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Identification of USP28/NMMHC IIA-interacting proteins in endothelial cells using immunoprecipitation-coupled mass spectrometry (IP–MS)

ABSTRACT: To identify protein interaction partners of USP28/NMMHC IIA in endothelial cells, we performed immunoprecipitation-coupled mass spectrometry (IP–MS) using human umbilical vein endothelial cells (HUVECs). Cell lysates were incubated with anti-USP28/NMMHC IIA antibody, followed by pulldown using Protein A/G agarose beads. Eluted proteins were subjected to SDS denaturation, trypsin digestion, and liquid chromatography–tandem mass spectrometry (LC–MS/MS) analysis. MS analysis was carried out on a Thermo Scientific Q Exactive mass spectrometer coupled to an Easy-nLC system. High-resolution MS1 scans and HCD-based MS2 fragmentation enabled sensitive detection of immunoprecipitated proteins. Data-dependent acquisition (DDA) was applied to generate peptide spectra. Raw data were processed using MaxQuant for peptide identification against the UniProt database and label-free quantification. Database comparison and quantitative analysis revealed a set of specifically enriched proteins in the anti-USP28/NMMHC IIA IP samples.

ORGANISM(S): Homo Sapiens

SUBMITTER: Junping Kou

PROVIDER: PXD071842 | iProX | Thu Dec 11 00:00:00 GMT 2025

REPOSITORIES: iProX

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