Elucidation of the disulfide bridge pattern of the recombinant human growth and differentiation factor 5 dimer and the interchain Cys/Ala mutant monomer

Trachsel, Christian; Kämpfer, Urs; Bechtold, Rolf; Schaller, Johann; Schürch, Stefan

doi:10.1016/j.ab.2009.04.024

Cited by 7 publications

(10 citation statements)

References 20 publications

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“…The XICs of these cysteine-containing peptides showed coeluting profiles with apex for all peptides at the same retention time (SI Figure S3). Lack of backbone digestion within the loop between the two pairs of closely spaced cysteines result in all peptides being connected by entangled disulfide bonds in a complex consisting of eight peptides due to the dimeric nature of GDF2 (Figure C) . Evident from the color-coded peptides in Figure A several missed digestion sites were observed, including a lysine residue between the closely spaced cysteines on the orange peptide, which otherwise upon digestion would have opened the ring structure.…”

Section: Resultsmentioning

confidence: 99%

Generic Workflow for Mapping of Complex Disulfide Bonds Using In-Source Reduction and Extracted Ion Chromatograms from Data-Dependent Mass Spectrometry

et al. 2018

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Disulfide bond mapping is a critical task in protein characterization as protein stability, structure, and function is dependent on correct cysteine connectivities. Mass spectrometry (MS) is the method of choice for this, providing fast and accurate characterization of simple disulfide bonds. Disulfide mapping by liquid chromatography tandem mass spectrometry (LC-MS/MS) is performed by identifying disulfide-bonded partner peptides following proteolytic digestion. With the recently introduced ability to assign complex disulfide patterns by online postcolumn partial disulfide reduction by in-source reduction (ISR) in a LC-ISR-MS/MS methodology, the main challenge is data analysis to ensure detection of both expected and unexpected disulfide species. In this study, we introduced a workflow for confident and unbiased mapping of complex disulfide bonds using the powerful combination of extracted ion chromatograms (XICs) of LC-ISR-MS/MS data. With postcolumn partial reduction, identical LC retention times of intact disulfide-bonded species, their constituting free peptides, and partially reduced variants were observed. Subsequent selective MS/MS fragmentation of all reduction products allowed confident identification of free cysteine-containing peptides using a classical shotgun proteomics database search. Matching XICs of the identified cysteine-containing peptides allowed identification of both predicted and unpredicted disulfide species, including unforeseen proteolytic specificities, missed cleavage sites, scrambled disulfide variants, and the presence of disulfide-entangled complexes. Applying this workflow, we successfully mapped the complex disulfide bonds of tertiapin and the epidermal growth factor (EGF) family members transforming growth factor α (TGFα) and EGF. In addition, we were able to characterize the disulfide patterns of the special disulfide fold of the TGFβ superfamily in an all-online methodology.

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Section: Resultsmentioning

confidence: 99%

Generic Workflow for Mapping of Complex Disulfide Bonds Using In-Source Reduction and Extracted Ion Chromatograms from Data-Dependent Mass Spectrometry

et al. 2018

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“…Samples were hydrolysed in the gas phase with 6 m hydrochloric acid containing 0.1% phenol (v/v) for 22 h at 115 °C under N 2 vacuum. The liberated amino acids were reacted with phenylisothiocyanate and the resulting phenylthiocarbamoyl amino acids were analysed by RP‐HPLC on a NovaPak C18 column (3.9 × 150 mm, 4 μm; Waters, Milford, MA, USA) on a Dionex HPLC system (Dionex, Olten, Switzerland) as described previously [45]. Disulfide‐containing peptides were identified by the presence of diphenylthiocarbamoyl‐cystine.…”

