A Native Nepenthesin Reactor for Improved Proteolytic Digestion of Intrinsically Disordered Proteins in Proteomics Workflows

Abstract

Intrinsically disordered proteins and proteins containing intrinsically disordered regions often harbor sequences that are difficult to digest with conventional proteases, such as trypsin, Asp-N, or pepsin. In particular, proline-rich regions (PRRs) resist efficient proteolysis and limit sequence coverage in proteomic workflows. Nepenthesins originate from pitcher plants, combining high catalytic activity and stability under acidic conditions with a broad substrate specificity. We describe a workflow for the extraction and purification of native nepenthesin (NEP-NAT) from greenhouse-cultivated Nepenthes species, followed by the enzyme's covalent immobilization on POROS-AL chromatographic material. The performance of the NEP-NAT reactor was evaluated in an online digestion liquid chromatography/tandem mass spectrometry setup for accelerated proteolysis, showing a high proteolytic activity for myoglobin, α-synuclein, and insulin-like growth factor 2 mRNA-binding protein 1. While commercial nepenthesin columns yielded broad coverage for structured proteins, the NEP-NAT reactor generated the largest number of peptides for the intrinsically disordered protein α-synuclein. Cleavages at Pro residues showed enhanced digestion in the PRR of the tumor suppressor protein p53, where conventional proteases show limited activity. These results confirm NEP-NAT as a potent protease in proteomics workflows, offering enhanced access to Pro-rich and disordered domains that are largely inaccessible to common proteases.

Keywords: intrinsically disordered proteins; mass spectrometry; nepenthesin; protease immobilization; proteomics.