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Combinatorial Peptide Ligand Library-based Photoaffinity Probe for the Identification of Phosphotyrosine-Binding Domain Proteins Jinying An, Guijin Zhai, Zhenchang Guo, Xue Bai, Pu Chen, Hanyang Dong, Shanshan Tian, Ding Ai, YuKui Zhang, and Kai Zhang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b04781 • Publication Date (Web): 05 Feb 2019 Downloaded from http://pubs.acs.org on February 11, 2019

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Analytical Chemistry

Combinatorial Peptide Ligand Library-based Photoaffinity Probe for the Identification of Phosphotyrosine-Binding Domain Proteins Jinying An,†, § Guijin Zhai,*,†, § Zhenchang Guo,†, § Xue Bai,† Pu Chen,† Hanyang Dong,† Shanshan Tian,† Ding Ai,‡ Yukui Zhang,∥ Kai Zhang*,† †2011

Collaborative Innovation Center of Tianjin for Medical Epigenetics, Tianjin Key Laboratory of Medical Epigenetics, Key Laboratory of Immune Microenvironment and Disease (Ministry of Education), Department of Biochemistry and Molecular Biology, Key Laboratory of Breast Cancer Prevention and Treatment (Ministry of Education), Cancer Institute and Hospital, Tianjin Medical University, Tianjin 300070, China ‡Tianjin

Key Laboratory of Metabolic Diseases, Department of Physiology and Pathophysiology, Tianjin Medical University, Tianjin 300070, China ∥Dalian

Institute of Chemical Physics, Chinese Academy of Sciences, 457 Zhongshan Road, Dalian 116023, China

§ Co-first

authors

* Corresponding

to [email protected] or [email protected]

ABSTRACT: Phosphotyrosine (pY) serves as docking site for the recognition proteins containing pY-binding (pYB) modules, such as the SH2 domain, to mediate cell signal transduction. Thus, it is vital to profile these binding proteins for understanding of signal regulation. However, identification of pYB proteins remains a significant challenge due to their low abundance and typically weak and transient interactions with pY sites. Herein, we designed and prepared a pY-peptide photoaffinity probe for the robust and specific enrichment and identification of its binding proteins. Using SHC1-pY317 as a paradigm, we showed that the developed probe enables to capture target protein with high selectivity and remarkable specificity even in a complex context. Notably, we expanded the strategy to a combinatorial pY-peptide-based photoaffinity probe by using combinatorial peptide ligand library (CPLL) technique and identified 24 SH2 domain proteins, which presents a deeper profiling of pYB proteins than previous reports using affinity probes. Moreover, the method can be used to mine putative pYB proteins and confirmed PKN2 as a selective binder to pY, expanding the repertoire of known domain proteins. Our approach provides a general strategy for rapid and robust interrogating pYB proteins and will promote the understanding of signal transduction mechanism.

Tyrosine phosphorylation is a critical protein modification that mediates signal transduction and regulation in living beings, governing cell proliferation, differentiation, development and so on.1,2 In fact, short peptides containing phosphorylated tyrosine enable to perform these functions by recruiting phosphotyrosine-binding (pYB) modules such as the Src homology 2 (SH2),3,4 the phosphotyrosine binding (PTB),5,6 and the C2 domains.7 Moreover, these domains have been found in various signaling enzymes and adaptor proteins and their dysregulation contribute to a host of human diseases.2,8 Thus, deciphering the pYB domain proteins is the key to understanding the phosphotyrosine (pY)-mediated diverse cellular processes. Nonetheless, a large number of pYB proteins remain poorly understood due to lack of sensitive detection. Phosphotyrosine peptide chip-based methods have been used for the profiling of SH2 domains.9,10 The approach used pY peptide chips to characterize the recognition specificity of a collection of expressed human SH2 domains. However, the strategy applied only to known domains and was not available for mining of putative domains. While the synthesis of numerous peptides and expression of various domain proteins makes the method time-consuming and complicated. In

