFA and 30% ACN/0.1%TFA, respectively. Finally, the bound peptides were eluted using 10% NH3H2O and dried down using a Speed-Vac. Tyrosine phosphorylated peptide enrichment The dried phospho-/peptides from Jurkat cell digests were dissolved in ice-cold immunoaffinity purification (IAP) buffer containing 50 mM MOPS-NaOH (pH 7.2), 50 mM NaCl and 10 mM Na2HPO4 and incubated with sepharose beads covalently immobilized SH2 superbinders (“SH2 sepharose beads” for short) at 4 °C overnight with rotation. Then the SH2 sepharose beads were washed four times with ice-cold IAP buffer. Finally, the bound peptides were eluted once with 0.2% TFA and once with 60% ACN/0.2%TFA at room temperature. The eluant components were collected, combined and lyophilized for RPLC-MS/MS analysis. To evaluate pTyr peptide recovery efficiency, we setup a semi-complex sample by mixing the serum digest with stable isotopic dimethyl labelled synthetic phosphotyrosyl peptides. Stable isotope dimethyl labeling

(2 × CH3, light labeled; 2 × CHD2, heavy

labeled) was performed as previously described (20), and recovery evaluation 7



experiment was performed as follows. Briefly, heavy labelled phosphotyrosyl peptides were spiked into serum digests and incubated with SH2 sepharose beads at 4°C overnight with rotation. After washing with ice-cold IAP buffer, the bound peptides were eluted twice, by 0.2% TFA and 60% ACN/0.2%TFA, respectively. Then the eluates were combined and equivalent light labeled standard phosphotyrosyl peptides were added. Afterwards, the obtained peptides were dried down using a Speed-Vac and saved at -20 oC until further LC-MS/MS analysis. MALDI-TOF MS analysis The MALDI-TOF mass experiments were carried out by AB Sciex 5800 MALDITOF/TOF mass spectrometer (AB Sciex, CA) equipped with a pulsed Nd/YAG laser at 355 nm. All spectra were obtained in the positive ion reflection mode with delayed ion extraction at a scan range of 800 - 2500 m/z. DHB (25 mg/mL in 50% (v/v) acetonitrile) containing 1% (wt/vol) phosphoric acid was used as the matrix. Aliquots of the sample (0.5 μL) and DHB matrix solution (0.5 μL) were sequentially spotted onto the MALDI plate and dried at room temperature prior to MALDI-TOF MS analysis. LC− MS/MS Analysis All Data were collected using a Q-Exactive mass spectrometer (Thermo Scientific, USA) coupled with a Dionex UltiMate 3000 RSLCnano system (Thermo Scientific, USA). The samples were resuspended in 0.1% FA/H2O solution and automatically loaded onto a C18 trap column at a flow rate of 5 μL/min. The capillary analytical column (150 μm i.d.) was packed in-house with 1.9 μm C18 ReproSil particles (Dr. Maisch GmbH). Peptides were separated with a binary buffer system of 0.1% FA (buffer A) and 80% ACN/0.1% FA (buffer B), at a flow rate of 600 nL/min. The RP gradient elution was performed as follows: 0-4% mobile phase B for 2 min; 4-35% B for 90 min; 35-45% B for 10 min; 45-90% B for 5 min; 90% B for 5 min and finally equilibration with mobile phase A for 15 min. The Q Exactive mass spectrometer was operated in data-dependent MS/MS 8


