Dataset Information

Proteome analysis of Mel426 melanoma cells with R2PD1 treatment

ABSTRACT: A label-free quantification experiment was performed to study the effect of R2PD1 chimeric protein treatment on the cellular proteome of Mel426 human melanoma cells. Peptides were separated by online reverse phase liquid chromatography and measured in an Exploris 480 Orbitrap mass spectrometer. Mass spectrometry raw data were processed by MaxQuant software package (version 2.1.3.0) using its built-in Andromeda search engine. Mass spectra were searched against a target-decoy database consisting of the forward and reverse sequences of UniProt human reference proteome (release 2022_04; 102,601 entries) and a list of 246 common contaminants. Trypsin/P specificity was chosen. The minimum peptide length was set to be 7 amino acids. False discovery rate (FDR) was set to 1% for both peptide and protein identifications. MaxLFQ algorithm was employed for protein quantification.

INSTRUMENT(S): Orbitrap Exploris 480

ORGANISM(S): Homo Sapiens (ncbitaxon:9606)

SUBMITTER: Christof Niehrs

PROVIDER: MSV000091500 | MassIVE | Thu Mar 16 08:11:00 GMT 2023

SECONDARY ACCESSION(S): PXD040916

REPOSITORIES: MassIVE

ACCESS DATA

Publications

ROTACs leverage signaling-incompetent R-spondin for targeted protein degradation.

Sun Rui R Meng Zibo Z Lee Hyeyoon H Offringa Rienk R Niehrs Christof C

Cell chemical biology 20230614 7

Proteolysis-targeting chimeras (PROTACs) are an emerging technology for therapeutic intervention, but options to target cell surface proteins and receptors remain limited. Here we introduce ROTACs, bispecific WNT- and BMP-signaling-disabled R-spondin (RSPO) chimeras, which leverage the specificity of these stem cell growth factors for ZNRF3/RNF43 E3 transmembrane ligases, to target degradation of transmembrane proteins. As a proof-of-concept, we targeted the immune checkpoint protein, programmed ...[more]

PMID: 37321224

Similar Datasets

Project description:Wild type yeast cells were metabolically labeled in SILAC medium. The heavy (H)-SILAC-labeled cells were then continuously exposed to 0.01% MMS to induce replication stress, and the light (L)-SILAC-labeled cells were mock-exposed. After 3 hours, heavy and light cells were harvested and lysed. To ensure reproducibility, the entire experiment was repeated, and the labels were swapped such that the (L)-SILAC-labeled wild type yeast cells were exposed to 0.01% MMS for 3 hours, and the (H)-SILAC-labeled wild type yeast cells were mock-exposed. Lysates from heavy and light cells were mixed 1:1 by protein mass, reduced and treated with iodoacetamide and then digested with trypsin. 5 mg of each protein lysate was fractionated by high-pH reverse phase (RP) liquid chromatography into 12 samples. For proteome analysis, 2% of each fraction was dried down and re-suspended for LC-MS/MS analysis. The remaining 98% was processed to enrich for phosphopeptides using immobilized metal affinity chromatography (IMAC), dried down, and re-suspended in 0.1% FA, 3% ACN for LC-MS/MS analysis. Global and phosphopeptide-enriched samples were analyzed by LC-MS/MS on a Thermo LTQ-Orbitrap Velos mass spectrometer. Raw MS/MS spectra were searched against version 3.69 of the Yeast International Protein Index (IPI) sequence database using three independent search engines (MaxQuant/Andromeda, Spectrum Mill, and xTandem). All searches were performed with the tryptic enzyme constraint set for up to two missed cleavages, oxidized methionine set as a variable modification, and carbamidomethylated cysteine set as a static modification. For MaxQuant, the peptide MH+ mass tolerances were set at 20 ppm. For X!Tandem, the peptide MH+ mass tolerances were set at ±2.0 Da with post-search filtering of the precursor mass to 50 ppm and the fragment MH+ mass tolerances were set at ±0.5 Da. For Spectrum Mill, peptide MH+ mass tolerances were set at 20 ppm and fragment MH+ mass tolerances were set at ±0.7 Da. The overall FDR was set at ≤3% based on a decoy database search. Phosphosite localization probabilities are reported in the MaxQuant results. Any site with a probability greater than 0.8 was considered to be localized. Quantification of the Heavy:Light ratios was performed using MaxQuant software, with a minimum ratio count of 2 and using unique + razor peptides for quantification.

