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Tolerogenic effect elicited by protein fraction derived from different formulas for dietary treatment of cow’s milk allergy in human cells

ABSTRACT: Comparative evaluation of tolerogenic effect elicited by the protein/peptide fraction of different formulas available for the dietary treatment of CMA. Peptides from digests of extensively hydrolyzed casein formula (EHCF) elicited tolerogenic effects. Peptides from EHCF and relevant digests (in vitro gastroduodenal simulated digestion with "infant" model) were charaterized by HPLC-MS/MS.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Bos Taurus (bovine)

TISSUE(S): Milk

SUBMITTER: Gianluca Picariello

LAB HEAD: Gianluca Picariello

PROVIDER: PXD023355 | Pride | 2021-02-09

REPOSITORIES: pride

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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.

			Action	DRS
	EHCF_1_replicate.raw	Raw
	EHCF_2_replicate.raw	Raw
	EHCF_3_replicate.raw	Raw
	EHCF_GD_1_replicate.raw	Raw
	EHCF_GD_2_replicate.raw	Raw

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Tolerogenic effect elicited by protein fraction derived from different formulas for dietary treatment of cow’s milk allergy in human cells

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