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acquisition mode. The full mass scan acquired in the Orbitrap mass analyzer was from m/z 400 to 2000 with a resolution of 60 000 (m/z 200). The 12 most intense parent ions with charge states ≥ 2 from the full scan were fragmented by higher-energy collisional dissociation (HCD). The MS/MS scans were also acquired by the Orbitrap with a 35 000 resolution (m/z 200), and the AGC target was set to 1×105 with a max injection time of 120 ms. A spray voltage of 2.0 kV was applied between the spray tip and MS interface. The temperature of the ion transfer capillary was set as 250 oC and the normalized collisional energy for HCD was set to 27%. Dynamic exclusion was set as 25 s. System control and data collection were carried out by Xcalibur software. Data Processing and Analysis Raw mass spectrometry files from the entire study were searched by MaxQuant (version 1.5.1.2) against a non-redundant Uniprot human database (containing 70 946 sequences, and downloaded from website of www.uniprot.org). Carbamidomethyl cysteine was set as a fixed modification, phosphorylation of serine, threonine, and tyrosine and oxidation of methionine were allowed as variable modifications. Trypsin was specified as the proteolytic enzyme with up to two missed cleavage sites allowed. Mass tolerances were 4.5 ppm for precursor ions and 0.5 Da for ITMS MS/MS ions. The false discovery rate (FDR) was controlled to < 0.01 at both peptide and protein level. The other settings were the same as the conventional search. The reverse hits and potential contaminant hits were removed for further analysis. The identified peptides showed in this work were screened by Score ≥ 40. The obtained phosphorylated sites were further filtered by Localization Probability ≥ 0.75 and Score Difference ≥ 5, which means high confident (Class I) of phosphorylated sites. And all these parameters are commonly used in phosphoproteomics studies(21, 22). The mass spectrometry data and Maxquant output files have been deposited onto the jPOST database (http://jpostdb.org/) with an accession code of JPST000539/PXD012268.

Results and Discussion The human SH2 domain family consist of 120 domains in 110 proteins, each has 9



different preference in recognizing the sequence context flanking pTyr residues (23). The SH2 superbinder is derived from the SH2 domain of Src protein and recognizes the same binding motif as wild-type Src SH2 does (11). However, selective substrate binding will result in biased pTyr site identification. To systematically investigate the substrate binding preference of SH2 superbinders, we synthesized a panel of eight mass spectrometry-amenable pTyr peptides with sequences covering representative SH2 domain motifs(12, 23) (Figure 1b). These pTyr peptides were directly subjected to SH2 superbinders enrichment followed by MALDI analysis. We identified all eight pTyr peptides with comparable intensity (Figure 1c), demonstrating SH2 superbinders indeed have little bias towards proximal amino acid sequences near pTyr residues. Therefore, SH2 superbinders can be used a pan-pTyr affinity purification reagent for tyrosine phosphoproteomics analysis. As previously discussed, the efficiency of pTyr peptide enrichment is critical for pTyr phosphoproteomics studies in physiologically relevant samples with low level tyrosine phosphorylation. To evaluate pTyr peptide recovery efficiency, we setup a semi-complex sample by mixing the serum digest with heavy labeled eight synthetic pTyr peptides (hereafter referred as Y1 to Y8) using the stable isotopic dimethyl labeling technique, a simple, efficient and cost-effective method for quantitative proteomics studies (20). We mixed 1 pmol of each heavy-labeled peptides with different amount of trypsin-digested human serum, which provides high nonphosphorylated background peptide interference. The pTyr peptide enrichment was performed by using the one-step method according to the workflow shown in Figure 1a. The same amount of light-labeled peptides were added to the enriched sample before LC-MS/MS analysis. Peptide recovery efficiencies were then determined by calculating the heavy to light ratio for each peptide according to the database searching results using Maxquant. Using this assay, we tested several factors that may affect pTyr peptide recovery, including the amount of non-phosphorylated interference from serum digests (molar ratio of pTyr peptide : serum = 1 : 10 or 1 : 100 ) and number of washing (from two to four times) after IP incubation. As shown in Figure S1, the recovery of the 10


