Skip to Content
MilliporeSigma
  • Proteomic and bioinformatic analysis of a nuclear intrinsically disordered proteome.

Proteomic and bioinformatic analysis of a nuclear intrinsically disordered proteome.

Journal of proteomics (2015-09-17)
Bozena Skupien-Rabian, Urszula Jankowska, Bianka Swiderska, Sylwia Lukasiewicz, Damian Ryszawy, Marta Dziedzicka-Wasylewska, Sylwia Kedracka-Krok
ABSTRACT

Intrinsically disordered proteins (IDPs) are biologically active and crucial for cell function although they do not possess defined three-dimensional architecture. IDPs are especially prevalent in eukaryotic proteomes, and large-scale experiments have shown that many IDPs are nuclear proteins. Bioinformatic analyses have also demonstrated that the vast majority of transcription factors contain extended regions of intrinsic disorder. In the current study, we isolated and functionally analyzed IDPs expressed in the nuclei of HEK293 human cells. According to the results of MS analysis followed by subsequent analysis with the bioinformatic tools IUPred and RAPID (regression-based accurate predictor of intrinsic disorder), a heat-treatment method was able to enrich the nuclear lysate in IDPs. For approximately 85% of the proteins obtained, IUPred predicted a sequence of 30 or more consecutive disordered residues (DRs), and for approximately 83% of the proteins RAPID reported a content of at least 25% DRs (compared to ~66% and 49%, respectively, for the nuclear lysate). Gene Ontology analysis in terms of molecular function revealed that the obtained fraction was generally enriched in proteins involved in the process of transcription and especially in transcription factors. We also showed experimentally that IDPs are overrepresented in the cell nucleus. Intrinsically disordered proteins (IDPs) are crucial cellular molecules and are especially numerous in eukaryotes. In particular, IDPs act as signaling and regulatory proteins, and impairment in their functioning may lead to serious diseases. Large-scale bioinformatic studies of IDPs have provided essential knowledge about this group of proteins. However, experimental data reflect the actual situation in living cells. Our study is the first large-scale proteomic analysis of nuclear IDPs. We showed experimentally that IDPs are overrepresented in the nucleus in comparison to the whole cell. Analysis of molecular function indicated that the nuclear intrinsically disordered proteome (IDP-ome) is enriched in proteins involved in transcription regulation and especially in transcription factors. The IDP isolation method from human cell nuclei presented in this article could be further applied in differential proteomic studies.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Phenylmethanesulfonyl fluoride, ≥99.0% (T)
Sigma-Aldrich
Phenylmethanesulfonyl fluoride, ≥98.5% (GC)
Sigma-Aldrich
Potassium phosphate monobasic, ≥99.5%
Sigma-Aldrich
Potassium phosphate monobasic, 99.99% trace metals basis
Sigma-Aldrich
Potassium phosphate monobasic, BioUltra, for molecular biology, anhydrous, ≥99.5% (T)
Sigma-Aldrich
Potassium phosphate monobasic, powder, suitable for cell culture, suitable for insect cell culture, suitable for plant cell culture, ≥99.0%
Sigma-Aldrich
Potassium phosphate monobasic, for molecular biology, ≥98.0%
Sigma-Aldrich
Potassium phosphate monobasic, ReagentPlus®
Sigma-Aldrich
Potassium phosphate monobasic, meets analytical specification of Ph. Eur., NF, E340, anhydrous, 98-100.5% (calc. to the dried substance)
Sigma-Aldrich
Potassium phosphate monobasic, buffer substance, anhydrous, puriss. p.a., ACS reagent, reag. ISO, reag. Ph. Eur., 99.5-100.5%
Sigma-Aldrich
Potassium phosphate monobasic, ACS reagent, ≥99.0%