Proteomic analysis of PNUTS-associated proteins immunopurified with an anti-HA antibody

Immunoprecipitated proteins on beads were washed once and resuspended in 50 mM ammonium bicarbonate, followed by the addition of 200 ng trypsin/Lys-C mix and an overnight incubation at 37ºC. The digests were reduced, alkylated, acidified, and desalted with GL-Tip SDB. Then, the eluates were evaporated and dissolved in 0.1% trifluoroacetic acid and 3% acetonitrile (ACN). The resultant peptides were analyzed by an EASY-nLC 1200 UHPLC connected to a Q Exactive Plus mass spectrometer in data-independent acquisition mode. Raw data were analyzed against an in silico spectral library generated from the UniProt database restricted to Drosophila melanogaster using DIA-NN 1.8.1. Missing values were imputed with random values sampled from the lower five percent of detected values.