Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

[HPP-2.0] Dynamic phosphotyrosine-dependent signaling profiling in living cells by two-dimensional proximity proteomics

ABSTRACT: Tyrosine phosphorylation (pTyr)-dependent signaling pathways play vital role in various biological processes, which are spatiotemporally assembled and dynamically regulated on minute-scale by pTyr in living cells. Studying these pTyr-mediated signaling complexes is therefore challenging due to the highly dynamic nature of the protein complexes and the low abundance of pTyr. In this study, we adopted minute-resolution APEX2-based proximity labeling (PL) in living cells and Src SH2 superbinder-based pTyr peptide enrichment for simultaneously profiling these protein complexes and associated pTyr sites from the same affinity-purified sample. Upon different time-course of EGF stimulation of the living cells stably expressing APEX2-FLAG-GRB2, we constructed two-dimensional time-course curves for both interactome and tyrosine phosphoproteome.

ORGANISM(S): Homo Sapiens

SUBMITTER: Ruijun Tian

PROVIDER: PXD049141 | iProX | Sat Feb 03 00:00:00 GMT 2024

REPOSITORIES: iProX

ACCESS DATA

Json Xml

Similar Datasets

Dynamic phosphotyrosine-dependent signaling profiling in living cells by two-dimensional proximity proteomics

Project description:Tyrosine phosphorylation (pTyr)-dependent signaling pathways play a vital role in various biological processes, which are spatiotemporally assembled and dynamically regulated on minute-scale by pTyr in living cells. Studying these pTyr-mediated signaling complexes is therefore challenging due to the highly dynamic nature of the protein complexes and the low abundance of pTyr. In this study, we adopted minute-resolution APEX2-based proximity labeling (PL) in living cells and Src superbinder-based pTyr peptide enrichment for simultaneously profiling these protein complexes and associated pTyr sites from the same affinity-purified sample. Upon different time-course of EGF stimulation of the living cells stably expressed with APEX2-fused adaptor protein GRB2, we constructed two-dimensional time-course curves for both GRB2 interactome and tyrosine phosphoproteome. Most of the known GRB2 interacting proteins and more than 40 pTyr sites were identified from each time point with acceptable quantification precision. Well-annotated pTyr signaling complexes in EGFR signaling and located at endosome were quantified with tightly correlated time-course curves for both interacting proteins and pTyr sites. Importantly, the correlated time-course curves for EGFR and endosomal HGS were well validated by PRM-based targeted MS analysis. Taking advantage of the high sensitivity of the PRM assay, the low abundant pTyr peptide EGFR pY1110 that cannot be identified in the DDA analysis could be well detected and quantified. Collectively, this two-dimensional proximity proteomic strategy is promising for comprehensively characterizing pTyr-mediated protein complexes with high sensitivity in living cells.

2022-07-06 | MSV000089826 | MassIVE

Alpha Factor arrest cell cycle synchronization time-course microarray in Glucose

Project description:Alpha Factor arrest cell cycle syncronization time-course microarray in Glucose. Time course from 0 to 60 minute.

2011-06-11 | GSE29893 | GEO

Alpha Factor arrest cell cycle synchronization time-course microarray in Galactose

Project description:Alpha Factor arrest cell cycle syncronization time-course microarray in Galactose. Time course from 0 to 40 minute.

2011-06-11 | GSE29892 | GEO

IgG-independent co-aggregation of FceRI and FcgRIIB results in LYN- and SHIP1-dependent tyrosine phosphorylation of FcgRIIB

Project description:We used tyrosine phosphorylation profiling by anti-pTyr-antibody mediated enrichment and subsequent analysis by mass spectrometry in order to determine the changes in early signalling after mast cell activation. Mast cells were stimulated with varying concentrations of antigen for one minute and the differences between optimal (20 ng/ml) and supra-optimal (2000 ng/ml) antigen mast cell activation were analysed by nanoLC-MS/MS.

2018-08-17 | PXD009318 | Pride

Photoaffinity engineered protein scaffold for systematically exploring native phosphotyrosine signaling complexes in tumor samples

Project description:Phosphotyrosine (pTyr)-regulated protein complexes play critical roles in cancer signaling. The systematic characterization of these protein complexes in tumor samples remains a challenge due to their limited access and the transient nature of pTyr-mediated interactions. We developed a hybrid chemical proteomic approach, termed Photo-pTyr-scaffold, by engineering Src homology 2 (SH2) domains, which specifically bind pTyr proteins, with both trifunctional chemical probes and genetic mutations to overcome these challenges. Dynamic SH2 domain-scaffolding protein complexes were efficiently crosslinked under mild UV light, captured by biotin tag and identified by mass spectrometry. This approach was successfully used to profile native pTyr protein complexes from breast cancer biopsies on a proteome scale with high selectivity, achieving about 100 times higher sensitivity for detecting pTyr signaling proteins than that afforded by traditional immunohistochemical methods. Among more than 1000 identified pTyr proteins, receptor tyrosine kinase PDGFRB expressed on cancer associated fibroblasts was validated as an important intercellular signaling regulator with poor expression correlation to ERBB2, and blockade of PDGFRB signaling could efficiently suppress tumor growth. The Photo-pTyr-scaffold approach may become a generic tool for profiling dynamic pTyr signaling complexes readily in clinical relevant samples.

2018-06-25 | MSV000082499 | MassIVE

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

Project description: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.

2021-03-29 | PXD019426 | Pride

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

Project description: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 acquisition 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 acquisition.

2022-02-16 | PXD021857 | Pride

Alpha Factor arrest cell cycle synchronization time-course microarray in Galactose

Project description:Alpha Factor arrest cell cycle syncronization time-course microarray in Galactose. Time course from 0 to 40 minute. Time-course experiment. Samples are hybridized onto the first timepoint.

2011-06-11 | E-GEOD-29892 | biostudies-arrayexpress

Alpha Factor arrest cell cycle synchronization time-course microarray in Glucose

Project description:Alpha Factor arrest cell cycle syncronization time-course microarray in Glucose. Time course from 0 to 60 minute. Time-course experiment. Samples are hybridized onto the first timepoint.

2011-06-11 | E-GEOD-29893 | biostudies-arrayexpress

Quantative pTyr protein complex

Project description:Our current research focuses on the complex-centric quantification of pTyr protein complexes. We initially constructed a tri-functional probe based on pTyr superbinder (SH2-S) for the enrichment of pTyr protein complexes. The pTyr protein complexes were separated by ion exchange chromatography (IEC). To further quantify the complexes, we developed an algorithm for co-fractionation/mass spectrometry (CF/MS) protein complex screening and quantification.

2024-05-31 | PXD052742 |

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data