Dataset Information

The role of ptpn21 in hematopoietic stem cells

ABSTRACT: To identify the downstream substrate/effector that mediates Ptpn21 function. We applied comprehensive phosphoproteomics analysis with Lin- cells isolated from Ptpn21-/- and Ptpn21+/+ mice. The analysis revealed many pTyr sites on molecules associated with cytoskeleton. Additionally, we present a list of Ptpn21-dependent targets and putative substrates including Septin1 with increased tyrosine phosphorylation.

ORGANISM(S): Mus Musculus

SUBMITTER: Chengkui Qu

PROVIDER: PXD011381 | iProX | Wed Oct 17 00:00:00 BST 2018

REPOSITORIES: iProX

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Publications

Ptpn21 Controls Hematopoietic Stem Cell Homeostasis and Biomechanics.

Ni Fang F Yu Wen-Mei WM Wang Xinyi X Fay Meredith E ME Young Katherine M KM Qiu Yongzhi Y Lam Wilbur A WA Sulchek Todd A TA Cheng Tao T Scadden David T DT Qu Cheng-Kui CK

Cell stem cell 20190314 4

Hematopoietic stem cell (HSC) quiescence is a tightly regulated process crucial for hematopoietic regeneration, which requires a healthy and supportive microenvironmental niche within the bone marrow (BM). Here, we show that deletion of Ptpn21, a protein tyrosine phosphatase highly expressed in HSCs, induces stem cell egress from the niche due to impaired retention within the BM. Ptpn21<sup>-/-</sup> HSCs exhibit enhanced mobility, decreased quiescence, increased apoptosis, and defective reconst ...[more]

PMID: 30880025

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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:Dynamic tyrosine phosphorylation is fundamental to a myriad of cellular processes. However, the inherently low abundance of tyrosine phosphorylation in the proteome and the inefficient enrichment of phosphotyrosine(pTyr)-containing peptides has led to poor pTyr peptide identification and quantitation, critically hindering researchers’ ability to elucidate signaling pathways regulated by tyrosine phosphorylation in systems where cellular material is limited. The most popular approaches to wide-scale characterization of the tyrosine phosphoproteome use pTyr enrichment with pan-specific, anti-pTyr antibodies from a large amount of starting material. Methods that decrease the amount of starting material and increase the characterization depth of the tyrosine phosphoproteome while maintaining quantitative accuracy and precision would enable the discovery of tyrosine phosphorylation networks in rarer cell populations. To achieve these goals, a novel method leveraging the multiplexing capability of tandem mass tags (TMT) and the use of trigger channels – cells treated with the promiscuous tyrosine phosphatase inhibitor pervanadate (PV) – selectively increased the relative abundance of pTyr-containing peptides. After PV facilitated selective fragmentation of pTyr-containing peptides, TMT reporter ions delivered sensitive quantitation of each peptide for the experimental samples while the quantitation from PV trigger channels was ignored. This method yielded up to 6.3-fold boost in quantification depth of statistically significant data derived from contrived ratios, compared to TMT without trigger channels or intensity-based label-free (LF) quantitation while maintaining quantitative accuracy and precision, allowing quantitation of over 2300 unique pTyr peptides from only 1 mg of T cell receptor-stimulated Jurkat T cells. The trigger channel strategy can potentially be applied in analyses of other post-translational modifications where treatments that broadly boost the levels of those modifications across the proteome are available.

Dataset Information

The role of ptpn21 in hematopoietic stem cells

Publications

Ptpn21 Controls Hematopoietic Stem Cell Homeostasis and Biomechanics.

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