MassIVE MSV000094251

Partial Public PXD050448

Targeting ERK-eIF4E Protein Translational Signaling in an Inflammatory Breast Cancer Model of Acquired Resistance and Reversal with an Altered Profile of Ribosomal Proteins Associated with Pathological Complete Response in Patients

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Description

Monolayer cells were lysed with RIPA buffer followed by a BCA assay. 25 micrograms of protein was normalized with 5% SDS (w/v) in TEAB, pH 8.5 reduction with 10 mM DTT at 80 degC for 15 min and alkylation with with 25 mM IAM ifor 30 min. SDS was removed, and samples were digested with 1:15 of Sequencing Grade Modified Trypsin (Promega) using an S-trap micro device (Protifi) at 47 degC for 1 h. Peptides were lyophilized, and equal volumes of reconstituted samples were mixed to generate a study pool quality control (SPQC) sample. 1D-LC-MS/MS was performed 0.75 micrograms of each sample in a block-randomized order as described in metadata. Samples were analyzed using a M-Class UPLC system (Waters) coupled to a coupled to a Fusion Lumos mass spectrometer (Thermo) via a Nanospray Flex ionization source. Samples were first trapped on a Symmetry C18 180 micrometer x 20 mm trapping column (5 microliters/min at 99.9/0.1 v/v H2O/MeCN) followed by an analytical sepa-ration using a 1.7 micrometer ACQUITY HSS T3 C18 75 micrometer x 250 mm column (Waters) with a 90 min gradient of 5 to 30% MeCN with 0.1% formic acid at a flow rate of 400 nl/min and column temperature of 55 degC. MS data was collected using BoxCar dataindependent acquisition (BoxCarDIA) with variable window BoxCar and DIA scans defined based on scouting runs. Each cycle utilized a full scan with 120,000 resolution from m/z 400 to 1200 with a normalized AGC target of 300% and 50 ms injection time (IT), two BoxCar scans at 120K resolution with 10 windows each, 100% AGC and auto IT, and a 23 window variable window DIA scan with 30K resolution, AGC target of 1000%, a 60 ms IT and NCE of 33. All data was collected in centroid mode. [doi:10.25345/C5ZW1938G] [dataset license: CC0 1.0 Universal (CC0 1.0)]

Keywords: BoxCar DIA ; triple negative ; data-independent acquisition

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Principal Investigators:
(in alphabetical order) 		Gayathri Devi, Duke University, USA
Submitting User: mwfoster
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Originally identified proteins that were automatically remapped by MassIVE to proteins in the SwissProt human reference database.

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