Analysis of a Nuclear Intrinsically Disordered Proteome.

Intrinsically disordered proteins (IDPs) play crucial roles in cell functioning, although they do not possess defined three-dimensional architecture. They are highly abundant in the cell nucleus, and the vast majority of transcription factors (TFs) contain extended regions of intrinsic disorder. IDPs do not respond to denaturing conditions in a standard manner, and this can be used for their separation from structured proteins. Here we describe a protocol for the isolation and characterization of nuclear IDPs in which heat treatment is used for enrichment of IDPs in samples. The whole workflow comprises the following steps: nuclei isolation from HEK293 (human embryonic kidney) cells, protein extraction, enrichment of IDPs, sample preparation for mass spectrometric analysis, liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis, in silico assessment of protein disorder, and Gene Ontology analysis.