Structural elucidation of polyamine containing natural products by HPLC-MS

Naturprodukte, die Polyamine enthalten, werden bei zahlreichen Tier- und Pflanzenarten gefunden. Zum Beispiel Spinnengifte - auf welche wir unsere Forschungen fokussierten - sind bekannt als reichhaltige Quellen von Acylpolyamin- Toxinen. Diese biologisch aktiven Substanzen beeinflussen z.B. Ionen-Kanäle im zentralen Nervensystem von Säugetieren und sind deshalb wertvoll für potenzielle therapeutische Zwecke im Zusammenhang mit Gehirnerkrankungen wie der Parkinson- oder Alzheimer-Krankheit. Spinnengifte stellen eine komplexe Mischung aus verschiedenen Typen von Verbindungen dar, wobei Polyamin-Toxine nur einen kleinen Teil davon ausmachen. Die untersuchten Spinnen waren Agelenopsis aperta, Hololena curta (Agelenidae), und Paracoelotes birulai (Amaurobiidae). Die Acylpolyamine wurden mittels Hochleistungs-Flüssigkeitschromatographie gekoppelt mit Massenspektrometrie (HPLC-MS) analysiert. In Kombination mit der Tandem- Massenspektrometrie (MS/MS oder MSn) wurde zusätzlich die Strukturaufklärung der Probemoleküle erlaubt. Um das Fragmentierungsverhalten verstehen zu lernen, mussten die MS/MS-Daten der Naturprodukte analysiert und mit Messdaten von bekannten und chemisch reinen Referenzverbindungen verglichen werden. Mehrere lineare Polyamine, die vermutlich die biosynthetischen Vorläufer der entsprechenden Acylpolyamine sind, wurden zusätzlich mit HPLC-MS/MS charakterisiert. Zwei neue Spermidin-Alkaloide, genannt Chisitin 1 und 2, aus dem Blätterextrakt des Baumes Chisocheton weinlandii (Meliaceae) wurden zusätzlich charakterisiert und deren Synthese durchgeführt. Polyamine-containing natural products are found in many animals and plants. For instance, spider venoms - whereon we focused our research - are known to be a rich source of acylpolyamine toxins. These biologically active compounds interact, e.g., with ion channels in the mammalian central nervous system and are thus of therapeutical interest for the study of brain disorders such as Parkinson's and Alzheimer's diseases. The toxins were found in the spider venoms within complex mixture of several types of compounds and they were available in trace amounts only. The spiders investigated were Agelenopsis aperta, Hololena curta (Agelenidae), and Paracoelotes birulai (Amaurobiidae). The acylpolyamines were analyzed by high- performance liquid chromatography coupled with mass spectrometry (HPLC-MS). In combination with tandem-mass spectrometry (MS/MS or MSn), efficient structural elucidation of the analytes was achieved. To rely the structural elucidation of the polyamine toxins, MS/MS data of structurally well defined and chemically pure reference compounds had to be collected and studied. Several linear polyamines, which are believed to be the biosynthetic precursors of the parent acylpolyamines, were also characterized by HPLC-MS/MS. In addition, two spermidine alkaloids named Chisitine 1 and 2 were characterized from the leaves of the tree Chisocheton weinlandii (Meliaceae) and their syntheses were also performed

Tandem mass spectrometric investigation of acylpolyamines of spider venoms and their 15N-labeled derivatives

The fragmentation mechanism of the acylpentamine toxins 1-4 found in the venom of the spider Agelenopsis aperta has been investigated in detail. To identify the origin of the two doublets of unexpected fragment ions at m/z 129/112 and m/z 115/98, three synthetic 15N-labeled analogs 5-7 have been prepared and subjected to CID fragmentation on a triple quadrupole mass spectrometer. It appears that the unexpected doublet of fragment ions arises from an internal portion of the polyamine backbone after either a transaminative Zip reaction or a sequential fragmentation of the quasi-molecular ion. The second option has been proven by in-source CID experiments. The detailed knowledge of acylpentamine fragmentation mechanisms is essential for the correct characterization of isomeric compounds, particularly for coeluting compounds within complex mixtures such as spider venoms

Solid-phase synthesis of 15N-labeled acylpentamines as reference compounds for the MS/MS investigation of spider toxins

