Dataset Information

Proteomics of NGF-TrkA signaling dynamics in neuroblastoma cells

ABSTRACT: We used quantitative mass spectrometry-based proteomics to unravel global nerve growth factor (NGF)-induced TrkA signaling dynamics at the interactome, phosphoproteome and proteome level. A tetracycline-inducible system for TrkA expression was generated in the human neuroblastoma cell line, SH-SY5Y. TrkA-induced cells were stimulated with NGF for different time points to follow phosphoproteome, interactome and proteome changes on a temporal scale. In a triple SILAC setup (Light: Lys0,Arg0; Medium: Lys4,Arg6; and Heavy: Lys8,Arg10), the samples were stimulated with NGF as indicated. Phosphoproteome: 0, 10, 45 min and 0, 120, 120 min+cycloheximide. Interactome: 0, 5, 10 min. Proteome: 0, 24, 48 h. All experiments were performed as biological replicates.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Epithelial Cell

DISEASE(S): Neuroblastoma

SUBMITTER: Kristina Bennet Emdal

LAB HEAD: Jesper V. Olsen

PROVIDER: PXD001115 | Pride | 2015-05-06

REPOSITORIES: Pride

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Publications

Temporal proteomics of NGF-TrkA signaling identifies an inhibitory role for the E3 ligase Cbl-b in neuroblastoma cell differentiation.

Emdal Kristina B KB Pedersen Anna-Kathrine AK Bekker-Jensen Dorte B DB Tsafou Kalliopi P KP Horn Heiko H Lindner Sven S Schulte Johannes H JH Eggert Angelika A Jensen Lars J LJ Francavilla Chiara C Olsen Jesper V JV

Science signaling 20150428 374

SH-SY5Y neuroblastoma cells respond to nerve growth factor (NGF)-mediated activation of the tropomyosin-related kinase A (TrkA) with neurite outgrowth, thereby providing a model to study neuronal differentiation. We performed a time-resolved analysis of NGF-TrkA signaling in neuroblastoma cells using mass spectrometry-based quantitative proteomics. The combination of interactome, phosphoproteome, and proteome data provided temporal insights into the molecular events downstream of NGF binding to ...[more]

PMID: 25921289

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Project description:Protein extraction and proteolytic digestion were performed using a Filter-Assisted Sample Preparation (FASP) protocol. In case of phosphopeptide enrichment immobilized Fe(III) affinity chromatography (Fe-IMAC) was performed. The peptides were fractionated using an HPLC system fitted with an SCX column. 10 sample datasets were used: 1. CRC_N: Normal tissue samples were obtained from 20 colorectal cancer patients, no quantification.Phosphopeptide enrichment was performed. 2. CRC_T: Human colorectal cancer tissue samples were obtained from 20 patients, no quantification. Phosphopeptide enrichment was performed. 3. CRC_iTRAQ: Human colorectal cancer tissue samples were obtained from 24 patients, iTRAQ quantification. 4. CRC_phospho_iTRAQ:Human colorectal cancer tissue samples were obtained from 24 patients, iTRAQ quantification.Phosphopeptide enrichment was performed. 5. HCT116_iTRAQ: Human colon cancer cell HCT116, iTRAQ quantification. 6. HCT116_phospho_iTRAQ: Human colon cancer cell HCT116, phosphopeptide enrichment, iTRAQ quantification. 7. SW_SILAC_HL: Human colon cancer cell SW480 and SW620, SW480 and SW620 were grown in SILAC media, containing l-arginine (Arg0) and l-lysine (Lys0) (SW480; light), or 13C615N4-l-arginine (Arg10) and 13C6-l-Lysine (Lys6) (SW620; heavy). 8. SW_SILAC_LH: SW480 and SW620 were grown in SILAC media, containing l-arginine (Arg0) and l-lysine (Lys0) (SW620; light), or 13C615N4-l-arginine (Arg10) and 13C6-l-Lysine (Lys6) (SW480; heavy). 9. SW_phospho_SILAC_HL: SW480 and SW620 were grown in SILAC media, containing l-arginine (Arg0) and l-lysine (Lys0) (SW480; light), or 13C615N4-l-arginine (Arg10) and 13C6-l-Lysine (Lys6) (SW620; heavy). Phosphopeptide enrichment was performed. 10. SW_phospho_SILAC_LH: SW480 and SW620 were grown in SILAC media, containing l-arginine (Arg0) and l-lysine (Lys0) (SW620; light), or 13C615N4-l-arginine (Arg10) and 13C6-l-Lysine (Lys6) (SW480; heavy). Phosphopeptide enrichment was performed. For creation of xml and mgf files, Proteome Discoverer 1.3 and Mascot v2.3 software were used.

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Dataset Information

Proteomics of NGF-TrkA signaling dynamics in neuroblastoma cells

Publications

Temporal proteomics of NGF-TrkA signaling identifies an inhibitory role for the E3 ligase Cbl-b in neuroblastoma cell differentiation.

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