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standard pTyr peptide panel was not decreased even with the non-phosphopeptide interference from serum digests increased to as high as ten folds and the number of washing increased up to four times (about 80 folds of bed volumes), indicating our method is highly robust. In addition to the recovery, we also investigated the specificity of this enrichment using the semi-complex sample of the eight synthetic pTyr peptide : serum =1 : 100. The base peak chromatograms for RPLC analysis of the samples before and after SH2 enrichment were compared. It is observed that the spectrum of sample before enrichment was much more complex than that of sample after enrichment because of the serious interferences from serum digests (Figure S2). The enrichment specificity was then obtained by calculating the ratio of spectral counts of pTyr peptides versus all spectral counts, which was about 19% (256/1358). The corresponding ratio for the sample without enrichment was only 0.8% (17/2180). Clearly these pTyr peptides were efficiently enriched by the SH2 superbinder, demonstrating the high efficiency of our one-step method for pTyr peptide isolation. A typical multi-step pTyr enrichment method can identify more than 1000 pTyr sites in a single LC-MS/MS run with up to 80% specificity(13). In these studies, the bound pTyr peptides were typically eluted from beads by using one buffer(9, 11, 13). To further increase the sample recovery, the bound peptides were eluted twice, by 0.2% TFA and 60% ACN/0.2%TFA, respectively, and then the eluates were combined for analysis in this one-step enrichment strategy. To characterize the one-step enrichment strategy, we systematically compare the performance of our method with a two-step method where a Ti4+-IMAC-mediated purification step was included before incubating with SH2 superbinders (13). We used resting and pervanadate-treated Jurkat cells as two model systems. This cell line has been extensively used as a model system in many mass spectrometry-based method developing studies (8). And for a fair comparison, the pervanadate-treated sample (1 mg) and the untreated sample (3mg) were analyzed by the two methods in a parallel way, respectively. After analysis, the phosphopeptide and phosphorylation-site mapping results were summarized in Table 1. In pervanadatetreated cells, the pTyr peptide enrichment specificity favors the two-step method (91% 11



for two-step method vs. 35% for one-step method), and more pTyr sites were identified by the two-step method (Table 1a). The result indicates that the two-step method is more efficient for hyper-phosphorylated samples, which is consistent with the results of our previous work(13). Remarkably, however, when lysates prepared from resting Jurkat cells were used, the one-step method identified 3-fold more pTyr peptides and sites than the two-step method did (Table 1b). Specifically, the one-step method identified a total of 313 pTyr sites when data from three independent MS experiments were combined, whereas the two-step method identified 96 sites (Figure S3). In order to investigate if this great difference of pTyr identifications was related with the amount of starting samples, we further compared the two methods using 5 mg protein digests from untreated Jurkat cells as the sample. The data summarized in Table 1b showed one-step method still enabled more pTyr identifications than two-step method. Besides, a great improvement of pTyr identification was observed in two-step method when the starting amount was increased from 3 mg to 5 mg, while not in one-step method. Hence, this implies that 3 mg protein digests are sufficient to serve as starting material for the pTyr analysis using one-step method and more samples are required for two-step method, highlighting the high recovery of our one-step method. The pTyr peptide enrichment specificity was always higher for the two-step method than the one-step method in all tested samples, however, the two-step method with higher enrichment specificity cannot always lead to more pTyr identifications. We supposed this could because the pre-enrichment process in the two-step method is a double-edged sword for pTyr identification. On one hand, it is helpful to improve the specificity which will reduce ion suppression from non-phosphopeptides during LCMS/MS analysis, thus facilitating the identification of pTyr peptides(24, 25). On the other hand, this additional enrichment procedure will result in sample loss, thus reducing the phosphosite identification. To verify this, we compared the average intensities of non-phosphorylated peptides, pSer/Thr peptides as well as pTyr peptides identified by the two methods (Figure 2). It is observed that the intensity distributions of phosphopeptides are distinctly different for the two sample models. For pervanadate12


