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Scalable Phosphotyrosine Enrichment with SH2 Superbinder Enables Deep Profiling of EGF Responses

ABSTRACT: Phosphotyrosine signaling plays a critical role in many biological processes, from cell proliferation to immune response. Despite its importance, proteomic studies of tyrosine phosphorylation have been limited in scale and throughput by costly reagents and labor-intensive protocols. To address this, we developed R2HaPpY, a phosphotyrosine enrichment method combining simplified superbinder reagent preparation and automated high-throughput enrichment. Our new reagent binds phosphotyrosine peptides at higher efficiency than existing enrichment reagents and reduces both cost and preparation time by 20-fold. We generalized R2HaPpY to samples of low and high phosphotyrosine levels, and benchmarked it on EGF signaling dynamics in HeLa cells. Using only ~1 mg of input peptides, we quantify 1,651 unique phosphotyrosine sites, including 878 regulated phosphotyrosine sites, many novel or not previously annotated as EGF-responsive. Our results reveal differential temporal regulation and represent the largest phosphotyrosine dataset of EGF stimulation response to date. This streamlined, cost-effective, and sensitive method enables quantitative mapping of tyrosine phosphorylation dynamics at the scale of hundreds of samples, facilitating integration of phosphotyrosine signaling into multiomic studies across diverse biological systems.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human) Saccharomyces Cerevisiae (baker's Yeast) Saccharomyces Cerevisiae

TISSUE(S): Cell Suspension Culture, Epithelial Cell, Cell Culture

DISEASE(S): Cervix Carcinoma,Disease Free

SUBMITTER: Alexis Chang

LAB HEAD: Judit Villen

PROVIDER: PXD062515 | Pride | 2026-07-03

REPOSITORIES: Pride

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			Action	DRS
	49301_x09885_AC080_EGF3min_1_tp_60mDDA_0_5ug_20ppm_2missedTryp_2024_03_21.mzML	Mzml
	49301_x09885_HeLaEGFtimecourse_EGF3min_rep1_TotalProteome.fasta	Fasta
	49301_x09885_HeLaEGFtimecourse_EGF3min_rep1_TotalProteome.mzid	Mzid
	49301_x09885_HeLaEGFtimecourse_EGF3min_rep1_TotalProteome.pepXML	Pepxml
	49301_x09885_HeLaEGFtimecourse_EGF3min_rep1_TotalProteome.raw	Raw

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Project description:Albeit much less abundant than Ser/Thr phosphorylation (pSer/pThr), Tyr phosphorylation (pTyr) is considered a hallmark in cellular signal transduction. However, its analysis remains a challenge. The conventional immunopurification (IP) approach using antibodies pan-specific to pTyr sites is known to have low sensitivity, poor reproducibility and high cost. SH2 domain-derived pTyr-superbinder is a good replacement of pTyr antibody for the specific enrichment of pTyr peptides for phosphoproteomics analysis. In this study, we aim to optimize the approach using SH2 superbinder for tyrosine phosphoproteome analysis. The present work covalently immobilized SH2 superbinders to agrose beads, designed and evaluated four different SH2 superbinder based experimental workflows for phosphotyrosine analysis. After made a head to head comparison of them, we observed that the combined strategy employing Ti4+-IMAC and covalently immobilized SH2 superbinder enrichment in sequence generated the largest pTyr peptide dataset (3519) with the best selectivity (90%). Next the optimal method was applied for pTyr profiling from normal mouse tissues, and resulted in the identification of 197 pTyr sites (of which 73 were novel) from 5 mg protein digests, which validated its high sensitivity. An comparison with antibody 4G10 by isolating pTyr peptides from 5 mg unstimulated Jurkat cell digests were made, our optimal strategy identified 343 while the antibody 4G10 based method identified 242 pTyr sites, respectively, further confirmed the robustness of the method. Finally, the optimal strategy was applied for quantitative pTyr phosphorylation analysis of EGF stimulated/unstimulated HeLa cells, 261 pTyr sites were successfully quantified from 5 mg stable-isotope dimethyl labling protein digests. In general, the combination of Ti4+-IMAC and SH2 superbinder enrichment in sequence generated a facial and robust strategy for qualitative and quantitative analysis of pTyr proteome.

