Native Structural and Functional Proteoform Characterization of the Prolyl-Alanyl-Specific Endoprotease EndoPro from Aspergillus niger
Journal of Proteome Research, vol. 20, pp. 4875–4885
Abstract
. EndoPro is mainly used for food applications but also as a protease in proteomics. In-depth characterization of this enzyme is essential to understand its structural features and functionality. However, there is a lack of analytical methods capable of maintaining both the structural and functional integrity of separated proteoforms. In this study, we developed an anion exchange (AEX) method coupled to native mass spectrometry (MS) for profiling EndoPro proteoforms. Moreover, we investigated purified EndoPro proteoforms with complementary MS-based approaches, including released N-glycan and glycopeptide analysis, to obtain a comprehensive overview of the structural heterogeneity. We showed that EndoPro has at least three sequence variants and seven N-glycosylation sites occupied by high-mannose glycans that can be phosphorylated. Each glycosylation site showed high microheterogeneity with ∼20 glycans per site. The functional characterization of fractionated proteoforms revealed that EndoPro proteoforms remained active after AEX-separation and the specificity of these proteoforms did not depend on N-glycan phosphorylation. Nevertheless, our data confirmed a strong pH dependence of EndoPro cleavage activity. Altogether, our study demonstrates that AEX-MS is an excellent tool to characterize complex industrial enzymes under native conditions.
Authors 10
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Leiden University Medical Center
Affiliation as printed
Leiden University Medical Center, Center for Proteomics and Metabolomics, Albinusdreef 2, 2333 ZA, Leiden, The Netherlands
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Leiden University Medical Center
Affiliation as printed
Leiden University Medical Center, Center for Proteomics and Metabolomics, Albinusdreef 2, 2333 ZA, Leiden, The Netherlands
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Leiden University Medical Center
Affiliation as printed
Leiden University Medical Center, Center for Proteomics and Metabolomics, Albinusdreef 2, 2333 ZA, Leiden, The Netherlands
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Affiliation as printed
DSM Biotechnology Center, Center for Enabling Innovation, Alexander Fleminglaan 1, 2613 AX, Delft, The Netherlands
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Affiliation as printed
DSM Biotechnology Center, Center for Enabling Innovation, Alexander Fleminglaan 1, 2613 AX, Delft, The Netherlands
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Affiliation as printed
DSM Biotechnology Center, Center for Enabling Innovation, Alexander Fleminglaan 1, 2613 AX, Delft, The Netherlands
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Leiden University Medical Center
Affiliation as printed
Leiden University Medical Center, Center for Proteomics and Metabolomics, Albinusdreef 2, 2333 ZA, Leiden, The Netherlands
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Utrecht University · Netherlands Proteomics Centre
Affiliation as printed
Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, University of Utrecht, Padualaan 8, 3584 CH, Utrecht, The Netherlands
Netherlands Proteomics Center, Padualaan 8, 3584 CH, Utrecht, The Netherlands
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Affiliation as printed
DSM Biotechnology Center, Center for Enabling Innovation, Alexander Fleminglaan 1, 2613 AX, Delft, The Netherlands
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Vojtěch Franc corresponding
Utrecht University · Netherlands Proteomics Centre
Affiliation as printed
Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, University of Utrecht, Padualaan 8, 3584 CH, Utrecht, The Netherlands
Netherlands Proteomics Center, Padualaan 8, 3584 CH, Utrecht, The Netherlands
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