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treated sample, the average intensities of phosphopeptides for two-step method are significantly higher than that of one-step method (Figure 2a). While for untreated sample, the average intensities of phosphopeptides obtained in two-step method are distinctly lower than that of one-step method (Figure 2b). These results indicate that the effect of reduced ion suppression can offset the influence of sample loss in two-step method for the analysis of pervanadate-treated sample. But for the sample with low tyrosine phosphorylation level, the influence of sample loss in two-step method was greater than the effect of reduced ion suppression, leading to a significantly reduced intensities of phosphopeptides and poor phosphosite identifications. The average intensity of non-phosphopeptides for one-step method was similar to that of two-step method for the pervanadate-treated sample, while it was significantly higher than that of one-step method for untreated samples. Clearly the improvement for pTyr analysis of untreated sample in one-step method was due to the reduced sample loss. Taken together, these results demonstrated that omitting the Ti4+-IMAC-mediated cleanup step in pTyr peptide enrichment procedure could significantly benefit site mapping in samples containing low level tyrosine phosphorylation. To further optimize the one-step method and investigate whether 3 mg starting material is optimal for pTyr peptide enrichment, aliquots of 1 mg, 2 mg, 3 mg or 4 mg total protein prepared from unstimulated Jurkat cells were subject to pTyr extraction using 100 μg SH2 superbinder and LC-MS/MS analysis. Number of identified pTyr peptides positively correlated with the total protein amount within the 1-3 mg range and reached plateau at 4 mg (Figure S4). Thus further increase of starting materials is not likely to enhance pTyr identification. Noteworthy, the increase of pTyr peptides identification appears to be associated with the decrease of pSer/Thr-containing peptides detection. Whether it reflects a weak binding affinity of SH2 superbinders towards phospho-Ser/Thr residues warrants further investigation. The intensity distributions of pTyr peptides in this experiment were as well analyzed. As observed from Figure S5, the average intensity of pTyr peptides increased with the increasing amount of sample from 1 mg to 3 mg, and it no longer increased from 3 mg to 4 mg. 13



This result indicated that 3 mg protein digests are sufficient to serve as starting material for the pTyr analysis using our one-step enrichment method. Then taking into account of the number of pTyr identified, the average intensity of pTyr peptides and the amount of starting materials consumed, we determined 3 mg as the optimal amount of sample for pTyr enrichment and phosphosite mapping. We also evaluated the reproducibility of our method by replicative experiments at the LC/MS/MS run level, the workflow level and the biological level (Figure 3a-d). As expected, the performance of LC-MS/MS analysis is the most reproducible while biological replicates being the least. By comparison, our workflow replicates also generated high degree overlapping data sets, as indicated by higher Pearson’s coefficient than that of a previous study (9). For example, 219 high confidence pTyr sites were observed in all three replicates, accounting for 54% of all identified pTyr sites. In addition, at least 70% of pTyr sites identified in one enrichment replicate can also be identified by the other two replicates, similar to our previous finding (13), demonstrating the robustness and high reproducibility of our one-step method. Upon method optimization, we explored the ability of this method in deciphering pTyr-dependent signaling in a pair of HER2-overexpressing breast cancer cell lines. HER2 is a member of the ERBB receptor tyrosine kinase (RTK) family and its overexpression is considered a prominent driver for breast cancer. Amplified HER2 gene expression is found in about 25% breast cancer patients and associates with poor prognosis (26, 27). Trastuzumab, a HER2-specific monoclonal antibody acts to block receptor activities via different mechanisms, has been shown to significantly improve the overall survival for early and advanced HER2-positive breast cancer patients. Nevertheless, the presence of intrinsic and acquired resistance to trustuzumab treatment in many patients remains a major clinic problem (28). Several mechanisms for resistance have been proposed including abnormal HER2 activity, upregulated HER2 downstream kinase signaling and the involvement of other RTKs such as insulin growth factor 1 receptor (IGF-1R) and other members of the HER family (i.g. EGFR and 14