A solid-phase route for synthesis of 15N-labeled acylpolyamines is described. Utilizing alkylation at benzylic N-atom as a key step, 15N-atoms are incorporated by stepwise construction of the polyamine framework on the solid support. The derivatives were used as reference compounds for the investigation of the MS/MS behavior of spider toxins. The stepwise construction of the polyamine framework on the solid support opens a way for selective incorporation of 15N-labeled atoms

A template approach for the characterization of linear polyamines and derivatives in spider venom

A combination of high-performance liquid chromatography (HPLC) and atmospheric-pressure chemical ionization mass spectrometry (APCI-MS and APCI-MS/MS) was used to detect and characterize linear polyamine derivatives in the venom of the spiders Agelenopsis aperta, Hololena curta and Paracoelotes birulai. The compounds were identified with a template approach, by which the collision- induced dissociation (CID) spectra of known compounds are directly compared and correlated with those of the analytes. To facilitate the perception of the spectra and the recognition of the structural features of the analytes, an ion nomenclature closely leaned on the accepted nomenclature for fragment ions of peptides or nucleic acids is introduced. The structure identification of polyamine derivatives by direct correlation of MS spectra is possible because such compounds show very distinctive fragmentation behavior. Of particular relevance is the fact that the signal patterns that are observed with analytes possessing different polyamine backbones are not only distinct with regard to mass distributions but also with regard to relative signal intensities, resulting in fingerprint-like signal patterns. The direct correlation of these patterns—more than the correlation of the ion distributions—was found to be of key significance. With this, the new approach is fundamentally different from the sequencing of peptides or nucleic acids, which are largely based on mass distributions. The method is more efficient and more reliable than the de novo interpretation of the MS data and it even allows the iden- tification of polyamine portions in compounds that are analyzed within mixtures

New linear polyamine derivatives in spider venoms

Linear free polyamines were characterized in the venom of the spiders Agelenopsis aperta, Hololena curta, and Paracoelotes birulai by RP-HPLC coupled to mass spectrometry. The several linear polyamines found were tetramine, pentamine, and hexamine derivatives. Some of these natural products were identified as N-hydroxylated, guanidylated, or acetylated compounds. In addition, the biosynthetical pathway leading to the formation of acylpolyamines in spider venoms is discussed

Two new spermidine alkaloids from Chisocheton weinlandii

The investigation of the MeOH extract of the leaves of Chisocheton weinlandii Harms (Meliaceae) revealed two new open-chain spermidine alkaloids, chisitine 1 (1) and chisitine 2 (2). Their structures were elucidated by NMR spectroscopy, tandem-mass spectrometry, and independant syntheses (Scheme 3). Detailed MS/MS fragmentation pathways are discussed for both compounds based on H/D exchange and 18O-labeling experiments (Schemes 1 and 2)

Alternative splicing liberates a cryptic cytoplasmic isoform of mitochondrial MECR that antagonizes influenza virus.

Viruses must balance their reliance on host cell machinery for replication while avoiding host defense. Influenza A viruses are zoonotic agents that frequently switch hosts, causing localized outbreaks with the potential for larger pandemics. The host range of influenza virus is limited by the need for successful interactions between the virus and cellular partners. Here we used immunocompetitive capture-mass spectrometry to identify cellular proteins that interact with human- and avian-style viral polymerases. We focused on the proviral activity of heterogenous nuclear ribonuclear protein U-like 1 (hnRNP UL1) and the antiviral activity of mitochondrial enoyl CoA-reductase (MECR). MECR is localized to mitochondria where it functions in mitochondrial fatty acid synthesis (mtFAS). While a small fraction of the polymerase subunit PB2 localizes to the mitochondria, PB2 did not interact with full-length MECR. By contrast, a minor splice variant produces cytoplasmic MECR (cMECR). Ectopic expression of cMECR shows that it binds the viral polymerase and suppresses viral replication by blocking assembly of viral ribonucleoprotein complexes (RNPs). MECR ablation through genome editing or drug treatment is detrimental for cell health, creating a generic block to virus replication. Using the yeast homolog Etr1 to supply the metabolic functions of MECR in MECR-null cells, we showed that specific antiviral activity is independent of mtFAS and is reconstituted by expressing cMECR. Thus, we propose a strategy where alternative splicing produces a cryptic antiviral protein that is embedded within a key metabolic enzyme

