Rafetzeder, J. (2022). Verification of a RiPP precursor on the peptide level [Diploma Thesis, Technische Universität Wien]. reposiTUm. https://doi.org/10.34726/hss.2022.91422
E164 - Institut für Chemische Technologien und Analytik
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Date (published):
2022
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Number of Pages:
78
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Keywords:
RiPP; secondary metabolites; fungi; mass spectrometry
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Abstract:
Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a group of secondary metabolites with unique biosynthetic pathways resulting in a wide variety of structures and bioactivities. RiPP biosynthesis is a two-stage process. First, linear precursor peptides consisting of an N-terminal leader sequence, a core sequence and an optional C-terminal recognition sequence are translated by classical ribosomal translation. After this, the linear precursor is heavily modified by an assembly of tailoring enzymes yielding the mature, often cyclized product. A set of potential RiPP precursors has been identified in the genome of the fungus Trichoderma reesei in a recent genome mining approach and gene cluster activity was demonstrated on the transcriptome level. The aim of this thesis was to determine whether the precursor/leader sequence mRNA is translated into the corresponding precursor/leader peptides. To this end, extracts of wild type and RiPP knockout strains of T. reesei were subjected to quantitative LCIMS-MS/MS analysis. Different protease digestion strategies were tested to maximize sequence coverage of the RiPP precursor. By employing a simple extraction procedure followed by nanoLC-IMS-MS/MS analysis and a common proteomics database search, the translation of the precursor mRNA into the corresponding peptide could be verified. Moreover, several extraction and analysis approaches were tested on model RiPPs produced in the fungus Aspergillus flavus to develop an efficient method for the analysis of mature RiPPs which will set a basis for the discovery and identification of RiPPs with yet unknown structure in T. reesei. The second part of this thesis compares the efficiency of different sample preparation methods for proteomics with a special focus on sub-microgram sample input. A recently published paramagnetic bead-based approach (single-pot, solid-phase enhanced sample preparation, SP3) for sample preparation was compared to an established in-solution digestion method and a commercially available kit for sample preparation (PreOmics). The SP3 approach was further optimized by adopting different peptide cleanup strategies in order to increase the number of identified proteins. It was shown that for protein amounts above 10 μg, an increase in protein identifications can be achieved by employing an additional desalting step.
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