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HER3). While it is difficult to pin down the exact mechanism for trastuzumab insensitive in a specific breast cancer cell type, we hypothesized that a comprehensive profiling of pTyr events may provide valuable clues. To test this hypothesis, we selected BT474 cells as the trastuzumab-sensitive model and HCC1954 cells as the acquired trastuzumab-resistant model. Enrichment of pTyr peptides was performed using the one-step method from these cells before and after EGF treatment. The EGF treatment was included to profile pTyr signaling of EGFR in the context of amplified HER2 signaling, which may play a role in regulating drug resistance. In unstimulated cells, we identified around 40% more pTyr sites in BT474 cells than in HCC1954 cells (Figure S6), in line with higher basal pTyr signaling in BT474 cells as estimated by the far-western blotting experiment using SH2 superbinders as the detection reagent (Figure S7). In addition, EGF treatment did not cause significant change of the overall pTyr phosphorylation in both cells, suggesting that the inherent cell type-specific signaling likely account for most of the differential pTyr signaling between cell lines. With improved pTyr peptide recovery by the one-step method, we were also able to quantify 87 pTyr sites in these cells with or without EGF treatment by label-free quantification (Figure 4). Many of these sites belongs to tyrosine kinases that are known to be involved in regulating cell growth in breast cancer, including YES1, EGFR, HER2/3 and TNK2. Notably, about one-fourth (23 of 87) of the pTyr sites were from proteins known to be essential components of the dynamic Shc1 signaling complex, in consistent with the scaffold protein Shc1 being a pivot mediator for HER protein signaling (29). By quantitative analysis of individual sites, we noted that HER2 was hyper-phosphorylated in BT474, but not in HCC1954, at several signature tyrosine residues, including the pTyr877, pTyr1023 and pTyr 1248 sites. The pTyr877 site is localized in the activation loop of HER2 kinase domain and its phosphorylation is correlated with ERBB2 activation, whereas pTyr1023 and pTyr1248 are proposed binding sites for downstream effectors such as PTPN11 and PTPN18 respectively, indicating high level of receptor activation in BT474 cells. Moreover, the Grb2-binding pTyr 427 site on Shc1 was also up-regulated regardless EGF treatment (Figure 4). 15



Page 16 of 27

These data suggest a systemic elevation of HER2 activity and its downstream signaling network in BT474 cells, forming basis for its sensitivity to HER2 blocking agent such as trastuzumab (30). While the overall pTyr level of HCC1954 cells is much lower than that of BT474 cells, we detected marked phosphorylation on PTPN11 at tyrosine residue Tyr542 (Figure 4, blue dots). PTPN11 is a protein tyrosine phosphatase (PTP) required for activation of Ras/MAPK by most RTKs including EGFR and PDGFR (31). It is the first bona fide PTP identified as an oncogene and its hyper-activation have been found in many cancers such as leukemia, breast cancer and anaplastic large cell lymphoma (ALCL) (32, 33). Most notably, recent studies suggest PTPN11 play a critical role with the development of intrinsic and acquired resistance to many RTK-driven cancer cell models including HCC1954, whereas the Tyr542 site might be used as a biomarker for predicating resistance(34-36). Therefore, pTyr sites and corresponding proteins identified by our method provides additional experimental supports to previous discoveries, demonstrating our method can be served as a powerful tool for mechanistic studies of pTyr-dependent signaling networks.

Conclusion Characterizing global tyrosine phosphorylation events under physiological and physiological conditions is fundamentally important in the illustration of regulatory mechanisms

of

cell

signaling.

However,

comprehensive

analysis

of

Tyr

phosphoproteome in cancer cells remains a significant challenge due to low abundance of most pTyr-containing peptides in physiologically-relevant samples and poor pTyr peptide enrichment efficiency. Especially, sample loss as a result of multiple sample handling steps during enrichment has an obvious negative impact on peptide recovery efficiency. To address this issue, we developed a one-step enrichment method relying on the high affinity and specificity of SH2 superbinders to pTyr peptides. By simply eliminating the global phosphopeptide enrichment step that are commonly adopted in 16


Page 17 of 27 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


current enrichment schemes, we achieved significant improvement in pTyr peptide purification, resulting in three-fold increase in the identification of corresponding pTyr sites. Through systematic comparison of our method with the conventional multiplestep method, where the Ti4+-IMAC-mediated enrichment step is added before pTyr extraction, we show that our strategy is especially more effective in capturing pTyr peptides from low abundant samples such as cells at the resting state. Specifically, we confidently identify more than 400 pTyr sites in Jurkat cells with high reproducibility. Applying our method to the quantitative analysis of two human breast cancer cell models, we successfully identified pTyr-dependent signaling properties underlining differential cellular behaviors. For example, in trastuzumab-sensitive BT474 cells, we detected elevated HER2 activation and related tyrosine-dependent downstream signaling, whereas in trastuzumab-resistant HCC1954 cells, we identified PTPN11, a proto-oncogene recently associated with the development of clinical drug resistance in RTK-driven cancers, being a prominently activated signaling molecule. Although our current works were accomplished on cell line models, it is the first and successful attempt to isolate pTyr peptides from samples with low level in vivo tyrosine phosphorylation using a simplified one-step SH2 based method. Owing to its high efficiency, robustness and high reproducibility, we believe this approach could be served as a powerful tool for illustrating the pTyr-dependent signaling networks. And the fact that our result in HER2 experiments is in high consistent with previous reports indicates our approach can be extended to study Tyr phosphoproteomics in breast tumor samples, which will provide new insights into the molecular pathogenesis and treatment of breast cancer.