Section: Methodsmentioning

confidence: 99%

Multicomponent venom of the spider Cupiennius salei: a bioanalytical investigation applying different strategies

et al. 2012

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The multicomponent venom of the spider Cupiennius salei was separated by three different chromatographic strategies to facilitate subsequent analysis of peptidic venom components by tandem mass spectrometry (MALDI-TOF-MS and ESI-MS), Edman degradation and amino acid analysis: (a) desalting of the crude venom by RP-HPLC only, (b) chromatographic separation of the crude venom into 42 fractions by RP-HPLC, and (c) multidimensional purification of the crude venom by size exclusion and cation exchange chromatography and RP-HPLC. A total of 286 components were identified in the venom of C. salei by mass spectrometry and the sequence of 49 new peptides was determined de novo by Edman degradation and tandem mass spectrometry; 30 were C-terminally amidated. The novel peptides were assigned to two main groups: (a) short cationic peptides and (b) Cys-containing peptides with the inhibitor cystine knot motif. Bioinformatics revealed a limited number of substantial similarities, namely with the peptides CpTx1 from the spider Cheiracantium punctorium and U3-ctenitoxin-Asp1a from the South American fishing spider (Ancylometes sp.) and with sequences from a Lycosa singoriensis venom gland transcriptome analysis. The results clearly indicate that the quality of the data is strongly dependent on the chosen separation strategy. The combination of orthogonal analytical methods efficiently excludes alkali ion and matrix adducts, provides indispensable information for an unambiguous identification of isomasses, and results in the most comprehensive repertoire of peptides identified in the venom of C. salei so far.

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“…The results revealed that cysteine knots were characteristic 3D structures found within each subunit. A disulfide bridge was formed between the two subunits, allowing the formation of an active homodimer [78,79,80]. …”

Section: Gdf-5: a Member Of The Bmp Familymentioning

confidence: 99%

Growth and Differentiation Factor-5 Contributes to the Structural and Functional Maintenance of the Intervertebral Disc

Feng

Liu

Yang

et al. 2015

Cell Physiol Biochem

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Intervertebral disc degeneration (IDD) is a widely recognized contributor to low back pain (LBP). The Prevention or reversal of IDD is a potential treatment for LBP. Unfortunately, current treatments for IDD are aimed at relieving symptoms rather than regenerating disc structure or function. Recently, the injection of growth factors and mesenchymal stem cell (MSC) transplantation have been shown to be promising biological therapies for IDD. Growth factors stimulate the proliferation of and matrix synthesis by intervertebral disc (IVD) cells, leading to the regeneration of degenerative discs. Growth factors, hypoxia and co-culture with nucleus pulposus (NP) cells induce MSCs to differentiate toward an NP-like phenotype, which can increase the number of functional cells in the IVD or enhance the function of endogenous disc cells to facilitate IVD regeneration. Therefore, the emerging roles of growth factors in IVD regeneration have piqued the interest of researchers. Growth factors including transforming growth factor-β (TGF-β), fibroblast growth factor (FGF), insulin-like growth factor-1 (IGF-1) and growth and differentiation factor-5 (GDF-5), among others, have been demonstrated to enhance anabolism in IVD cells and to induce NP-like differentiation of MSCs. However, the injection of TGF, IGF and FGF into human IVDs may induce unwanted blood vessel ingrowth, which accelerates the process of IDD, the injection of GDF-5 may not have the same effect. This finding suggests that GDF-5 is a preferable growth factor for use in IDD treatment compared with TGF, IGF and FGF. The GDF-5 gene is one of the few growth factor genes that have been found to be associated with IDD thus far; moreover, the GDF-5 gene defects lead to collagen and proteoglycan abnormalities in discs in mice, suggesting that GDF-5 contributes to the structural and functional maintenance of the IVD. This review is focused on the functions of GDF-5 in the IVD and on the association between GDF-5 and a genetic predisposition to IDD. The effects of GDF-5 on IVD regeneration and on MSC differentiation are also discussed. GDF-5 plays a crucial role in the pathogenesis of IDD and is a promising therapeutic agent for IDD. Additionally, stem cell transplantation has been shown to be a promising biological therapy for IDD.

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Elucidation of the disulfide bridge pattern of the recombinant human growth and differentiation factor 5 dimer and the interchain Cys/Ala mutant monomer

Cited by 7 publications

References 20 publications

Generic Workflow for Mapping of Complex Disulfide Bonds Using In-Source Reduction and Extracted Ion Chromatograms from Data-Dependent Mass Spectrometry

Generic Workflow for Mapping of Complex Disulfide Bonds Using In-Source Reduction and Extracted Ion Chromatograms from Data-Dependent Mass Spectrometry

Multicomponent venom of the spider Cupiennius salei: a bioanalytical investigation applying different strategies

Growth and Differentiation Factor-5 Contributes to the Structural and Functional Maintenance of the Intervertebral Disc

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