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IP-LC-MS/MS to identify proteins co-precipitating with HDP4 in Arabidopsis

ABSTRACT: 1-week-old seedlings of HDP4-YFP/hdp4-1 and GFP/col-WT transgenic plants under normal growth conditions were used for IP analysis. The GFP-Trap beads containing immunoprecipitated HDP4-YFP or GFP were washed twice with 1× PBS and dissolved in lysis buffer with 8M urea, 5 mM dithiothreitol and incubated at 50 °C for 30 min. Proteins were alkylated in 15 mM iodoacetamide for 1 h in the dark at 25 °C and then digested with trypsin (1:50) overnight at 37 °C. After digestion, chromatographic separation was performed using an Easy nLC 1200 chromatographic system (Thermo Fisher). Then, the peptides were analysed with the targeted parallel reaction monitoring method using a Velos LTQ-Orbitrap mass spectrometer (Thermo Fisher). The raw files were searched directly against Arabidopsis thaliana database (53,270 entries; UniprotKB) with no redundant entries using SEQUEST algorithm on Proteome Discoverer (Version 1.4; Thermo Fisher).

ORGANISM(S): Arabidopsis Thaliana

SUBMITTER: Chunzhao Zhao

PROVIDER: PXD050797 | iProX | Wed Mar 20 00:00:00 GMT 2024

REPOSITORIES: iProX

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