Acknowledgments This work was supported, in part, by funds from the National Key R&D Program of China (2016YFA0501402, 2017YFA0505004, 2016YFA0501404), the National Natural Science Foundation of China (21535008, 21605140, 91753105, 31670839), the innovation program (DICP TMSR201601)of science and research from the DICP, CAS, 17



the Natural Science Foundation of Beijing (5172012). M.Y. is a recipient of the National Natural Science Fund of China for Distinguished Young Scholars (21525524).

Competing financial interests: The authors declare no competing financial interest.

Supporting information The following files are available free of charge at ACS website http://pubs.acs.org: Figure S1. Optimizing the recovery efficiency of one-step method using synthetic pTyr peptides under different conditions. Figure S2. Base peak chromatograms for RPLC analysis of the sample (molar ratio of pTyr peptide : serum = 1 : 100) before and after enrichment using one-step method. Figure S3. Overlap of identified pTyr site in unstimulated Jurkat cell samples using two methods. Figure S4. The number of identified peptides from unstimulated Jurkat cells with different amounts of starting material. Figure S5. Log2 intensity distribution of the identified pTyr peptides from unstimulated Jurkat cells with different amounts of starting material. Figure S6. Total number of pTyr site identified in two human breast cancer cell lines with or without EGF stimulation using one-step enrichment method. Figure S7. Characterizing global tyrosine phosphorylation level by far western blotting in two breast cancer cell lines. References 1.

