Disulfide Connectivity Analysis of Peptides Bearing Two Intramolecular Disulfide Bonds Using MALDI In-Source Decay

Massonnet, Philippe; Haler, Jean; Upert, Grégory; Smargiasso, Nicolas; Mourier, Gilles; Gilles, Nicolas; Quinton, Loïc; Pauw, Edwin De

doi:10.1007/s13361-018-2022-y

Cited by 10 publications

(7 citation statements)

References 36 publications

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“…In the absence of this information, cysteine residues were randomly oxidized and the vast majority of toxins presented a unique peak in LC, suggesting that they adopted only one conformation, hopefully the natural one. In the case where LC profiles presented two or three peaks with the good expected oxidized mass, we assumed that it could be due to the generation of peptide isomers 32 (Figures S1 and S2).…”

Section: ■ Resultsmentioning

confidence: 99%

A Venomics Approach Coupled to High-Throughput Toxin Production Strategies Identifies the First Venom-Derived Melanocortin Receptor Agonists

et al. 2020

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Animal venoms are rich in hundreds of toxins with extraordinary biological activities. Their exploitation is difficult due to their complexity and the small quantities of venom available from most venomous species. We developed a Venomics approach combining transcriptomic and proteomic characterization of 191 species and identified 20,206 venom toxin sequences. Two complementary production strategies based on solid-phase synthesis and recombinant expression in Escherichia coli generated a physical bank of 3597 toxins. Screened on hMC4R, this bank gave an incredible hit rate of 8%. Here, we focus on two novel toxins: N-TRTX-Preg1a, exhibiting an inhibitory cystine knot (ICK) motif, and N-BUTX-Ptr1a, a short scorpion-CSαβ structure. Neither N-TRTX-Preg1a nor N-BUTX-Ptr1a affects ion channels, the known targets of their toxin scaffolds, but binds to four melanocortin receptors with low micromolar affinities and activates the hMC1R/Gs pathway. Phylogenetically, these two toxins form new groups within their respective families and represent novel hMC1R agonists, structurally unrelated to the natural agonists.

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Section: ■ Resultsmentioning

confidence: 99%

A Venomics Approach Coupled to High-Throughput Toxin Production Strategies Identifies the First Venom-Derived Melanocortin Receptor Agonists

et al. 2020

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“…In addition, the conotoxin Que-0.1 may contain seven pairs of disulfide bonds, while the other four conotoxins (κ-PVIIA, Kwon et al, 2016;GeXIV, Zhangsun et al, 2017;and ω-CVID Adams et al, 2003) have only three pairs of disulfide bonds. Disulfide connectivity in the peptides bearing intramolecular disulfide bonds is highly important for the structure and the biological activity of the peptides (Massonnet et al, 2018). It may be that Que-0.1 is more stable and can prevent degradation during in vivo administration.…”

Section: Discussionmentioning

confidence: 99%

Identification of Novel Conopeptides and Distinct Gene Superfamilies in the Marine Cone Snail Conus quercinus

Zhang

Wang²,

Yang³

et al. 2021

Front. Mar. Sci.

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Conopeptides from the marine cone snails are a mixture of cysteine-rich active peptides, representing a unique and fertile resource for neuroscience research and drug discovery. The ConoServer database includes 8,134 conopeptides from 122 Conus species, yet many more natural conopeptides remain to be discovered. Here, we identified 517 distinct conopeptide precursors in Conus quercinus using de novo deep transcriptome sequencing. Ten of these precursors were verified at the protein level using liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS). The combined gene and protein analyses revealed two novel gene superfamilies (Que-MNCLQ and Que-MAMNV), and three other gene superfamilies (N, P, and I1) were reported for the first time in C. quercinus. From the Que-MAMNV superfamily, a novel conotoxin, Que-0.1, was obtained via cloning and prokaryotic expression. We also documented a new purification process that can be used to induce the expression of conopeptides containing multiple pairs of disulfide bonds. The animal experiments showed that Que-0.1 strongly inhibited neuroconduction; the effects of Que-1.0 were 6.25 times stronger than those of pethidine hydrochloride. In addition, a new cysteine framework (CC-C-C-C-C-C-CC-C-C-C-C-C) was found in C. quercinus. These discoveries accelerate our understanding of conopeptide diversity in the genus, Conus and supply promising materials for medical research.

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“…For stable crosslinks such as di-Tyr, this has recently been accomplished by ultraviolet photodissociation (UVPD) fragmentation [ 141 ]. For more labile crosslinks, such as disulfides, in-source fragmentation can be utilized [ 204 , 205 ]. Disulfides can also be probed indirectly through MS analysis of alkylated Cys thiol groups released after in vitro disulfide reduction using chemical (e.g., dithiothreitol) or enzymatic (e.g., thioredoxin [ 206 ]) treatment.…”

Section: Detection Of Crosslinks Including Advantages and Disadvantages Of Different Methodsmentioning

confidence: 99%

Oxidative Crosslinking of Peptides and Proteins: Mechanisms of Formation, Detection, Characterization and Quantification

et al. 2021

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Covalent crosslinks within or between proteins play a key role in determining the structure and function of proteins. Some of these are formed intentionally by either enzymatic or molecular reactions and are critical to normal physiological function. Others are generated as a consequence of exposure to oxidants (radicals, excited states or two-electron species) and other endogenous or external stimuli, or as a result of the actions of a number of enzymes (e.g., oxidases and peroxidases). Increasing evidence indicates that the accumulation of unwanted crosslinks, as is seen in ageing and multiple pathologies, has adverse effects on biological function. In this article, we review the spectrum of crosslinks, both reducible and non-reducible, currently known to be formed on proteins; the mechanisms of their formation; and experimental approaches to the detection, identification and characterization of these species.

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Disulfide Connectivity Analysis of Peptides Bearing Two Intramolecular Disulfide Bonds Using MALDI In-Source Decay

Cited by 10 publications

References 36 publications

A Venomics Approach Coupled to High-Throughput Toxin Production Strategies Identifies the First Venom-Derived Melanocortin Receptor Agonists

A Venomics Approach Coupled to High-Throughput Toxin Production Strategies Identifies the First Venom-Derived Melanocortin Receptor Agonists

Identification of Novel Conopeptides and Distinct Gene Superfamilies in the Marine Cone Snail Conus quercinus

Oxidative Crosslinking of Peptides and Proteins: Mechanisms of Formation, Detection, Characterization and Quantification

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