Sensitive analysis of protein tyrosine phosphorylation
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo Sapiens (human)
SUBMITTER: Mingliang Ye
PROVIDER: PXD007998 | JPOST Repository | Sat Oct 20 00:00:00 BST 2018
REPOSITORIES: jPOST
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SH2_beads_1000ug_1.raw | Raw | |||
| SH2_beads_1000ug_2.raw | Raw | |||
| SH2_beads_100ug_1.raw | Raw | |||
| SH2_beads_100ug_2.raw | Raw | |||
| SH2_beads_200ug_1.raw | Raw |
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Project description: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.
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Project description: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.
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Project description:To demonstrate the usability of our SIL peptides (JPT GmbH)on an MS application, 4 standard peptides were synthesized at 95% purity and each spiked at an amount 133 fmol into protein extracts of wild type HEK cells and EZH2-knockout cells, respectively. The spiked peptides in this experiment are histone H3 K27-containing heavy-arginine versions of the peptide ARKSAPATGGVKKPH-R, where K27 was synthetically modified to carry either no methylation, or one or two or three (me3) methyl groups, respectively. Previously to trypsination, the HEK samples were spiked with the 4 SIL peptides. Because the standards have 10 Da greater mass given by the heavy R*, they can be distinguished from their light natural versions in the MS1 spectra. Furthermore, the standards were each spiked at a concentration of 133 fmol and thus, their MS intensities were used to normalize the intensities of the endogenous peptides and quantify them in absolute terms.
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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.
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