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A single amino acid in the Salmonella effector SarA/SteE triggers supraphysiological activation of STAT3 for anti-inflammatory target gene expression

ABSTRACT: Immunoprecipitates were adjusted to 5% (w/v) SDS and 50 mM TEAB, pH 8.5, reduced and alkylated, and digested with 1 microgram of wild-type alpha lytic protease using an S-trap micro device (Protifi). Eluents were lyophilized and reconstituted in twenty microlites of 0.1% formic acid, and 7.5 microliters of each sample was loaded onto Evotip Pure disposable C18 trap columns and analyzed using an Evosep One LC interfaced to an Thermofisher Orbitrap Astral MS. Each sample was analyzed twice using a 30 sample-per-day method, and MS/MS used data-dependent (DDA) and data-independent acquisition (DIA). Database searching using a Uniprot human database (downloaded on 08/17/24) appended with transgenes and common contaminants (20,472 total entries) was performed with Spectronaut 19.2 using the Pulsar search engine with semispecific WALP cleavage specificity (cleavage C-terminal to T,A,S or V with non-specific cleavage on either C- or N-terminus) and variable phosphorylation on S/T/Y. The DIA acquisition was used for identification and quantification of peptide precursors and protein groups at a 1% false discovery rate. Local normalization was applied, and protein group quantities were determined using the MaxLFQ algorithm. Phosphorylation site localization used a probability cut-off of 0.75.

INSTRUMENT(S): Orbitrap Astral

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

SUBMITTER: Dennis C. Ko

PROVIDER: MSV000096120 | MassIVE | Thu Oct 17 14:09:00 BST 2024

SECONDARY ACCESSION(S): PXD056916

REPOSITORIES: MassIVE

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