Aspergillus niger Prolyl Endoprotease for Hydrogen–Deuterium Exchange Mass Spectrometry and Protein Structural Studies

Abstract

To monitor the structural integrity of therapeutic proteins, hydrogen–deuterium exchange mass spectrometry (HDX-MS) is increasingly utilized in the pharmaceutical industry. The successful outcome of HDX-MS analyses depends on the sample preparation conditions, which involve the rapid digestion of proteins at 0 °C and pH 2.5. Very few proteases are able to withstand such harsh conditions, with pepsin being the best-known exception, even though its activity is also strongly reduced at 0 °C. Here, we evaluate the usage of a prolyl endopeptidase from Aspergillus niger (An-PEP) for HDX-MS. What makes this protease very attractive is that it cleaves preferentially the hardest to digest amino acid, proline. To our surprise, and in contrast to previous reports, An-PEP activity was found optimal around pH 2.5 and could be further enhanced by urea up to 40%. Under typical HDX-MS conditions and using small amounts of enzyme, An-PEP generated an equivalent number of peptides as pepsin, as exemplified by using the two model systems tetrameric human hemoglobin (Hb) and human IgG4. Interestingly, because An-PEP peptides are shorter than pepsin-generated peptides, higher sequence resolution could be achieved, especially for Pro-containing protein regions in the alpha subunit of Hb, revealing new protected Hb regions that were not observed with pepsin. Due to its Pro-preference and resistance to low pH, we conclude that An-PEP is an archetype enzyme for HDX-MS, highly complementary to pepsin, and especially promising for structural studies on Pro-rich proteins or proteins containing Pro-rich binding domains involved in cellular signaling.

Structural Dynamics of Lytic Polysaccharide Monooxygenase during Catalysis

“…However, in contrast to the previously used guanidine hydrochloride, we switched to urea, which provided similar sequence coverage but avoided adverse effects on LC analysis. In addition, from the studies published so far, it is evident that in contrast to guanidine hydrochloride, even a high concentration of urea has much less deleterious or even enhancing effects on the protease activity [46][47][48]. We also tested various proteolytic setups (protease columns, flow rates) with serial coupling of nepenthesin-1 and rhizopuspepsin columns operated at 200 µL min −1 , providing the best sequence coverage and spatial resolution.…”

Hydrogen-Deuterium Exchange Mass Spectrometry: A Novel Structural Biology Approach to Structure, Dynamics and Interactions of Proteins and Their Complexes

“…In most research groups, either commercial (fully automated) or in-house-constructed systems are used, where the enzyme is ligated onto a chromatographic matrix; such pre-packed columns are now commercially available. For home-packed columns, Protease XIII and Aspergillus niger Prolyl Endo-protease are also frequently used [ 63 , 64 ]. Recently, in the James A. Carroll laboratory, the concept of a dual protease column was introduced and used for the analysis of monoclonal antibodies [ 65 ].…”

ProAlanase is an Effective Alternative to Trypsin for Proteomics Applications and Disulfide Bond Mapping

“…An-PEP has also been tested in hydrogen-deuterium exchange experiments and protein structural analysis in 2017 and showed high suitability in that application 29 . In 2019, the An-PEP protease, also named EndoPro, was characterized by Laarse et al 30 and showed both complementarity to trypsin and high efficiency in phosphorylation profiling in selectively enriched phosphorylated peptides.…”

“…In 2009, an acidic prolyl-endopeptidase An-PEP from Aspergillus niger fungi was tested by Šebela et al for possible proteomic applications, showing potential for in-solution protein digestion ( 28 ). An-PEP has also been tested in hydrogen-deuterium exchange experiments and protein structural analysis in 2017 and showed high suitability in that application ( 29 ). In 2019, the An-PEP protease, also named EndoPro, was characterized by Laarse et al ( 30 ) and showed both complementarity to trypsin and high efficiency in phosphorylation profiling in selectively enriched phosphorylated peptides.…”