<div class="csl-bib-body">
<div class="csl-entry">Rafetzeder, J. (2022). <i>Verification of a RiPP precursor on the peptide level</i> [Diploma Thesis, Technische Universität Wien]. reposiTUm. https://doi.org/10.34726/hss.2022.91422</div>
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dc.identifier.uri
https://doi.org/10.34726/hss.2022.91422
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dc.identifier.uri
http://hdl.handle.net/20.500.12708/20037
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dc.description
Abweichender Titel nach Übersetzung der Verfasserin/des Verfassers
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dc.description.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.
en
dc.language
English
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dc.language.iso
en
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dc.rights.uri
http://rightsstatements.org/vocab/InC/1.0/
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dc.subject
RiPP
en
dc.subject
secondary metabolites
en
dc.subject
fungi
en
dc.subject
mass spectrometry
en
dc.title
Verification of a RiPP precursor on the peptide level
en
dc.title.alternative
Nachweis eines RiPP-Precursors auf Peptid-Ebene
de
dc.type
Thesis
en
dc.type
Hochschulschrift
de
dc.rights.license
In Copyright
en
dc.rights.license
Urheberrechtsschutz
de
dc.identifier.doi
10.34726/hss.2022.91422
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dc.contributor.affiliation
TU Wien, Österreich
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dc.rights.holder
Julia Rafetzeder
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dc.publisher.place
Wien
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tuw.version
vor
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tuw.thesisinformation
Technische Universität Wien
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dc.contributor.assistant
Schittmayer-Schantl, Matthias
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tuw.publication.orgunit
E164 - Institut für Chemische Technologien und Analytik
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dc.type.qualificationlevel
Diploma
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dc.identifier.libraryid
AC16515047
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dc.description.numberOfPages
78
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dc.thesistype
Diplomarbeit
de
dc.thesistype
Diploma Thesis
en
dc.rights.identifier
In Copyright
en
dc.rights.identifier
Urheberrechtsschutz
de
tuw.advisor.staffStatus
staff
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tuw.assistant.staffStatus
staff
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tuw.advisor.orcid
0000-0003-3950-0312
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item.languageiso639-1
en
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item.fulltext
with Fulltext
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item.openaccessfulltext
Open Access
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item.mimetype
application/pdf
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item.openairetype
master thesis
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item.grantfulltext
open
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item.openairecristype
http://purl.org/coar/resource_type/c_bdcc
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item.cerifentitytype
Publications
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crisitem.author.dept
E164-01-3 - Forschungsgruppe Bioanalytik
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crisitem.author.parentorg
E164-01 - Forschungsbereich Imaging und Instrumentelle Analytische Chemie