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High-density monitoring of tyrosine phosphorylation signaling targets in human tumors using internal standard triggered targeted quantitation

ABSTRACT: SureQuant pTyr, a targeted, tyrosine phosphorylation (pTyr) mass spectrometry method, was applied to profile the pTyr signatures of human colorectal tumor samples. Using internal standard guided aquisition with the SureQuant acquisition mode on the Oribtrap Exploris 480 mass spectrometer (Thermo Scientific), the real-time detection of trigger peptides during analysis initiates the sensitive and selective quantitation of endogenous pTyr targets. This framework allows for reliable quantification of several hundred commonly dysregulated pTyr targets with high quantitative accuracy and improves the robustness and usability of targeted aquisition.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture, Colon

DISEASE(S): Colon Cancer

SUBMITTER: Lauren Stopfer

LAB HEAD: Forest White

PROVIDER: PXD019426 | Pride | 2021-03-29

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
	A549_1_pTyr_SQ.raw	Raw
	A549_2_pTyr_SQ.raw	Raw
	A549_3_pTyr_SQ.raw	Raw
	A549_pTyr_SQ_Search.csv	Csv
	Tumor10_pTyr_SQ.raw	Raw

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High-density monitoring of tyrosine phosphorylation signaling targets in human tumors using internal standard triggered targeted quantitation

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