MassIVE.quant: a community resource of quantitative mass spectrometry-based proteomics datasets

MassIVE.quant is a repository infrastructure and data resource for reproducible quantitative mass spectrometry-based proteomics, which is compatible with all mass spectrometry data acquisition types and computational analysis tools. A branch structure enables MassIVE.quant to systematically store raw experimental data, metadata of the experimental design, scripts of the quantitative analysis workflow, intermediate input and output files, as well as alternative reanalyses of the same dataset.This work was supported in part by NSF CAREER award no. DBI-1054826, grant no. NSF DBI-1759736 and the Chan-Zuckerberg foundation to O.V., grant no. NIH-NLM 1R01LM013115 to N.B. and O.V., NSF award no. ABI 1759980, NIH award nos. P41GM103484 and R24GM127667 to N.B. and the Personalized Health and Related Technologies (grant no. PHRT 0-21411-18) strategic focus area of ETH to B.W. The CRG/UPF Proteomics Unit is part of the Spanish Infrastructure for Omics Technologies (ICTS OmicsTech) and it is a member of the ProteoRed PRB3 consortium that is supported by grant no. PT17/0019 of the PE I+D+i 2013–2016 from the Instituto de Salud Carlos III (ISCIII) and ERDF. We acknowledge support from the Spanish Ministry of Science, Innovation and Universities, ‘Centro de Excelencia Severo Ochoa 2013–2017’, SEV-2012–0208 and Secretaria d’Universitats i Recerca del Departament d’Economia i Coneixement de la Generalitat de Catalunya (grant no. 2017SGR595). This project has received funding from the European Union’s Horizon 2020 research and innovation program under grant agreement no. 823839 (EPIC-XS). Y.P.-R. acknowledges the Wellcome Trust (grant no. 208391/Z/17/Z)

Secreted retrovirus-like GAG-domain-containing protein PEG10 is regulated by UBE3A and is involved in Angelman syndrome pathophysiology

Angelman syndrome (AS) is a neurodevelopmental disorder caused by the loss of maternal UBE3A, a ubiquitin protein ligase E3A. Here, we study neurons derived from patients with AS and neurotypical individuals, and reciprocally modulate UBE3A using antisense oligonucleotides. Unbiased proteomics reveal proteins that are regulated by UBE3A in a disease-specific manner, including PEG10, a retrotransposon-derived GAG protein. PEG10 protein increase, but not RNA, is dependent on UBE3A and proteasome function. PEG10 binds to both RNA and ataxia-associated proteins (ATXN2 and ATXN10), localizes to stress granules, and is secreted in extracellular vesicles, modulating vesicle content. Rescue of AS patient-derived neurons by UBE3A reinstatement or PEG10 reduction reveals similarity in transcriptome changes. Overexpression of PEG10 during mouse brain development alters neuronal migration, suggesting that it can affect brain development. These findings imply that PEG10 is a secreted human UBE3A target involved in AS pathophysiology

Secreted retrovirus-like GAG-domain-containing protein PEG10 is regulated by UBE3A and is involved in Angelman syndrome pathophysiology

Angelman syndrome (AS) is a neurodevelopmental disorder caused by the loss of maternal UBE3A, a ubiquitin protein ligase E3A. Here, we study neurons derived from patients with AS and neurotypical individuals, and reciprocally modulate UBE3A using antisense oligonucleotides. Unbiased proteomics reveal proteins that are regulated by UBE3A in a disease-specific manner, including PEG10, a retrotransposon-derived GAG protein. PEG10 protein increase, but not RNA, is dependent on UBE3A and proteasome function. PEG10 binds to both RNA and ataxia-associated proteins (ATXN2 and ATXN10), localizes to stress granules, and is secreted in extracellular vesicles, modulating vesicle content. Rescue of AS patient-derived neurons by UBE3A reinstatement or PEG10 reduction reveals similarity in transcriptome changes. Overexpression of PEG10 during mouse brain development alters neuronal migration, suggesting that it can affect brain development. These findings imply that PEG10 is a secreted human UBE3A target involved in AS pathophysiology