Dataset Information

Rose petal whole protein and proteomics of phosphorylation and ubiquitination modification

ABSTRACT: The 4D label-free quantitative proteomics (4D-LFQ) was used for quantification of proteome changes. Enrichment of phosphorylated peptides using immobilized metal ion affinity chromatography (IMAC) and enrichment of ubiquitinated peptides using anti-diglycine remnant (anti-K-ε-GG) antibodywere performed in phosphoproteome and ubiquitylome, respectively. Phosphorylation and ubiquitination quantifications were normalized by protein quantifications to eliminate the interference caused by protein abundance differences.

ORGANISM(S): Rosa Chinensis

SUBMITTER: Yulin Cheng

PROVIDER: PXD040139 | iProX | Wed Feb 15 00:00:00 GMT 2023

REPOSITORIES: iProX

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Publications

Integrated proteomic analysis reveals interactions between phosphorylation and ubiquitination in rose response to Botrytis infection.

Li Rui R Yao Juanni J Ming Yue Y Guo Jia J Deng Jingjing J Liu Daofeng D Li Zhengguo Z Cheng Yulin Y

Horticulture research 20231114 1

As two of the most abundant post-translational modifications, phosphorylation and ubiquitination play a significant role in modulating plant-pathogen interactions and increasing evidence indicates their crosstalk in plant immunity. Rose (Rosa sp.) is one of the most important ornamental plants and can be seriously infected by Botrytis cinerea. Here, integrated proteomics analysis was performed to detect global proteome, phosphorylation, and ubiquitination changes in rose upon B. ...[more]

PMID: 38222823

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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.

Dataset Information

Rose petal whole protein and proteomics of phosphorylation and ubiquitination modification

Publications

Integrated proteomic analysis reveals interactions between phosphorylation and ubiquitination in rose response to <i>Botrytis</i> infection.

Similar Datasets

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