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A tyrosine phosphoproteome analysis approach enabled by selective dephosphorylation with protein tyrosine phosphatase

ABSTRACT: Protein tyrosine phosphorylation (pTyr) plays a prominent role in signal transduction and regulation in all eukaryotic cells. In conventional immunoaffinity purification (IP) methods, phosphotyrosine peptides are isolated from the digest of cellular protein extracts with a phosphotyrosine-specific antibody and are identified by tandem mass spectrometry. However, the low sensitivity, poor reproducibility and cost-prohibitive are universal concerns for IP approaches. In this study, we presented an antibody-free approach to identify phosphortyrosine peptides by using protein tyrosine phosphatase (PTP). It was found that most of PTPs including PTP1B, TCPTP and SHP1 can efficiently and selectively dephosphorylated phosphotyrosine peptides. We then designed a workflow by combining two Ti4+-IMAC-based phosphopeptide enrichment steps with PTP catalyzed dephosphorylation for tyrosine phosphoproteomics analysis. This workflow was firstly validated by selectively detection of phosphotyrosine peptides from semi-complex sample and then applied to analyze the tyrosine phosphoproteome of Jurkat T cells. Around 1000 putative former phosphotyrosine peptides were identified from less than 500 μg of cell lysate. The tyrosine phosphosites on majority of these peptides could be unambiguously determined for over 70% of them possess only one tyrosine residue. It was also found that the tyrosine sites identified by this method was highly complementary to these identified by SH2 superbinder based method. Therefore, the combination of Ti4+-IMAC enrichment with PTP dephosphorylation provides an alternative and cost-effective approach for tyrosine phosphoproteomics analysis.

ORGANISM(S): Homo Sapiens

SUBMITTER: Mingliang Ye

PROVIDER: PXD045682 | iProX | Tue Sep 26 00:00:00 BST 2023

REPOSITORIES: iProX

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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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A tyrosine phosphoproteome analysis approach enabled by selective dephosphorylation with protein tyrosine phosphatase

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