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RPE-1 and RPE-1 trisomic AHA p-c and steady state data

ABSTRACT: We developed a pulse-chase method in where fully SILAC (heavy, medium-heavy and Light) labelled cells were pulsed with Azidohomoalanine (AHA) and then chased for different length of times. In addition, steady state protein levels comparing the RPE- and RPE-1 trisomic cells by standard SILAC labeling was also acquired. These datasets contain data for the human RPE-1 and RPE-1 trisomic cell lines.

INSTRUMENT(S): Q Exactive

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

SUBMITTER: Matthias Selbach

PROVIDER: MSV000080831 | MassIVE | Fri Mar 31 15:22:00 BST 2017

SECONDARY ACCESSION(S): PXD004915

REPOSITORIES: MassIVE

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Kinetic Analysis of Protein Stability Reveals Age-Dependent Degradation.

McShane Erik E Sin Celine C Zauber Henrik H Wells Jonathan N JN Donnelly Neysan N Wang Xi X Hou Jingyi J Chen Wei W Storchova Zuzana Z Marsh Joseph A JA Valleriani Angelo A Selbach Matthias M

Cell 20161006 3

Do young and old protein molecules have the same probability to be degraded? We addressed this question using metabolic pulse-chase labeling and quantitative mass spectrometry to obtain degradation profiles for thousands of proteins. We find that >10% of proteins are degraded non-exponentially. Specifically, proteins are less stable in the first few hours of their life and stabilize with age. Degradation profiles are conserved and similar in two cell types. Many non-exponentially degraded (NED) ...[more]

PMID: 27720452

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RPE-1 and RPE-1 trisomic AHA p-c and steady state data

Project description:We developed a pulse-chase method in where fully SILAC (heavy, medium-heavy and Light) labelled cells were pulsed with Azidohomoalanine (AHA) and then chased for different length of times. In addition, steady state protein levels comparing the RPE- and RPE-1 trisomic cells by standard SILAC labeling was also acquired. These datasets contain data for the human RPE-1 and RPE-1 trisomic cell lines.

2016-10-06 | PXD004915 | Pride

NIH 3T3 AHA p-c, SILAC p-c, inhibitor, and enrichment control experiments

Project description:We developed a pulse-chase method in where fully SILAC (heavy, medium-heavy and Light) labelled cells were pulsed with Azidohomoalanine (AHA) and then chased for different length of times. These experiments were performed with 0, 1, 2, 4, 8, 16 and 13 h of chase. Data from 3 biological replicates are provided. Each replicate contains data from 3 experiments (0, 1, 2 and 0, 4, 8 and 0, 16, 32 hours chase) in where the 0 h time point is always heavy labelled followed by the medium and light labelled time points. In addition, AHA p-c experiments were performed using only 0, 4 and 8 h chase times in combination with different inhibitor combinations (MG132, Actinomycin D and Wortmanninin + Bafilomycin A1) or control (DMSO). Also, a control enrichment data set was created. Here heavy SILAC labelled cells were pulsed with AHA before being mixed with light cells that were not pulsed with AHA. This was followed by the click reaction and enrichment of AHA containing proteins. Label-swap experiments were also performed. Finally, a SILAC p-c data set is provided. Here light cells were pulsed with heavy (Arg10, Lys8) amino acids and then split in two. One half of the cells was chased in medium (Arg6, Lys4) amino acids and the other part was directly frozen. Label-swap experiments and experiments using different pulse and chase times were also performed. All provided datasets are from mouse fibroblast cells (NIH 3T3).

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HEK293T cell degradome (pulse chase) analysis upon 12h Torin1 treatment. Related to Supplementary Table 3 from An et al (2020)

Project description:HEK293T Azidohomoalanine (AHA) Pulse Chase (degradome) experiment, were cells from various genetic background (WT, ATG7-/-, RB1CC1-/-) are treated or not with Torin1 for 12h. Samples are Biotin-click, and streptavidin enriched.

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HEK293T cell degradome (pulse chase) time course (5h, 10, 15h) analysis upon Torin1 treatment. Related to Supplementary Table 3 from An et al (2020)

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HEK293T cell degradome (pulse chase) time course (5h, 10, 15h) analysis upon Torin1 treatment. Related to Supplementary Table 3 from An et al (2020)

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semi-automated enrichment of AHA-containing newly-synthesized proteins with magnetic alkyne agarose beads

Project description:Development of an improved workflow for newly synthesized proteome analysis, based on combined pulsed metabolic labelling with L-azidohomoalanine (AHA) and semi-automated click chemistry-based enrichments with magnetic alkyne agarose beads.

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Thiouracil Pulse-chase, Tachyzoites and Bradyzoites mRNA stability data

Project description:Pulse chase measurements using thiouracil (DTU) labeling via UPRT and chasing with uracil Data from tachyzoites is labeled "DTU Pulse Chase". Two independent pulse chase experiments were performed in tachyzoites, pulse chase 1 and 2. Duplicate arrays at each timepoint were performed for pulse chase 2 (2 a and b). Data from bradyzoites are labeled "DTU Bradyzoite Pulse Chase". Two independent pulse chase experiments were performed in bradyzoites and a single set of arrays were performed for each experiment. Just one chase timepoint was used in the bradyzoite experiments, the 2 hour chase. An RNA stablity experiment design type examines stability and/or decay of RNA transcripts. Keywords: RNA_stability_design

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pSILAC mass spectrometry reveals ZFP91 as novel IMiD dependent substrate of the CRL4CRBN ligase

Project description:Thalidomide and its derivatives lenalidomide and pomalidomide (IMiDs) are effective treatments of hematologic malignancies. It was shown that IMiDs impart gain of function properties to the CUL4-RBX1-DDB1-CRBN (CRL4CRBN) ubiquitin ligase that enable binding, ubiquitination and degradation of key therapeutic targets such as IKFZ1, IKZF3 and CSNK1A1. While these substrates have been implicated as efficacy targets in multiple myeloma (MM) and 5q deletion associated myelodysplastic syndrome (del(5q)-MDS), other targets likely exist. Using a pulse-chase SILAC mass spectrometry-based proteomics approach, we demonstrate that lenalidomide induces the ubiquitination and degradation of ZFP91. We establish that ZFP91 is a bona fide IMiD dependent CRL4CRBN substrate and further show that ZFP91 harbors a zinc finger (ZnF) motif, related to the IKZF1/3 ZnF, critical for IMiD dependent CRBN binding. These findings demonstrate that single time point pulse-chase SILAC mass spectrometry-based proteomics (pSILAC-MS) is a sensitive approach for target identification of small molecules inducing selective protein degradation.

2017-03-01 | GSE94728 | GEO

Proteome wide measurements of protein half-lives in Mycoplasma pneumoniae

Project description:We obtained proteome wide measurements of protein half-lives in M. pneumoniae by performing pulse-chase experiments following SILAC methods. We estimated degradation rates of individual proteins by measuring the increase in light protein compared to heavy protein labeling along the growth curve.

2020-11-24 | PXD016386 | Pride