NEDD8 subtrates from NEDP1 knockout cells

The aim of the study was to identify proteins that are modified by NEDD8. The NEDD8 specific protease NEDP1/SENP8 was deleted from HEK 293 cells via Crispr/CAS9 gene editing to allow for the accumulation of NEDD8 modified proteins. Lysates from NEDP1 KO cells were then enriched via pulldown with a catalytically inactivated NEDP1 (C162A) fused to the HALO protein. For a negative control a mutated NEDP1 (DAGC) with reduced binding to NEDD8 was also fused to the HALO protein for pulldown. Pulldowns were resolved by SDS-PAGE and bands were excised and subjected to in gel trypsin digestion followed by mass spectrometry analysis.

本研究旨在鉴定受NEDD8修饰的蛋白质。研究人员通过CRISPR/Cas9基因编辑技术敲除HEK 293细胞中的NEDD8特异性蛋白酶NEDP1/SENP8，以实现NEDD8修饰蛋白的积累。随后，采用融合HALO蛋白的催化失活型NEDP1（C162A）进行亲和下拉富集，从NEDP1敲除细胞的裂解液中纯化目标蛋白。为设置阴性对照，另构建了与NEDD8结合能力减弱的突变体NEDP1（DAGC），同样融合HALO蛋白用于亲和下拉实验。所得亲和下拉产物经SDS-PAGE分离后，切取凝胶条带进行胶内胰蛋白酶酶解，随后开展质谱分析。