Analysis of Cisplatin-Induced Changes in P53 Complexes in HEK293 Cells

CaptiVate Shotgun Proteomics Services - Service Procedure Example: Analysis of cisplatin-induced changes in p53 complexes in HEK293 cells

Part I: Bait construction and stable cell line generation p53 cDNA •• Bait: human full-length p53 •• Stable expression of Strep-tagged p53 in HEK293 cells p53-HEK293 line

Service Procedure - Part I Part II: Cisplatin treatment, affinity purification and quantitative LC-MS/MS analysis

•• Treatment of cells with cisplatin, non-treated control Non-treated Treated •• Cell lysis •• Strep-tag, high-speed affinity purification of p53 complexes •• Quantitative analysis of 3 replicates each •• Data filtering against protein frequency library •• Quantitative description of protein complex changes upon cisplatin treatment

Results

•• 91 candidate interactors identified

•• 59 interactors after data filtering (including 21 known interactors) Unchanged Upregulated Known interactors (selected): Protein name Protein description Downregulated Mdm2 Isoform 11 of E3 ubiquitin-protein ligase Mdm2 Cisplatin treatment HSP90AB1 Heat shock protein HSP 90-beta Service Procedure - Part II HNGS1 Isoform 1 of Double-strand break repair protein MRE11A HNGS1 POH1 ERR1 RPL11 Isoform 1 of 60S ribosomal protein L11 new PSMB7 new CHIP CUL9 281 kDa protein;Isoform 1 of Cullin-9;Isoform 2 of Cullin-9 MDM2 ABCD3 HSP90AB1 PSMC1 PDHX HSP90AB2P HSPA1 Heat shock 70 kDa protein 1 CUL9 PSMB3 HSP70B Heat shock 70 kDa protein 6 FIGC new AIP3 CUL7 CCT5 T-complex protein 1 subunit epsilon BAG5 RAD50 new SCP1 UBC ubiquitin C;39 kDa protein ADRBK2 HSC70 GRP75 Stress-70 protein, mitochondrial new MYH3 MSTP030 60S ribosomal protein L5 KIAA1027 CAPE HSP90AB4 new CCT3 P53 new Novel interactors (selected): CCT4 HSP90A Protein name Protein description CCT7 CCT5 CCT6 KIAA BAG2 BAG family molecular chaperone regulator 2 99D8.1 CCT1 STUB1 Isoform 1 of STIP1 homology and U box-containing protein 1 DNAJC7 STIP1 ATG13 TRIM28 Isoform 1 of Transcription intermediary factor 1-beta HSP90AB1 CPR3 DAAP21 IGKV4-1 Microfibrillar protein 2 HSPA1 AIP AKT1 FAM10A1 ERH Enhancer of rudimentary homolog AOF2 AIG6 RPS6KB1 KPNA1 DNAJ2 new HSP70B SUGT1 MCCB Isoform 1 of Methylcrotonoyl-CoA carboxylase beta chain, mitochondrial BAG2 EDD PCCB 62 kDa protein;Propionyl Coenzyme A carboxylase, beta polypeptide, DAAP NUDC LYAR isoform CRA_c MCCA Methylcrotonoyl-CoA carboxylase subunit alpha, mitochondrial known new TCOF1 Isoform 4 of Treacle protein -20 20 ACAC Isoform 4 of Acetyl-CoA carboxylase 1;268 kDa protein Cytoscape network graph DNAJA1 DnaJ homolog subfamily A member 1 RAD50 Isoform 2 of DNA repair protein RAD50 AKT1 RAC-alpha serine/threonine-protein kinase IRS4 Insulin receptor substrate 4 EDD E3 ubiquitin-protein ligase UBR5

Upregulated proteins: CAPE, SCP1, EDD Downregulated proteins: ATG13, SUGT1, DNAJ2