Sensitive analysis of protein tyrosine phosphorylation
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo Sapiens (human)
SUBMITTER: Mingliang Ye
PROVIDER: PXD007998 | JPOST Repository | Sat Oct 20 00:00:00 BST 2018
REPOSITORIES: jPOST
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SH2_beads_1000ug_1.raw | Raw | |||
| SH2_beads_1000ug_2.raw | Raw | |||
| SH2_beads_100ug_1.raw | Raw | |||
| SH2_beads_100ug_2.raw | Raw | |||
| SH2_beads_200ug_1.raw | Raw |
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Project description:A comprehensive analysis of the phosphoproteome is essential for understanding molecular mechanisms of human diseases. However, current tools to enrich phosphotyrosine are limited in their applicability and scope. Here, we engineered new superbinder SH2 domains that enrich diverse sets of phosphotyrosine peptides. We used phage display to select a Fes SH2 domain variant with high affinity for phosphotyrosine (superFes-SH2, sFes1) and solved the structure of sFes1 bound to a phosphopeptide. We performed systematic structure-function analyses of the superbinding mechanisms of sFes1 and superSrc-SH2 (sSrc1), another SH2-superbinder. We grafted the superbinder motifs from sFes1 and sSrc1 into 17 additional SH2 domains and confirmed increased binding affinity for specific phosphopeptides. Using mass spectrometry, we demonstrated that SH2 superbinders have distinct specificity profiles and superior capabilities to enrich phosphotyrosine peptides. Finally, combinations of SH2 superbinders as affinity purification tools showed that unique subsets of phosphopeptides can be enriched with unparalleled depth and coverage.
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Project description:Tyrosine phosphorylation plays a major role in regulating cell signaling pathways governing diverse biological functions such as proliferation and differentiation. Systemically mapping phosphotyrosine (pTyr) sites is the key to understand molecular mechanisms underlining pTyr-dependent signaling. Although mass spectrometry-based technologies have been widely used for pTyr site profiling and quantification, their applications are often hindered by the poor efficiency in current multi-step enrichment procedures for inherently low abundance pTyr peptides, especially under physiological conditions. Taking advantage of the sequence-independent high affinity of SH2 superbinder towards pTyr residues, we have developed a simplified one-step pTyr peptide enrichment method that uses immobilized SH2 superbinder for unbiased and robust enrichment of endogenous pTyr peptides from biological samples.
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Project description:LC-MS/MS was used to profile total phosphotyrosine phosphatase in acute myeloid leukemia (AML) in order to find potential biomarkers, drug targets, signatures for AML classification, and its relationship with total phosphotyrosine amount in the samples.
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Project description:Phosphorylation is a crucial component of signaling cascades in a cell. It controls various biological cellular functions such as cell growth and apoptosis. Due to the low stoichiometry of phosphorylaion, enrichment of phosphopeptide prior to LC-MS/MS is necessary for comprehensive phosphoproteome analysis and typical quantitative phosphoproteomic workflows are often limited by the amount of sample input. To address this issue, we developed an easy-to-establish, widely applicable and reproducible strategy to amplify phosphoproteome signals from a small amount of sample without phosphoenrichment step. Taking advantage of the multiplexing nature of isobaric labeling to achieve merged signal from multiple samples, and using a large amount of enriched phosphopeptides as a carrier, we were able to amplify a trace amount of phosphopeptide in the unpurified sample to an identifiable level and quantify it with the reporter ion intensity of the isobaric tag. Our result showed that >1400 phosphopeptide were quantified from 250 ng of tryptic peptides of cell extract. Through proof-of-concept experiments of our strategy, we distinguished 3 types of lung cancer cell line based on their quantitative phosphoproteome data and also identified phosphoproteome changes upon cellular perturbation achieved by drug treatment.
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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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Project description: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.
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Each participating laboratory will receive two sample vials. The vial labeled 'Phosphopeptide' contains a set of synthesized phosphorylated (Ser, Thr, or Tyr) peptides of human sequence origin at various concentrations, mixed with their non-phosphorylated counterparts. For some peptides, there is more than one phosphorylated form. The peptide sequences are included in the attached Excel file. The second vial labeled 'Phosphopeptide-Yeast' contains the same peptides in a background matrix consisting of 6 ug of trypsin-digested yeast lysate. Each vial is provided dry. Resuspension in 100 uL will result in synthetic peptide concentrations of 3 fmol/uL to 30 fmol/uL.
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1. Identify the peptide sequences in the vial and determine the number and location of phosphorylation sites on each peptide (Phosphopeptide).
2. Determine the relative abundance of phosphorylation at each modified site by comparison with its non-phosphorylated counterpart (Phosphopeptide).
3. Enrich for phosphorylated peptides from the sample containing the yeast background matrix and re-analyze by MS (Phosphopeptide-Yeast).
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