Arabidopsis thaliana whole-proteome BIN2 kinase assay

ABSTRACT: Finely ground leaf tissue of wild-type columbia-0 ecotype was used. Protein extraction was performed in buffer-saturated phenol and high sucrose buffer. Extracted proteins were incubated with purified recombinant GST or GST-BIN2 kinase. Filter-assisted sample preparation (FASP) columns in presence of 8M urea were used to further purify proteins. Purified proteins were reduced with TCEP, alkylated with iodoacetamide and digested into peptides with trypsin and lys-C. Digested peptides were desalted with c18 columns and labelled with 11-plex Tandem Mass Tag (TMT11) reagents. Phosphoenrichment was performed in a two-steps tandem pipeline: High-Select TiO2 Phosphopeptide Enrichment kit (Thermo Scientific) was used first and High Select Fe-NTA Phosphoptide Enrichment kit (Thermo Scientific) was used on the flow-through obtained from first enrichment.

INSTRUMENT(S): Q Exactive Plus

ORGANISM(S): Arabidopsis Thaliana (ncbitaxon:3702)

SUBMITTER: Justin Walley  

PROVIDER: MSV000086462 | MassIVE | Tue Nov 17 07:41:00 GMT 2020

REPOSITORIES: MassIVE