Human HEK293 cells for SIALC-based quantitaiton
Ontology highlight
ABSTRACT:
INSTRUMENT(S): Orbitrap Fusion
ORGANISM(S): Homo Sapiens (human)
TISSUE(S): Early Embryonic Cell
SUBMITTER: 
Sheng Zhang
LAB HEAD: SHENG ZHANG
PROVIDER: PXD017774 | Pride | 2022-01-14
REPOSITORIES: Pride
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Project description:We used mass spectrometry-based proteomics to unravel anaplastic lymphoma kinase (ALK) signaling in the ALK and MYCN amplified neuroblastoma cell line, NB1. We specifically measured the ALK interactome, phosphoproteome, phosphotyrosine interactome and proteome as outlined below. ALK Interactome and phosphoproteome: For each experiment, three SILAC conditions (‘Light’: Lys0,Arg0, ‘Medium’: Lys4,Arg6, ‘Heavy’: Lys8,Arg10) of NB1 cells were treated for 30 minutes with each of the following ALK-targeting small-molecule inhibitors: TAE684 (100 nM), crizotinib (500 nM), and LDK378 (250 nM). The experiments were performed as specified below: Experiment 1: Light: 500 nM crizotinib, Medium: 100 nM TAE684, Heavy: 0.1% DMSO Experiment 2: Light: 100 nM TAE684, Medium: 250 nM LDK378, Heavy: 0.1% DMSO Experiment 3: Light: 250 nM LDK378, Medium: 500 nM crizotinib, Heavy: 0.1% DMSO These three triple-SILAC experiments allowed the effect of each inhibitor to be measured in duplicates. ALK phosphotyrosine interactome: To identify phosphotyrosine-specific interaction partners of ALK we used a label-free mass spectrometry-based proteomics approach. Interacting proteins to the peptide sequences (phosphorylated and non-phosphorylated peptide) indicated in the table below were identified by a peptide pull-down assay and mass spectrometry. Peptide pull-downs were performed on NB1 cell lysate prepared from cells treated for 30 minutes with LDK378 (250 nM) or DMSO. Each peptide pull-down was performed on lysates from two biological replicates. Sample name (raw file) Gene Uniprot Site Peptide sequence A1 ALK Q9UM73 pY1078 ELQSPEpYKLSKLR A2 ALK Q9UM73 pY1092 RTIMTDpYNPNYSF A3 ALK Q9UM73 pY1096 TDYNPNpYSFAGKT A4 ALK Q9UM73 pY1131 GAFGEVpYEGQVSG A5 ALK Q9UM73 pY1278 GMARDIpYRASYYR A6 ALK Q9UM73 pY1282 DIYRASpYYRKGGS A7 ALK Q9UM73 pY1283 IYRASYpYRKGGSA A8 ALK Q9UM73 pY1359 RSPGPVpYRIMTQS A9 ALK Q9UM73 pY1507 RLWNPTpYGSWFTE A10 ALK Q9UM73 pY1584 RSGNVNpYGYQQQG A11 ALK Q9UM73 pY1604 APGAGHpYEDTILK A12 ALK Q9UM73 Y1078 ELQSPEYKLSKLR B1 ALK Q9UM73 Y1092 RTIMTDYNPNYSF B2 ALK Q9UM73 Y1096 TDYNPNYSFAGKT B3 ALK Q9UM73 Y1131 GAFGEVYEGQVSG B4 ALK Q9UM73 Y1278 GMARDIYRASYYR B5 ALK Q9UM73 Y1282 DIYRASYYRKGGS B6 ALK Q9UM73 Y1283 IYRASYYRKGGSA B7 ALK Q9UM73 Y1359 RSPGPVYRIMTQS B8 ALK Q9UM73 Y1507 RLWNPTYGSWFTE B9 ALK Q9UM73 Y1584 RSGNVNYGYQQQG B10 ALK Q9UM73 Y1604 APGAGHYEDTILK C3 IRS2 Q9Y4H2 pY675 SSRSDDpYMPMSPA C4 IRS2 Q9Y4H2 pY742 SPEDSGpYMRMWSG C5 IRS2 Q9Y4H2 pY978 RSPLSDpYMNLDFS C6 IRS2 Q9Y4H2 Y675 SSRSDDYMPMSPA C7 IRS2 Q9Y4H2 Y742 SPEDSGYMRMWSG C8 IRS2 Q9Y4H2 Y978 RSPLSDYMNLDFS Additional explanations to MS raw files: -1 extension indicates Control (DMSO lysate) replicate 1. -2 extension indicates LDK378 (treated lysate) replicate 1. -3 extension indicates Control (DMSO lysate) replicate 2. -4 extension indicates LDK378 (treated lysate) replicate 2. Proteome: The NB1 cell line proteome was measured by a label-free mass spectrometry-based approach. The analysis was performed in two biological replicates.