addition, mimic-peptides as baits for affinity pull-down experiment have been adopted for the identification of pY binders.11-13 In particular, the screening approach could be used to identify novel pYB proteins. However, it is still a big challenge for the low abundance of pYB proteins due to the non-covalent interactions between pY peptides and domains are usually weak and transient. Photoaffinity labeling (PAL) is a powerful technique used for the fishing of those weak affinity interactors as it can use a chemical probe to covalently bind target proteins by light activation.14,15 Thus it is possible by the incorporation of a photo-reactive group within a chemical probe to trap partners of small molecules, RNA or peptides. Salisbury et al.16 designed an activity-based probe, in which a photo-reactive group was incorporated into an HDAC inhibitor to enrich its binders. Besides, Arguello and coworkers17 developed an approach relying upon photo-cross-linking with RNA probes to profile RNA-protein interactions. However, to our best knowledge, it has not been reported that pYB domain proteins are profiled by combining photoaffinity technique and pYpeptide probes. It has been known that the peptide motif surrounding pY has an important effect on the recognition of pYB modules to pY.

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Analytical Chemistry

EXPERIMENTAL SECTION Capturing of pYB Domain Protein Using pY-peptide Photoaffinity Probe. The developed probe (0.8 nmol) was mixed with SRCSH2 (4 M) in 50 L binding buffer (50 mM Tris-HCl, 150 mM NaCl, 1% NP-40) and the mixture was incubated on a rotary mixer at 4 °C overnight. Then, the mixture was irradiated at 365 nm on ice for 30 min using UVP crosslinker (Analytik Jena). Next, the mixture was transferred into prewashed streptavidin agarose beads at 4 °C for 4 h. The beads were washed with wash buffer 1 (2 M NaCl, 1 mM EDTA, 10 mM HEPES, 0.01% Triton X100, pH=7.4) twice followed by wash buffer 2 (2 M NaCl, 1 mM EDTA, 10 mM HEPES, 0.1% Triton X100, pH=7.4) once to remove noncrosslinked protein. Finally, the loading buffer was added to the beads and incubated for 10 min at 95 °C. Then, the mixture was centrifuged at 500 g for 1 min and the supernatant was loaded onto the gel to analyze by SDS-PAGE. Enrichment of Endogenous pYB Proteins from Cell Lysate by Combinatorial pY-peptide Photoaffinity Probe. We extracted proteins from Hela cells using RIPA lysis reagents (further details are included in Supporting Information). The prepared cell proteins (10 mg) were incubated with combinatorial pY-peptide photoaffinity probe (2.5 nmol) at 4 °C overnight. Then, the mixture was irradiated at 365 nm on ice for 30 min by UVP crosslinker to trap target proteins covalently. Next, we transfer the mixture into prewashed beads at 4 °C for 4 h. Then, the beads were washed 3 times as described above. Similarly, the loading buffer was added to the beads and incubated for 10 min at 95 °C. Then, the mixture was centrifuged at 500 g for 1 min and the supernatant was digested followed by LC-MS/MS analysis. The same procedure was also applied for Probe 1 to enrich its endogenous targets from cell lysate.

