Unveiling the unfolded proteome: evaluation and comparison of methods for the enrichment of intrinsically disordered proteins

Intrinsically disordered proteins (IDPs) comprise a significant fraction of the eukaryotic proteome and are characterised by the absence of stable secondary or tertiary structures under physiological conditions. Despite their functional importance, comprehensive proteomic analysis of IDPs remains a challenge, mainly because they are poorly represented in conventional proteomics workflows. This underrepresentation is due to their susceptibility to proteolytic degradation, their limited crystallisability and, most critically, their lower abundance compared to structured proteins, which often dominate mass spectrometric analyses due to their higher solubility and stability. To address these limitations, we systematically evaluated and compared two widely used pre-fractionation methods: heat shock treatment (HEAT) and perchloric acid extraction (PCA). For structured proteins, harsh conditions such as elevated temperatures or extreme pH values induce denaturation, which exposes hydrophobic residues and leads to aggregation and precipitation. In contrast, IDPs, which are characterised by low hydrophobicity and an abundance of charged residues, generally remain soluble under these conditions. This different behaviour forms the basis for both enrichment strategies. Using MDA-MB-231 breast cancer cells as a model system, we have optimised and applied two extraction protocols: HEAT and PCA.