Synthese Sulfoxid-basierter Reagenzien für die Proteomik und deren Anwendung

Während der Doktorarbeit wurden neuartige, Sulfoxid-basierte chemische Reagenzien entwickelt, welche als anreicherbare Crosslinker und als einfach spaltbare Quantifizierungsreagenzien erfolgreich angewandt wurden.Novel sulfoxide-based chemical reagents were developed during the PhD thesis, which were successfully applied as enrichable crosslinkers and as easily cleavable quantification reagents

A Click‐Chemistry‐Based Enrichable Crosslinker for Structural and Protein Interaction Analysis by Mass Spectrometry

Mass spectrometry is the method of choice for the characterisation of proteomes. Most proteins operate in protein complexes, in which their close association modulates their function. However, with standard MS analysis, information on protein–protein interactions is lost and no structural information is retained. To gain structural and interactome data, new crosslinking reagents are needed that freeze inter‐ and intramolecular interactions. Herein, the development of a new reagent, which has several features that enable highly sensitive crosslinking MS, is reported. The reagent enables enrichment of crosslinked peptides from the majority of background peptides to facilitate efficient detection of low‐abundant crosslinked peptides. Due to the special cleavable properties, the reagent can be used for MS2 and potentially for MS3 experiments. Thus, the new crosslinking reagent, in combination with high‐end MS, should enable sensitive analysis of interactomes, which will help researchers to obtain important insights into cellular states in health and diseases

Prebiotic methylations and carbamoylations generate non-canonical RNA nucleosides as molecular fossils of an early Earth

The RNA world hypothesis assumes that life on earth started with small RNA molecules that catalyzed their own formation. Vital to this hypothesis is the need for prebiotic routes towards RNA. Contemporary RNA, however, is not only constructed from the four canonical nucleobases (A, C, G and U), but it contains in addition many chemically modified (non-canonical) bases. A yet open question is if these non-canonical bases were formed in parallel to the canonical bases (chemical origin), or whether they were created later, when life demanded higher functional diversity (biological origin). Here we show that isocyanates in combination with sodium nitrite establish methylating and carbamoylating reactivity compatible with early Earth conditions. This chemistry leads to the formation of methylated and amino acid modified nucleosides that are still extant. Our data provide a plausible scenario for the chemical origin of certain non-canonical bases, which suggests that they are fossils of an early Earth

A sulfoxide-based isobaric labelling reagent for accurate quantitative mass spectrometry

Modern proteomics requires reagents for exact quantification of peptides in complex mixtures. Peptide labelling is most typically achieved with isobaric tags that consist of a balancer and a reporter part that separate in the gas phase. An ingenious distribution of stable isotopes provides multiple reagents with identical molecular weight but a different mass of the reporter groups, allowing relative quantification of multiple samples in one measurement. Current generation reagents require a high fragmentation energy for cleavage, leading to incomplete fragmentation and hence loss of signal intensity. Here we report a new isobaric labelling reagent, where the balancer and the reporter are linked by a sulfoxide group, which, based on the sulfoxide pyrolysis, leads to easy and asymmetric cleavage at low fragmentation energy. The fragmentation of our new design is significantly improved, yielding more intense complementary ion signals, allowing complementary ion cluster analysis as well

Orchestrating the biosynthesis of an unnatural pyrrolysine amino acid for its direct incorporation into proteins inside living cells

We here report the construction of an E. coli expression system able to manufacture an unnatural amino acid by an artificial biosynthesis. This can be orchestrated with incorporation into protein by amber stop codon suppression inside a living cell. In our case an alkyne bearing pyrrolysine amino acid was biosynthesized and incorporated site-specifically allowing orthogonal double protein labeling

Ein Sulfoxid-basierendes isobares Derivatisierungs-Reagenz für präzise quantitative Massenspektrometrie

Die moderne Proteomik verlangt nach Reagenzien für eine akkurate Quantifizierung von Peptiden in komplexen Proben. Peptide werden üblicherweise mit isobaren Markierungen versehenen, die aus einem Massenausgleicher und einem Reporter bestehen, welche in der Gasphase gespalten werden. Eine geschickte Verteilung der stabilen Isotope liefert mehrere Reagenzien mit identischem Molekulargewicht, jedoch mit einer abweichenden Masse der Reportergruppen, was die relative Quantifizierung mehrerer Proben in einer einzigen Messung ermöglicht. Gegenwärtige Reagenzien benötigen eine hohe Fragmentierungsenergie für die Spaltung, was zu einer unvollständigen Fragmentierung und infolgedessen zu einer Minderung der Signalintensität führt. In dieser Arbeit stellen wir ein neues isobares Derivatisierungs-Reagenz vor, in dem der Massenausgleicher und der Reporter über eine Sulfoxidgruppe verbunden sind, was basierend auf einer Sulfoxid-Pyrolyse zu einer einfachen und asymmetrischen Spaltung bei niedriger Energie führt. Das neue Design unseres Reagenzes zeigt eine signifikante Verbesserung der Spaltung, was zu intensiveren Signalen der komplementären Ionen führt und somit auch die Analyse der komplementären Ionen-Cluster ermöglicht

The cGMP-Dependent Protein Kinase 2 Contributes to Cone Photoreceptor Degeneration in the Cnga3-Deficient Mouse Model of Achromatopsia

Mutations in the CNGA3 gene, which encodes the A subunit of the cyclic guanosine monophosphate (cGMP)-gated cation channel in cone photoreceptor outer segments, cause total colour blindness, also referred to as achromatopsia. Cones lacking this channel protein are non-functional, accumulate high levels of the second messenger cGMP and degenerate over time after induction of ER stress. The cell death mechanisms that lead to loss of affected cones are only partially understood. Here, we explored the disease mechanisms in the Cnga3 knockout (KO) mouse model of achromatopsia. We found that another important effector of cGMP, the cGMP-dependent protein kinase 2 (Prkg2) is crucially involved in cGMP cytotoxicity of cones in Cnga3 KO mice. Virus-mediated knockdown or genetic ablation of Prkg2 in Cnga3 KO mice counteracted degeneration and preserved the number of cones. Analysis of markers of endoplasmic reticulum stress and unfolded protein response confirmed that induction of these processes in Cnga3 KO cones also depends on Prkg2. In conclusion, we identified Prkg2 as a novel key mediator of cone photoreceptor degeneration in achromatopsia. Our data suggest that this cGMP mediator could be a novel pharmacological target for future neuroprotective therapies