Dataset Information

Engineering human SH2 domains for targeted phospho-proteomics

ABSTRACT: A comprehensive analysis of the phosphoproteome is essential for understanding molecular mechanisms of human diseases. However, current tools to enrich phosphotyrosine are limited in their applicability and scope. Here, we engineered new superbinder SH2 domains that enrich diverse sets of phosphotyrosine peptides. We used phage display to select a Fes SH2 domain variant with high affinity for phosphotyrosine (superFes-SH2, sFes1) and solved the structure of sFes1 bound to a phosphopeptide. We performed systematic structure-function analyses of the superbinding mechanisms of sFes1 and superSrc-SH2 (sSrc1), another SH2-superbinder. We grafted the superbinder motifs from sFes1 and sSrc1 into 17 additional SH2 domains and confirmed increased binding affinity for specific phosphopeptides. Using mass spectrometry, we demonstrated that SH2 superbinders have distinct specificity profiles and superior capabilities to enrich phosphotyrosine peptides. Finally, combinations of SH2 superbinders as affinity purification tools showed that unique subsets of phosphopeptides can be enriched with unparalleled depth and coverage.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture

SUBMITTER: Ulrike Kusebauch

LAB HEAD: Robert L. Moritz

PROVIDER: PXD030038 | Pride | 2023-01-13

REPOSITORIES: Pride

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Dataset's files

Source:

			Action	DRS
	2021_04_16_Lumos_EvoSep_PepSep_25cm_1_5um_ReSyn_K562_1.mzML	Mzml
	2021_04_16_Lumos_EvoSep_PepSep_25cm_1_5um_ReSyn_K562_1.pep.xml	Pepxml
	2021_04_16_Lumos_EvoSep_PepSep_25cm_1_5um_ReSyn_K562_1.raw	Raw
	2021_04_30_Lumos_EvoSep_PepSep_25cm_1_5um_SB13.mzML	Mzml
	2021_04_30_Lumos_EvoSep_PepSep_25cm_1_5um_SB13.pep.xml	Pepxml

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Publications

Engineered SH2 Domains for Targeted Phosphoproteomics.

Martyn Gregory D GD Veggiani Gianluca G Kusebauch Ulrike U Morrone Seamus R SR Yates Bradley P BP Singer Alex U AU Tong Jiefei J Manczyk Noah N Gish Gerald G Sun Zhi Z Kurinov Igor I Sicheri Frank F Moran Michael F MF Moritz Robert L RL Sidhu Sachdev S SS

ACS chemical biology 20220525 6

A comprehensive analysis of the phosphoproteome is essential for understanding molecular mechanisms of human diseases. However, current tools used to enrich phosphotyrosine (pTyr) are limited in their applicability and scope. Here, we engineered new superbinder Src-Homology 2 (SH2) domains that enrich diverse sets of pTyr-peptides. We used phage display to select a Fes-SH2 domain variant (superFes; sFes<sup>1</sup>) with high affinity for pTyr and solved its structure bound to a pTyr-peptide. We ...[more]

PMID: 35613471

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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.