Mass Spectrometry Analysis of Isolated Proteins. The proteins captured by probe were separated and digested with trypsin. Then, the tryptic digest was injected into a Nano-LC system equipped with a C18 column and electrosprayed into an Orbitrap Q-Exactive Plus mass spectrometer. The mass spectrometric analysis was carried out in a data-independent mode (DIA) and the resulting files were searched using Spectronaut Pulsar X. For the detailed parameters of LCMS/MS and data searching, please see Supporting Information. RESULTS AND DISCUSSION Analytical Strategy for Efficient Enrichment of the pYB Domain Protein by Photoaffinity Probes. Considering the weak and transient interactions between pY peptides and recognizated domains, as well as the effect of peptide motif surronding pY on their interactions, we develped a combintoral pY-peptide photoaffinity probe via CPLL technique to improve the enrichment of pYB proteins. And then the proteomic profiling of pYB proteins was carried out followed by probe enrichment and mass spectrometry analysis. A brief description of the strategy is shown in Figure 1. The probe contained a combinatorial pY peptide sequence and a photo-reactive group as well as a biotin tag for pull-down experiment. First, the probe was incubated with cell lysates for the specific recognition with its target proteins. Second, the above mixture was exposed to UV irradiation, which could convert weak non-covalent interactions into irreversible covalent bonds between cross-linkers and binders, strengthening the capture of pYB proteins. Third, harsh washing buffer was used to remove non-crosslinked proteins, enabling to selective trap interactors. Finally, the captured proteins were isolated via its biotin tag for gel imaging or HPLC-MS/MS detection followed by bioinformatics analysis.

Cell lysates Targets

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And therefore it indicates that a single peptide probe might result in loss of a number of pYB domain proteins. Thus the combinatorial peptide probe may increase the capturing probability of binders. To date, combinatorial peptide ligand library (CPLL) has been developed into a unique technology for digging deeper proteome and revealing low abundance species.18,19 The methodology utilizes diverse ligand library generated by combinatorial chemistry for affinity enrichment of various binding proteins, which allows us to build a pY library containing different peptide motifs. Additionally, phosphoproteomics has revealed the probability of peptide sequence around pY, which further provides a guide to rationalize the peptide sequence of pY probe.9,20-22 In the present work, we sought to take advantage of photocrosslinking technique and CPLL strategy to design a combinatorial pY-peptide-based photoaffinity probe for the effective enrichment and deep profiling of pYB domain proteins. Using pY317-peptide of SHC-transforming protein 1 (SHC1) as a paradigm, we first developed the SHC1-pY317 photoaffinity probe to achieve a robust and selective enrichment of the targeted pYB protein even in a complex proteome. Furthermore, we designed and synthesized combinatorial peptide photoaffinity probes to proteomic profile the pYB proteins. Our results showed that the novel approach can perform a deep mapping of pYB proteome and further allows us to uncover putative pY-binders.

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m/z pYB domain proteins X1X2YX3X4X5 P

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Figure 1. Workflow of the enrichment and identification of pYB domain proteins by combinatorial pY-peptide photoaffinity probe.

Designed and Prepared of Probes. Phosphorylation of tyrosine-317 of SHC-transforming protein 1 (SHC1) (SHC1pY317) is found to mediate proliferation signals in cancer cells and is one of the well-established pY sites.23 Thus, we choose SHC1-pY317 as a paradigm to design photoaffinity probe in this study. Proto-oncogene tyrosine-protein kinase Src (SRC) was reported to bind SHC1-pY317 through its SH2 domain with Kd value of 2.2 M.24 We therefore expressed the SH2 domain of SRC (SRCSH2), which was analyzed and confirmed by mass

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Analytical Chemistry spectrometry (Figure S1), as a target to assess the ability of the developed probe for capturing pYB domain proteins. Next, we prepared the photoaffinity peptide probe 1: FDBpa-PS(pY)VNVQNLK-biotin. The probe carry SHC1-pY317 peptide (-PS(pY)VNVQNLK) as a bait, benzophenone (Bpa) as a photo-cross-linker, and biotin as an affinity handle for protein enrichment. Since SH2 domain typically interacts with pY through amino acid residues between P+3 and P-2 surrounding pY,25 we therefore designed pY-peptide probes where a Bpa group inserted in the position of P-3 (in deed, the enrichment yield of probe 1 (P+4) is lower (about 30%) than probe 1 (P-3), see Figture S2 for detail). Thus we anticipated that the Bpa radicals generated by UV irradiation were proximal to the binding proteins while did not disturb their interactions. The synthesis of probe was further characterized and confirmed by the detection of MALDI-TOF MS (Figtures S3 – S5).