Project description:Cysteine is a rare and conserved amino acid involved in most cellular functions. The thiol group of cysteine can be subjected to diverse oxidative modifications that regulate most physio-pathological states. Here, a Cysteine-specific Phosphonate Adaptable Tag (CysPAT) was synthesized to selectively label cysteine-containing peptides (Cys peptides) followed by enrichment with titanium dioxide (TiO2) and mass spectrometric analysis. The CysPAT strategy was developed using a synthetic peptide, a standard protein and subsequently the strategy was applied to Hela cells lysate, achieving high specificity and enrichment efficiency. In particular,From 500µg of Hela cell lysate, the method led to the identification of 9229 unique Cys peptides starting from 500µg of Hela cell lysate . the CysPAT labelled Cys peptides showed increased hydrophobicity. Finally, the method was further developed to simultaneously enrich Cys peptides and phosphorylated peptidesThe method was further developed to simultaneously enrich Cys peptides and phosphorylated peptides from SILAC labelled Hela cells subjected to 5 min epidermal growth factor (EGF) stimulation. In total, of 22972 unique reversibly modified Cys peptides (3886 proteins) and 18549 unique phosphopeptides (2257 proteins) were simultaneously identified by Orbitrap Fusion. Significant regulation was observed in both phosphorylation and reversible Cys modification of proteins interacted with EGF receptor (EGFR)-binding proteins and proteins involved in EGFR signaling pathway. EGF stimulation can activate the phosphorylation of EGFR and downstream signaling molecules, such as mitogen-activated protein kinases (MAPK1 and MAPK3), meanwhile, it also leads to the activation of multiple protein tyrosine phosphatases (PTPs) by reduction of the catalytic Cys site in the conserved putative phosphatase HC(X)5R motif, Llater the activated PTPs would dephosphorylate and inactivate the molecules activated by EGF stimulation. Overall, the CysPAT strategy is a promising tool for studying redox proteomics and the simultaneous enrichment strategy offers an excellent solution for characterization of cross-talk between phosphorylation and redox induced cysteine modifications.

Project description:Epidermal growth factor (EGF) is a potent mitogenic agent promoting cell differentiation, proliferation, and growth. The kidney thick ascending limb of Henle’s loop and distal convoluted tubule were identified as major sites of EGF synthesis. As expected, EGF can modulate the functioning of kidney collecting duct including osmotic water permeability (Pf). Here, we found that EGF (0.1 uM) reduced the vasopressin-stimulated Pf by 26% in isolated perfused rat inner medullary collecting duct (IMCD). Immunoblotting using IMCD suspensions prepared in similar condition showed that EGF significantly reduced phosphorylation of AQP2 at Ser264 and Ser269 but had not affected on Ser256 or proline-directed Ser261 site. Quantitative phosphoproteomics was carried out to investigate other effects of EGF. Rat IMCD suspensions were treated with 1 M of EGF or vehicle for 30 min. Samples from three replicates of experiment were analyzed by TMT isobaric labeling quantitative protein mass spectrometry. We identified 23859 phosphopeptides with unique phosphorylation sites in EGF-treated rat IMCD samples, with 25 proteins increased and 100 proteins decreased in phosphorylation as determined by dual statistical analysis. EGF induced phosphorylation of multiple residues at the C-terminus of EGF receptor, EGFR or Erbb1, and mildly activated the classical MAPK pathway, RAF-MEK-ERK. EGF also affected phosphorylation of several proteins in PI3K/Akt pathway involved in cap-dependent translation initiation (Eif4ebp1, Gigyf2, Eif3b, Eif4g3, Pdcd4) and translation elongation (Eef2k, Eef2), predicted an overall translation repression. In comparison with a dDAVP-induced phosphoproteome, we found that the majority of proteins had phosphorylation change on specific site by either EGF or dDAVP but not both, suggesting a highly selectivity of phosphorylation modification induced by these two agents. Among the 25 peptides that had phosphorylation change on the same site by both EGF and dDAVP, AQP2 at Ser264/Ser269 was changed in opposite direction. We concluded that the short-term effect of EGF is insufficient to cause an effect on Ser261 phosphorylation of AQP2 in normal rat IMCD.

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Scalable Phosphotyrosine Enrichment with SH2 Superbinder Enables Deep Profiling of EGF Responses

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