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& Cellular Proteomics 2008, 7, (4), 768-784. 24. Heemskerk, A. A.; Busnel, J. M.; Schoenmaker, B.; Derks, R. J.; Klychnikov, O.; Hensbergen, P. J.; Deelder, A. M.; Mayboroda, O. A., Ultra-low flow electrospray ionization-mass spectrometry for improved ionization efficiency in phosphoproteomics. Anal Chem 2012, 84, (10), 4552-9. 25. Ishihama, Y., Proteomic LC–MS systems using nanoscale liquid chromatography with tandem mass spectrometry. Journal of Chromatography A 2005, 1067, (1-2), 73-83. 26. Korkaya, H.; Kim, G. I.; Davis, A.; Malik, F.; Henry, N. L.; Ithimakin, S.; Quraishi, A. A.; Tawakkol, N.; D'Angelo, R.; Paulson, A. K.; Chung, S.; Luther, T.; Paholak, H. J.; Liu, S.; Hassan, K. A.; Zen, Q.; Clouthier, S. G.; Wicha, M. S., Activation of an IL6 inflammatory loop mediates trastuzumab resistance in HER2+ breast cancer by expanding the cancer stem cell population. Mol Cell 2012, 47, (4), 570-84. 27. Li, B.; Meng, Y.; Zheng, L.; Zhang, X.; Tong, Q.; Tan, W.; Hu, S.; Li, H.; Chen, Y.; Song, J.; Zhang, G.; Zhao, L.; Zhang, D.; Hou, S.; Qian, W.; Guo, Y., Bispecific antibody to ErbB2 overcomes trastuzumab resistance through comprehensive blockade of ErbB2 heterodimerization. Cancer Res 2013, 73, (21), 6471-83. 28. Burris, H. A., 3rd; Tibbitts, J.; Holden, S. N.; Sliwkowski, M. X.; Lewis Phillips, G. D., Trastuzumab emtansine (T-DM1): a novel agent for targeting HER2+ breast cancer. Clin Breast Cancer 2011, 11, (5), 275-82. 29. Zheng, Y.; Zhang, C.; Croucher, D. R.; Soliman, M. A.; St-Denis, N.; Pasculescu, A.; Taylor, L.; Tate, S. A.; Hardy, W. R.; Colwill, K.; Dai, A. Y.; Bagshaw, R.; Dennis, J. W.; Gingras, A. C.; Daly, R. J.; Pawson, T., Temporal regulation of EGF signalling networks by the scaffold protein Shc1. Nature 2013, 499, (7457), 166-71. 30. Hao, Q.; McKenzie, R.; Gan, H.; Tang, H., Protein kinases D2 and D3 are novel growth regulators in HCC1806 triple-negative breast cancer cells. Anticancer Res 2013, 33, (2), 393-9. 31. Nichols, R. J.; Haderk, F.; Stahlhut, C.; Schulze, C. J.; Hemmati, G.; Wildes, D.; Tzitzilonis, C.; Mordec, K.; Marquez, A.; Romero, J.; Hsieh, T.; Zaman, A.; Olivas, V.; McCoach, C.; Blakely, C. M.; Wang, Z.; Kiss, G.; Koltun, E. S.; Gill, A. L.; Singh, M.; Goldsmith, M. A.; Smith, J. A. M.; Bivona, T. G., RAS nucleotide cycling underlies the SHP2 phosphatase dependence of mutant BRAF-, NF1- and RAS-driven cancers. Nat Cell Biol 2018, 20, (9), 1064-1073. 32. Xu, R.; Yu, Y.; Zheng, S.; Zhao, X.; Dong, Q.; He, Z.; Liang, Y.; Lu, Q.; Fang, Y.; Gan, X.; Xu, X.; Zhang, S.; Dong, Q.; Zhang, X.; Feng, G. S., Overexpression of Shp2 tyrosine phosphatase is implicated in leukemogenesis in adult human leukemia. Blood 2005, 106, (9), 3142-9. 33. Zhou, X.; Coad, J.; Ducatman, B.; Agazie, Y. M., SHP2 is up-regulated in breast cancer cells and in infiltrating ductal carcinoma of the breast, implying its involvement in breast oncogenesis. Histopathology 2008, 53, (4), 389-402. 34. Fedele, C.; Ran, H.; Diskin, B.; Wei, W.; Jen, J.; Geer, M. J.; Araki, K.; Ozerdem, U.; Simeone, D. M.; Miller, G.; Neel, B. G.; Tang, K. H., SHP2 Inhibition Prevents Adaptive Resistance to MEK Inhibitors in Multiple Cancer Models. Cancer Discov 2018, 8, (10), 1237-1249. 35. Prahallad, A.; Heynen, G. J.; Germano, G.; Willems, S. M.; Evers, B.; Vecchione, L.; Gambino, V.; Lieftink, C.; Beijersbergen, R. L.; Di Nicolantonio, F.; Bardelli, A.; Bernards, R., PTPN11 Is a Central Node in Intrinsic and Acquired Resistance to Targeted Cancer Drugs. Cell Rep 2015, 12, (12), 1978-85. 36. Lee-Hoeflich, S. T.; Pham, T. Q.; Dowbenko, D.; Munroe, X.; Lee, J.; Li, L.; Zhou, W.; Haverty, P. M.; Pujara, K.; Stinson, J.; Chan, S. M.; Eastham-Anderson, J.; Pandita, A.; Seshagiri, S.; Hoeflich, K. P.; Turashvili, G.; Gelmon, K. A.; Aparicio, S. A.; Davis, D. P.; Sliwkowski, M. X.; Stern, H. M., PPM1H is a p27 phosphatase implicated in trastuzumab resistance. Cancer Discov 2011, 1, (4), 326-37. 20