Figure 2. The effect of experiment conditions for promoting SRCSH2 enrichment using photoaffinity probe. (A) The effects of various probe concentrations on enrichment yield. (B) Investigation of UV illumination time for capturing protein. (C) The effects of different binding buffer on target enrichment. The probe (16 M) was first incubated with SRCSH2 (4 M) and exposed to UV irradiation. After removing non-crosslinked proteins, the resultant targets were isolated and visualized using gel staining.

Effect of Experiment Conditions for Promoting Targets Enrichment. To acquire optimal photo-crosslinked targets, we first assessed the recognition of SRCSH2 and a series of concentrations of the probes, which incubated with a defined concentration of target protein. As shown in Figures 2A and S6A, an increase in the concentration of probe leads to an improved enrichment yield of target, which was saturated at 16 M probe. Thus, we chose 16 M as the preferred probe concentration for the following experiments. In addition, we observed probe effectively bound target in a dose-dependent fashion with Kd values of 3.3 M (Figure S7), which is similar to the reported Kd values of 2.2 M for the native pY-peptide. This interesting result demonstrated that the photoaffinity probe can effectively mimic the affinity interaction between pY and binding protein. We next investigated the effects of varied UV irradiation time on the enrichment efficiency. As shown in Figures 2B and S6B, the amounts of target products grow obviously with the UV light exposure time up to 30 min. when irradiation time was more than 30 min, the yield of target does not improved significantly. Notably, compared with the negative control without UV crosslinking, the probe demonstrated a

substantial increase in target enrichment yield, implying UVdependent protein traping. Together, a 30 min illumination time was used in the following study. Moreover, the salt concentration of binding buffer has been shown to have a significant effect on specific protein-protein interactions.26 So the influence of different binding buffers on the enrichment yield of target was investigated. As shown in Figure 2C and S6C, a remarkable increase of captured target appears with the salt concentration goes up to 1 × binding buffer. Therefore, we chose 1×binding buffer as the preferred reaction medium in the subsequent experiments. Photoaffinity Probes Show High Enrichment Efficiency for Target Protein. To explore the feasibility of the probe for effective capturing target, we further designed a series of parallel experiments (Figure 3). Besides negative control with no UV irradiation, probe C1 (contains an unmodified tyrosine, but is otherwise identical to probe 1) and probe NC1 (no Bpa included) were used in the following control experiment. The probe was first incubated with SRCSH2 and subsequently irradiated via UV light. After discarding non-crosslinked proteins, the resultant targets were visualized using gel staining following SDS-PAGE separation. The results indicated that the target captured by probe 1 is significantly improved compared with control without UV cross linking, implying UV-dependent protein traping (Figure 3 Lanes 1 and 3, Figure S8). Notably, there is barely detectable target observed using probe C1, indicating phospho-selective recognition of the probe. Moreover, the enrichment yield of probe NC1 is dramatically lower than probe1, which suggest the probe allow to enrich target effectively compared with traditional affinity method (Figure 3, Figure S8). Collectively, The results demonstrated that the pY-peptide photoaffinity probe was able to capture its targeted binder with high efficiency. Probe 1 Probe C1 Probe NC1 SrcSH2 UV Lane

+ + + 1

+ + + 2

+ + 3

+ + 4

+ 5

KD

46 35 target

Figure 3. Enrichment of SrcSH2 using probes 1, C1 and NC1 (resolved by SDS-PAGE). Lane 1: cross-linked SrcSH2 enriched by probe 1, Lane 2: cross-linked SrcSH2 captured by probe C1, Lane 3: negative control experiment with no UV irradiation, Lane 4: enriched BPTFPHD by probe NC1, Lane 5: SrcSH2 input as a control.

