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Protection of beta cells against pro-inflammatory cytokine stress by GDF15-ERBB2 signaling

ABSTRACT: TMT-labeled proteomic data from human islets treated with or without GDF15 for 24 h followed by cytokine (IL1beta & IFNgamma) treatment for 24 h. There are 4 biological replicates. Islets were collected and dissolved in 50 mM NH4HCO3 containing 8 M urea, digested with trypsin and labeled with a 16-plex TMT kit. Peptides were multiplexed, fractionated by high pH reverse phase chromatography, and analyzed by LC-MS/MS on an Acquity M-Class Nano UHPLC system connected to a Q-Exactive mass spectrometer. LC-MS/MS data were processed with MaxQuant, by searching against a human SwissProt database (2017-04-12 with 20,198 proteins). The default settings for precursor and fragment mass tolerance were used. Peptide searching was performed with specific trypsin digestion with a maximum of two missed cleavage sites. Carbamidomethylation of cysteine was set as a fixed modification; acetylation of protein N-terminus and oxidation of methionine residues were set as variable modifications. The false discovery rate (FDR) was set to 1% at the protein and peptide levels. The dataset was log2 transformed, and sample level quality control was performed to ensure that all of the samples had data of high enough quality for analyses. With no sample-level issues identified, the data was normalized to total abundance. Statistical analyses utilized a standard Analysis of Variance (ANOVA) model.

INSTRUMENT(S): Q Exactive Plus

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

SUBMITTER: Ernesto S. Nakayasu

PROVIDER: MSV000093466 | MassIVE | Wed Nov 22 10:26:00 GMT 2023

REPOSITORIES: MassIVE

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Project description:To identify low abundance autocrine growth factors in CHO cell conditioned media, we utilized a label-free shotgun proteomics approach. CHO cell conditioned media were harvested from fed batch bioreactors and concentrated using methanol/chlorofrom precipitation. Proteins in the samples were then subjected to proteolysis with trypsin, and then subjected to primary fractionation using a SCX column, followed by RP liquid chromatography MS (LC-MS) with a LTQ Orbitrap Velos instrument using the data dependent acquisition (DDA) method. The MS system was set up and run with a method which enabled fast acquisitions of high quality peptide precursor and fragment ion data, with the desired precursor mass accuracy of ±5 ppm. For the LTQ Orbitrap Velos MS, the data-dependent MS/MS analytical workflow in positive ion mode was used. Each precursor survey scan (m/z: 300 to 1800) by the Orbitrap mass analyzer (resolution = 60,000 FWHM) was linked to 10 MS/MS events using the 2D ion trap CID approach, with dynamic ion exclusion set at 60 s. This value was determined based on the observed mean peptide chromatographic peak width. All other instrument parameters were set up according to the manufacturer’s suggested values for complex peptide samples. The nano-ESI source was fitted with a 30-µm stainless steel nano-bore emitter (Thermo Fisher Scientific) with 1.7 kV applied near the tip. Raw data files from the LTQ Orbitrap Velos MS were processed using the Proteome Discoverer 1.3 software (Thermo Fisher Scientific). The LC-MS data were searched against the human (Homo sapiens; UniProtKb, updated in August 2012, 45 848 entries), mouse (Mus musculus; UniProtKb, updated in August 2012, 31 528 entries) and chinese hamster (Cricetulus griceus; UniprotKb, updated in August 2012, 24 609 entries) protein databases using the Sequest search engine for the LTQ Orbitrap Velos LC-MS data, assuming tryptic digestion with precursor ions to fall within 10 ppm of projected m/z values and a fragment ion mass tolerance of 0.5 m/z. The specified search parameters were carbamidomethylation of cysteine as fixed modification, oxidation of methionine as dynamic modification and a maximum of two missed cleavage events. Reverse database searching resulted in a specific false discovery rate (FDR) of 1% at the peptide and protein level.

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Protection of beta cells against pro-inflammatory cytokine stress by GDF15-ERBB2 signaling

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