2018-11-21 | PXD006404 | Pride
Project description:We have presented a protocol that utilizes the triple malignant brain tumor domains of L3MBTL1 (3xMBT) to enrich proteins modified with mono- and di-methylated lysine from cell lysate. Cells in culture are grown with amino acids containing light or heavy stable isotopic labels. Methylated proteins are enriched by incubating cell lysates with 3xMBT, or with the binding-null D355N mutant as a negative control. Quantitative liquid chromatography and tandem mass spectrometry (LC-MS/MS) is then used to identify proteins that are specifically enriched by 3xMBT pull-down. Addition of a third isotopic label allows comparison of protein methylation between biological conditions. As an example yeast cells were prepared in SILAC media under three conditions. In two independent experiments either light or heavy cells are Rkm1 knock-out, while the other two are wild-type. 3xMBT was used to enrich methylated proteins from the light/heavy cells, with 3xMBT_D355N as a negative control using the medium cell lysate. Proteins were separated into three fractions by SDS-PAGE and analyzed by LC-MS/MS using an Orbitrap Velos. Data was analyzed with MaxQuant version 1.3.0.5 using default parameters except that mono- and di-methylation of lysine were included as variable modifications, the modification-specific false-discovery was set to 10%, and minimum peptide count for quantitative data set to 1. The amount of ribosomal protein Rpl23, a known substrate of Rkm1, is greatly decreased in 3xMBT pull-down from Rkm1 knock-out cells relative to wild-type. The level of heat shock protein SSA4 is also greatly reduced, suggesting that it may also be methylated by Rkm1.
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Project description:C10:0 and C14:0 fatty acids were conjugated onto agarose beads, and used for pull-down with stable isotope (light: K0R0, heavy: K8R10) labeled Hela whole cell extract. C2:0 conjugated beads were used as control. Both forward (C2:0-light, C10:0 or C14:0-heavy) and reverse (C2:0-heavy, C10:0 or C14:0-light) labeling and pull-down experiments were performed.
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Project description:Histone H3 peptides with acyl modification on either K9 or K27 were used for peptide pull-down with stable isotope (light: K0R0, heavy: K8R10) labeled Hela nuclear extract. The acyl modifications are: acetyl (Ac), propionyl (Pro), Butanoyl (But), crotonyl (Cr), 3-hydroxy butanoyl (bHB), succinyl (Su), Pentanoyl (Vl), Hexanoyl (Xho) and Myristoyl (Myr). Each modified peptide is compared with an unmodified peptide with the same amino acid sequence. Both forward (unmodified-light, acyl-heavy) and reverse (unmodified-heavy, acyl-light) labeling and pull-down experiments were performed.
2019-10-02 | PXD015233 | Pride
Project description:triple SILAC phosphoproteomics experiment of Flp-In T-Rex HEK293 cells stably expressing either FLAG empty, PINK1-FLAG Kinase dead or PINK1-FLAG wild type were grown in ‘light’ (K0R0), ‘medium’ (K4R6) and ‘heavy’ (K8,R10) SILAC media, respectively.
2015-09-16 | PXD002127 | Pride
Project description:Triple Silac approach to analyze cellline specific protein expression. Hek293 (Heavy, Lys8 and Arg10), K526 (Medium, DMEM with Lys 4 and Arg6), HeLa (Light).
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Project description:Triple Silac approach to analyze cellline specific protein expression. Hek293 (Heavy, Lys8 and Arg10), K526 (Medium, DMEM with Lys 4 and Arg6), HeLa (Light).
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Project description:Regular iPOND: 707789: C3-4 EdU, 707790: C3-4 Thymidine chase, 707791: A3-6 EdU, 707792: A3-6 Thymidine chase. SILAC iPOND: 1002641. Regular iPOND: WT MEF cell line (C3-4) and P286R MEF cells (A3-6) were labeled with EdU with (C3-4/A3-6 Thymidine chase) or without thymidine chase (C3-4/A3-6 EdU). Click reaction was performed to link a biotin to the EdU followed by pull down with streptavidin beads.
SILAC iPOND: P286R MEF cells (abundance 1002641 Light) were cultured in "light" medium containing [12C14N]-L-lysine and [12C14N]-L-arginine, and WT MEF cells (Abundance 1002641 Heavy) were cultured in "heavy" medium containing [13C15N]-L-lysine [13C15N]-L-arginine. After more than 99% cells were labeled with isotopes, equal amount of WT and P286R cells were combined followed by EdU labeling and click reaction.
Proteins captured were dissolved on SDS/PAGE and subjected to MS analysis.
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