Project description:The covalent attachment of methyl groups to the side-chain of arginine residues is known to play essential roles in regulation of transcription, protein function and RNA metabolism. The specific N-methylation of arginine residues is catalyzed by a small family of gene products known as protein arginine methyltransferases; however, very little is known about which arginine residues become methylated on target substrates. Here we describe an unbiased methodology that combines single-step immunoenrichment of methylated peptides with high-resolution mass spectrometry to identify endogenous arginine mono-methylation (MMA) sites. We thereby identify 1,027 site-specific MMA sites on 494 human proteins, discovering numerous novel mono-methylation targets and confirming the majority of currently known MMA substrates. Nuclear RNA-binding proteins involved in RNA processing, RNA localization, transcription, and chromatin remodeling are prominently found modified with MMA. Despite this, MMA sites prominently are located outside RNA-binding domains as compared to the proteome-wide distribution of arginine residues. Quantification of arginine methylation in cells treated with Actinomycin D uncovers strong site-specific regulation of MMA sites during transcriptional arrest. Interestingly, several MMA sites are down-regulated after a few hours of under transcriptional arrest. In contrast, the corresponding di-methylation or protein expression level is not altered in expression, confirming that MMA sites contain regulated functions on their own. Collectively, we present a site-specific MMA dataset in human cells and demonstrate for the first time that MMA is a dynamic post-translational modification regulated during transcriptional arrest by a hitherto uncharacterized arginine demethylase. Data analysis: All raw data analysis was performed with MaxQuant software suite version 1.2.6.20 supported by the Andromeda search engine. Data was searched against a concatenated target/decoy (forward and reversed) version of the UniProtKB Human database encompassing 71,434 protein entries. Mass tolerance for searches was set to maximum 7 ppm for peptide masses and 20 ppm for HCD fragment ion masses. Data was searched with carbamidomethylation as a fixed modification and protein N-terminal acetylation, methionine oxidation and mono-methylation on lysine and arginine as variable modifications. A maximum of three mis-cleavages was allowed while requiring strict trypsin specificity, and only peptides with a minimum sequence length of seven were considered for further data analysis. Peptide assignments were statistically evaluated in a Bayesian model on the basis of sequence length and Andromeda score. Only peptides and proteins with a false discovery rate (FDR) of less than 1% were accepted, estimated on the basis of the number of accepted reverse hits. Protein sequences of common contaminants such as human keratins and proteases used were added to the database.

Project description:Two datasets: StREM1.3: methyl-beta cyclodextrin-dependent distribution of StREM1.3 to detergent resistant and detergent soluble membrane fractions. SLAH3: localization of SLAH3 to detergent resistant and detergent soluble fractions dependent on co-expression with CPK21 and ABI1. Data analysis: Protein identiﬁcation and ion intensity quantitation was carried out by MaxQuant version 1.3.0.5. Spectra were matched against the Arabidopsis proteome (TAIR10, 35,386 entries) using Andromeda. Thereby, carbamidomethylation of cysteine was set as a ﬁxed modiﬁcation; oxidation of methionine as well as phosphorylation of serine, threonine, and tyrosine was set as variable modiﬁcations. Because label-free quantitation based on ion intensities was used, multiplicity was set to 1. Mass tolerance for the database search was set to 20 ppm on full scans and 0.5 Da for fragment ions. Retention time matching between runs was chosen within a time window of 2 min. Peptide false discovery rate (FDR), protein FDR was set to 0.01, and site FDR was set to 0.05. Contaminants and reverse hits identiﬁed by MaxQuant were excluded from further analys Protein identiﬁcation and ion intensity quantitation was carried out by MaxQuant version 1.3.0.5. Spectra were matched against the Arabidopsis proteome (TAIR10, 35,386 entries) using Andromeda. Thereby, carbamidomethylation of cysteine was set as a ﬁxed modiﬁcation; oxidation of methionine as well as phosphorylation of serine, threonine, and tyrosine was set as variable modiﬁcations. Because label-free quantitation based on ion intensities was used, multiplicity was set to 1. Mass tolerance for the database search was set to 20 ppm on full scans and 0.5 Da for fragment ions. Retention time matching between runs was chosen within a time window of 2 min. Peptide false discovery rate (FDR), protein FDR was set to 0.01, and site FDR was set to 0.05. Contaminants and reverse hits identiﬁed by MaxQuant were excluded from further analysis. PXD000211 is also assoicated with the same study/paper.

Dataset Information

Proteome analysis of Mel426 melanoma cells with R2PD1 treatment

Publications

ROTACs leverage signaling-incompetent R-spondin for targeted protein degradation.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure