5 years ago

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

Aspergillus niger Prolyl Endoprotease for Hydrogen–Deuterium Exchange Mass Spectrometry and Protein Structural Studies
Maurien Olsthoorn, Liana Tsiatsiani, Albert J. R. Heck, Michiel Akeroyd
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.

Publisher URL: http://dx.doi.org/10.1021/acs.analchem.7b01161

DOI: 10.1021/acs.analchem.7b01161

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