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Mass spectrometry to screen potential ORMs-interacting proteins

ABSTRACT: To screen potential ORMs-interacting proteins, HEK293T cells were transfected with plasmids containing HA-tagged ORM1, HA-tagged ORM2, HA-tagged ORM3 and HA-Empty vector, respectively. Then, protein lysates were harvested for immunoprecipitation. Mass spectrometry combined with high-performance liquid chromatography (HPLC-MS) was performed by Cloud‐Seq Biotech (Shanghai, China) using Q Exactive Orbitrap Mass Spectrometer (Thermo Fisher Scientific, Waltham, MA, USA). Proteins were digested with trypsin (trypsin: protein = 1:50 (w/w)) at 37°C for 16 hours. After digestion, the protein fragments were separated by high pH reverse-phase HPLC. The peptides were then analyzed by an EASY-nLC 1000 system connected to an Q Exactive mass spectrometer and Orbitrap Fusion mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA). In raw file, MaxQuant 1.6.0.16 was used to analyze the data, and MaxQuant own algorithm was used to identify peptides and proteins.

ORGANISM(S): Homo Sapiens

SUBMITTER: Yan Lu

PROVIDER: PXD035373 | iProX | Fri Jul 15 00:00:00 BST 2022

REPOSITORIES: iProX

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