Proteomics

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Chromatin proteomics of glioblastoma post-treatment with AsiDNA, Belinostat and/or radiation


ABSTRACT: Cells were solubilized with 8M urea followed by reduction with DTT and alkylation with iodoacetamide and overnight digestion with trypsin. After solid phase extraction, samples were labeled with TMT reagents as shown in metadata. Two pooled samples per plate were used for bridging two TMT sets. Reactions were quenched and samples mixed and desalted using high pH SPE. One ug of each TMT set was analyzed twice using a Waters nanoACQUITY interfaced to ThermoFisher Fusion Lumos. Trapping used a Symmetry C18 20 mm x 180 um trapping column (5 ul/min at 99.9/0.1 v/v water/acetonitrile), after which the analytical separation was performed using a 1.8 um Acquity HSS T3 C18 75 um x 250 mm column (Waters) with a 90 min linear gradient of 5 to 30% MeCN with 0.1% FA at a flow rate of 400 nL/min with a column temperature of 55C. Data collection Data-dependent acquisition used a r=120,000 (at m/z 200) full MS scan from m/z 375 - 1600 with a target AGC value of 2e5 ions. MS/MS scans were acquired at r=50,000 (at m/z 200) at a target AGC value of 1e5 ions and 105 ms. Precursor ions with a charge state of 2 and charge states of 3-6 had isolation windows of 1.2 and 0.7, respectively. A 20s dynamic exclusion was employed.

INSTRUMENT(S): Orbitrap Fusion Lumos

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

SUBMITTER: Mahesh Chandrasekharan  

PROVIDER: MSV000095293 | MassIVE | Wed Jul 10 15:52:00 BST 2024

REPOSITORIES: MassIVE

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