Unknown

Dataset Information

0

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.

SUBMITTER: Tsiatsiani L 

PROVIDER: S-EPMC5541327 | BioStudies | 2017-01-01

REPOSITORIES: biostudies

Similar Datasets

2017-07-12 | PXD006766 | Pride
| S-EPMC3553288 | BioStudies
2013-01-01 | S-EPMC3567866 | BioStudies
2020-01-01 | S-EPMC7295189 | BioStudies
2019-01-01 | S-EPMC6593480 | BioStudies
2012-01-01 | S-EPMC3424325 | BioStudies
2018-01-01 | S-EPMC6050989 | BioStudies
2012-01-01 | S-EPMC3319197 | BioStudies
2014-01-01 | S-EPMC4592603 | BioStudies
1000-01-01 | S-EPMC2583057 | BioStudies