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Captions Table 1. Comparison of one-step and two-step methods for the analysis of Tyr phosphoproteome in Jurkat cells. Ti4+-IMAC+SH2_1, _2, _3 and SH2_1, _2, _3 refer to three replicate LC-MS/MS runs of samples (pervanadate-treated samples, 1 mg; untreated samples, 3 mg) prepared using two-step and one-step methods, respectively. The Ti4+-IMAC+SH2_5mg and SH2_5mg refer to another analysis using starting material of 5 mg. Figure 1. Selectively enrichment of pTyr peptides by covalently immobilized SH2 superbinders with the one-step method. a) the workflow, b) Sequences of a panel of eight synthetic pTyr peptides with pTyr residue flanking sequences covering representative SH2 domain recognition motifs, c) A representative MALDI-TOF MS spectrum showing eight synthetic peptides purified from semi-complex peptide mixture using the one-step pTyr enrichment method. Figure 2. Comparison of intensity distributions for the peptides identified in Jurkat cells with (a) and without (b) pervanadate treatment using two methods. The average intensities across three independent LC-MS/MS runs were log2 transformed and significance was assessed using Mann-Whitney U (unpaired and nonparamatric) test. p-value < 0.05 was considered statistically significant (NS, No significant; **, p-value < 0.01; ***, p-value < 0.001; ****, p-value < 0.0001). Figure 3. Reproducibility evaluation of the one-step method. Scatter plots depicting the log2 transformed pTyr site intensities of two representative LC-MS/MS replicates (a), biological replicates (b) and workflow replicates (c). Venn diagram shows the overlap of pTyr site identifications from three workflow replicates (d). All sites have a localization probability ≥ 0.75 and Score Difference ≥ 5. Figure 4. Comparison of pTyr sites identified from two human breast cancer cell lines by the one-step enrichment method. a) B0 and H0 refer to BT474 and HCC1954 cells without treatments, respectively; b) B5 and H5 refer to BT474 and HCC1954 cells treated with EGF at 100 ng/ml for 5 minutes, respectively. Four replicative LC–MS/MS runs were performed for each sample and the results were submitted to label-free quantification analysis. All sites have a localization probability ≥ 0.75 and Score Difference ≥ 5.

21



Page 22 of 27

Table1. (a) pervanadate-treated Jurkat cell sample All

pTyr

% pTyr

All p-

pTyr

% pTyr

Sample

peptides

Phosphopeptide

peptide

peptide

sites

site

site

Ti4+-IMAC+SH2_1

2606

2563

2374

91.10%

2154

1869

86.77%

Ti4+-IMAC+SH2_2

2598

2557

2377

91.49%

2127

1855

87.21%

Ti4+-IMAC+SH2_3

2647

2616

2432

91.88%

2171

1890

87.06%

SH2_1

6499

2432

2249

34.61%

2004

1735

86.58%

SH2_2

6528

2469

2272

34.80%

2007

1726

86.00%

SH2_3

6051

2151

1994

32.95%

1783

1536

86.15%

(b) untreated Jurkat cell sample All

pTyr

% pTyr

All p-

pTyr

% pTyr

Sample

peptides

Phosphopeptide

peptide

peptide

sites

site

site

Ti -IMAC+SH2_1

220

147

93

42.27%

133

71

53.38%

Ti -IMAC+SH2_2

218

142

103

47.25%

124

80

64.52%

Ti4+-IMAC+SH2_3

201

127

95

47.26%

109

73

66.97%

Ti4+-IMAC+SH2_5mg

857

646

275

32.09%

496

220

44.35%

SH2_1

7364

354

321

4.36%

285

247

86.67%

SH2_2

7164

339

307

4.29%

279

242

86.74%

SH2_3

7172

343

319

4.45%

280

247

88.21%

SH2_5mg

2324

428

344

14.80%

340

254

74.71%

4+ 4+

22


Page 23 of 27 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


a

b

c

Figure 1.

23



Log2 intensity

a

36

Non-phosphopeptides

32

b

36

pSer/Thr peptides

****

32

NS

36 32

28

28

28

24

24

24

20

20

20

16

16

16

one-step

Log2 intensity

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 24 of 27

36 32

two-step

Non-phosphopeptides

one-step 36

pSer/Thr peptides

32

***

two-step 36

28

24

24

24

20

20

20

16

16

16

one-step

two-step

Figure 2.

24


two-step

pTyr peptides

**

32

**

28

two-step

****

one-step

28

one-step

pTyr peptides

one-step

two-step

Page 25 of 27 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 3.

25



Figure 4.

26


Page 26 of 27

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For TOC Only

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One-Step SH2 Superbinder-Based Approach for Sensitive Analysis of

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