Native Liquid Chromatography and Mass Spectrometry to Structurally and Functionally Characterize Endo-Xylanase Proteoforms |
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| Authors: | Guusje van Schaick,Nadi el Hajjouti,Simone Nicolardi,Joost den Hartog,Romana Jansen,Rob van der Hoeven,Wim Bijleveld,Nicolas Abello,Manfred Wuhrer,Maurien M. A. Olsthoorn,Elena Domí nguez-Vega |
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| Affiliation: | 1.Center for Proteomics and Metabolomics, Leiden University Medical Center, Albinusdreef 2, 2333 ZA Leiden, The Netherlands; (N.e.H.); (S.N.); (M.W.); (E.D.-V.);2.Center for Analytical Innovation, DSM, Alexander Fleminglaan 1, 2613 AX Delft, The Netherlands; (J.d.H.); (R.J.); (R.v.d.H.); (W.B.); (N.A.); (M.M.A.O.) |
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| Abstract: | Xylanases are of great value in various industries, including paper, food, and biorefinery. Due to their biotechnological production, these enzymes can contain a variety of post-translational modifications, which may have a profound effect on protein function. Understanding the structure–function relationship can guide the development of products with optimal performance. We have developed a workflow for the structural and functional characterization of an endo-1,4-β-xylanase (ENDO-I) produced by Aspergillus niger with and without applying thermal stress. This workflow relies on orthogonal native separation techniques to resolve proteoforms. Mass spectrometry and activity assays of separated proteoforms permitted the establishment of structure–function relationships. The separation conditions were focus on balancing efficient separation and protein functionality. We employed size exclusion chromatography (SEC) to separate ENDO-I from other co-expressed proteins. Charge variants were investigated with ion exchange chromatography (IEX) and revealed the presence of low abundant glycated variants in the temperature-stressed material. To obtain better insights into the effect on glycation on function, we enriched for these species using boronate affinity chromatography (BAC). The activity measurements showed lower activity of glycated species compared to the non-modified enzyme. Altogether, this workflow allowed in-depth structural and functional characterization of ENDO-I proteoforms. |
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| Keywords: | native protein analysis, endo-1,4-β -xylanase, boronate affinity chromatography, ion exchange chromatography, size exclusion chromatography, mass spectrometry, glycation |
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