Tucher, J., Koudelka, T., Schlenk, J. and Tholey, Andreas (2016) From top-down to bottom-up: Time-dependent monitoring of proteolytic protein degradation by LC-MS. Journal of Chromatography B-Analytical Technologies in the Biomedical and Life Sciences, 1015 . pp. 111-120. DOI 10.1016/j.jchromb.2016.02.021.

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Abstract

The understanding of proteolytic processes includes manifold aspects, ranging from the characterization of proteases and their corresponding substrates to the localization of cleavage sites. The analysis of protease-catalyzed reactions at a single time-point in many cases excludes the identification of intermediate cleavage products of potential biological function. To overcome this problem, proteolysis has to be monitored over time. For that purpose, we established a straight-forward two-step approach. First, Tricine-SDS-PAGE separation of the proteolytic products is applied to survey the proteolytic reaction. In the second step, the reaction mixture is analyzed by an LC-MS set-up. An optimized chromatographic separation coupled to electrospray Orbitrap mass spectrometry allowed the simultaneous monitoring of intact substrates, intermediates and cleavage products of lower molecular weight. The applicability of the strategy was shown on the example of the gastric protease pepsin and its physiologically relevant substrate hen egg white lysozyme, one of the major egg allergens. While lysozyme-derived cysteine-free peptides were cleaved comparatively fast, disulfide bonds protected connected peptides from rapid peptic proteolysis. Two previously identified potential IgE-binding motifs were observed as disulfide-linked cleavage products. In summary, the presented approach is not only ideally suited for the simulation of gastro-intestinal digestion, which is of high interest in food research, but can be transferred to any protease-substrate pair of interest. Furthermore the strategy can be exploited to deduce the effect of post-translational modifications on proteolysis. (C) 2016 Elsevier B.V. All rights reserved.

Document Type:	Article
Additional Information:	Times Cited: 0 Tucher, Joanna Koudelka, Tomas Schlenk, Jana Tholey, Andreas
Keywords:	Proteolysis, Cleavage product, Lysozyme, LC-MS, Disulfide bond, Intermediates
Research affiliation:	Kiel University > Kiel Marine Science OceanRep > The Future Ocean - Cluster of Excellence
Refereed:	Yes
Open Access Journal?:	No
Publisher:	Elsevier
Projects:	Future Ocean
Date Deposited:	01 Mar 2017 08:29
Last Modified:	23 May 2019 11:50
URI:	https://oceanrep.geomar.de/id/eprint/36353

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