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A Native Nepenthesin Reactor for Improved Proteolytic Digestion of Intrinsically Disordered Proteins in Proteomics Workflows
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DOI：10.1002/cbic.202500832.png)
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Abstract
En 中文
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, alpha-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 alpha-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
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