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Probe Shows Remarkable Specificity for Targets in Complex Proteomes. To examine whether the probe could be used to specifically trap target proteins in a complex context, we performed enrichment experiments by mixing SRCSH2 with excess cell lysates. After enrichment by probe 1, the isolated proteins were separated by SDS-PAGE and detected using gel imaging. As shown in Figure 4 Lane 1, probe 1 enables to effectively enrich target proteins with little interference in the complex environment, and it revealed robust capturing ability toward binders in an UV-dependent manner (the capturing yield of the probe without UV irradiation is less than 5% of the probe with UV cross-linking Figure 4 Lane 2). Collectively, the result showed remarkable specificity of the probe to its target protein even in a competitive environment. Probe 1 Probe 2 UV Lysate SrcSH2 Lane

+ + + + 1

+ + + 2

+ + + + 3

+ + + 4

+ + 5

+ 6

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Figure 4. Enrichment of SrcSH2 mixed in cell lysates using probe 1 and 2 (resolved by SDS-PAGE). Lanes 1 and 3: SrcSH2 enriched by probe 1 and 2, Lanes 2 and 4: negative control experiment with no UV irradiation, Lanes 5 and 6: negative control experiment without enrichment.

Preparation and Characterization of the Combinatorial pY-peptide Photoaffinity Probes. To expand the application of our strategy and explore a deeper proteome of pY-binders, we designed probe 2 on the basis of the probability of sequence motif surrounding pY (TA-Bpa-X1X2(pY)X3X4X5biotin, X1: PNVLT, X2: PEILVY, X3: EDSVIYTL, X4: NMTEDL, X5: LVIPME.).9,10,20-22 and thus the combinatorial photoaffinity probe was prepared via CPLL strategy. The probe was generated based on standard solid-phase synthesis, by designing and introducing randomized amino acid residues into five key positions in peptide motif (the amount of every amino acid added in one position is identical), to create libraries of combinatorial peptide ligands. The sensitivity of the probe was also assessed and the target can be captured while the concentration of the probe is about 0.1 M (Figure S9). The probe 2 was also used to capture target from mixture of SRCSH2 and excess cell lysates and the results were shown in Figure 4 Lanes 3. It is clear that probe 2 also demonstrated high specificity to its target protein in the complex proteomes,

which indicates the recognition of SRCSH2 mainly focuses on pY site. High Efficient Identification of Endogenous pYB Proteins from Cell Lysates. Inspired by the successful application of the probes in target enrichment, we further investigate the applicability of our probe to capture endogenous pYB domain proteins. We set up comparative proteomics experiments with probe 1 and C1 to effective enrich binding proteins from Hela cell lysates. In addition, UV-control experiment (the same conditions except no UV cross-linking) was performed to eliminate false-positive targets from indirect interaction. The probes were incubated with cell lysates and subsequently subject to UV irradiation, followed by LC-MS/MS analysis to quantify protein abundance (Figure 5A). The data-independent acquisition (DIA) approach, which has recently been introduced as preferred method for quantitative protein profiling due to its high reproducibility and precision,27 was used to quantify difference between probes. After excluding proteins with both ratios of probe 1/probe C1 and UV/-UV 1.5 (both ratios of probe 2/C2 and UV/-UV) are considered as potential binders. Ratios >20 are approximated as 20. Known SH2 domain proteins are colored in red. (C) Visualization of several proteins containing SH2, PTB and C2 domain identified by probe 2 (see Table S1 for details). (D) Venn diagrams showing the number of SH2 domain proteins identified by probe 2 and other reports (ref.12 and ref.28). (E) Western blotting analysis for the captured binders by probe 2 and C2 respectively. Cell lysate was photo-crosslinked with the probe and enriched proteins were detected by STAT3 and PKN2 antibodies, respectively. Equal loading is validated by SDS-PAGE of enriched samples before immunoblotting (Figure S10).

CONCLUSION In summary, we have developed an efficient pY-peptide photoaffinity probe for robust and selective enrichment of its binding proteins. By inserting the photo-cross-linker in the appropriate position, the probe enables to convert weak and transient pY mediated protein-protein interactions into covalent ones, which greatly promotes the capture of those low affinity and low abundance target proteins. Indeed, our

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