αδ-Bungarotoxins, a novel group of long-chain α-neurotoxins, manifest different affinity to two agonist/competitive antagonist binding sites of muscle-type nicotinic acetylcholine receptors (nAChRs), being more active at the interface of α-δ subunits. Three isoforms (αδ-BgTx-1-3) were identified in Malayan Krait () from Thailand by genomic DNA analysis; two of them (αδ-BgTx-1 and 2) were isolated from its venom. The toxins comprise 73 amino acid residues and 5 disulfide bridges, being homologous to α-bungarotoxin (α-BgTx), a classical blocker of muscle-type and neuronal α7, α8, and α9α10 nAChRs.
View Article and Find Full Text PDFElectrospray ionization mass spectrometry is a valuable tool to probe noncovalent interactions. However, the integrity of the interactions in the gas-phase is heavily influenced by the ionization process. Investigating oligomerization and ligand binding of transthyretin (TTR) and the chaperone domain from prosurfactant protein C, we found that dimethyl sulfoxide (DMSO) can improve the stability of the noncovalent interactions during the electrospray process, both regarding ligand binding and the protein quaternary structure.
View Article and Find Full Text PDFRapid Commun Mass Spectrom
January 2014
Rationale: Insulin, islet amyloid polypeptide (IAPP), and the C-peptide part of proinsulin are co-secreted from the pancreatic beta cell granules. IAPP aggregation can be inhibited by insulin and insulin aggregation by C-peptide, but different binding and disaggregating interactions may apply for the peptide complexes. A more detailed knowledge of these interactions is necessary for the development strategies against diabetic complications that stem from peptide aggregations.
View Article and Find Full Text PDFPostpartum, infants have not yet established a fully functional adaptive immune system and are at risk of acquiring infections. Hence, newborns are dependent on the innate immune system with its antimicrobial peptides (AMPs) and proteins expressed at epithelial surfaces. Several factors in breast milk are known to confer immune protection, but which the decisive factors are and through which manner they work is unknown.
View Article and Find Full Text PDFUnlabelled: Proinsulin processing into insulin and C-peptide in the secretory granules of the pancreatic β-cells occurs under mildly acidic conditions and at high peptide concentrations (> 10 mm). Mature insulin has reduced solubility and a propensity to adopt an amyloid-like structure, but is physiologically released as a mixture of a zinc-containing core and a zinc-free, C-peptide-rich fluid phase. C-peptide is known to function in the insulin secretion, but its exact mode of interaction is not established.
View Article and Find Full Text PDFBiochem Biophys Res Commun
December 2010
Evidence has emerged that proinsulin C-peptide has at least three types of functional interactions in addition to its role during synthesis and secretion of insulin. Thus, C-peptide has been shown (i) to bind to cell membranes triggering G-protein-mediated intracellular signaling; (ii) to be internalized into cells and nuclei promoting transcription of rRNA and expression of particular genes; and (iii) to interact with peptides, including insulin, causing desaggregation of insulin oligomers like a chaperone, and with itself, causing homo-oligomers potentially capable of forming aggregates and deposits. In this work, we studied the insulin-C-peptide interactions by monitoring desaggregation and binding effects of C-peptide fragments on insulin.
View Article and Find Full Text PDFSpider dragline silk, one of the strongest polymers in nature, is composed of proteins termed major ampullate spidroin (MaSp) 1 and MaSp2. The N-terminal (NT) domain of MaSp1 produced by the nursery web spider Euprosthenops australis acts as a pH-sensitive relay, mediating spidroin assembly at around pH 6.3.
View Article and Find Full Text PDFThree principally different sites of action have been reported for proinsulin C-peptide, at surface-mediated, intracellular, and extracellular locations. Following up on the latter, we now find that (i) mass spectrometric analyses reveal the presence of the C-peptide monomer in apparent equilibrium with a low-yield set of oligomers in weakly acidic or basic aqueous solutions, even at low peptide concentrations (sub-muM). It further shows not only C-peptide to interact with insulin oligomers (known before), but also the other way around.
View Article and Find Full Text PDFThe C-terminal domain of lung surfactant protein C (CTC) precursor (proSP-C) is involved in folding of the transmembrane segment of proSP-C. CTC includes a Brichos domain with homologs in cancer- and dementia-associated proteins. Mutations in the Brichos domain cause misfolding of proSP-C and hence amyloid fibril formation in interstitial lung disease.
View Article and Find Full Text PDFJ Chromatogr B Analyt Technol Biomed Life Sci
May 2009
The present work investigates qualitative and quantitative variability for rapid liquid chromatography-mass spectrometry (LC-MS) analyses of tryptic digests of total mammalian cell lysate. Experimental variability is characterized in a global manner across technical replicates using label-free quantification software (DeCyder MS 2.0).
View Article and Find Full Text PDFA two-dimensional (2D) separation method was used to decrease sample complexity in analysis of tryptic peptides from glomerular membrane proteins by tandem mass spectrometry (MS/MS). The first dimension was carried out by electrocapture (EC), which fractionates peptides according to electrophoretic mobility. The second dimension was reverse-phase liquid chromatography (RP-LC), in which EC fractions were further separated and analyzed online by MS/MS.
View Article and Find Full Text PDFIdentification of peptides from a complex mixture can be difficult because of the wide concentration range and the different ionization efficiencies of peptides during analysis by electrospray ionization (ESI) mass spectrometry (MS). Preconcentration methods are necessary to allow low-abundance and low-intensity peptides to reach the ionization threshold of the mass spectrometer. Here we demonstrate peptide enrichment based on electroimmobilization.
View Article and Find Full Text PDFIn humans, the brain accounts for about 20% of the body's free cholesterol, most of which is synthesized de novo in brain. To maintain cholesterol balance throughout life, cholesterol becomes metabolized to 24S-hydroxycholesterol, principally in neurons. In mouse, rat, and probably human, metabolism to 24S-hydroxycholesterol accounts for about 50% of cholesterol turnover; however, the route by which the remainder is turned over has yet to be elucidated.
View Article and Find Full Text PDFLithocholic acid (LCA) is a potent hepatotoxic compound. Fetal LCA may have a role in the pathogenesis of neonatal cholestasis/extrahepatic biliary atresia (EHBA). Fetal liver efficiently hydroxylates LCA in several positions.
View Article and Find Full Text PDFOxysterols are oxygenated derivatives of cholesterol. They are intermediates in cholesterol excretion pathways and may also be regarded as transport forms of cholesterol. The introduction of additional hydroxyl groups to the cholesterol skeleton facilitates the flux of oxysterols across the blood brain barrier, and oxysterols have been implicated in mediating a number of cholesterol-induced metabolic effects.
View Article and Find Full Text PDFNeutral steroids have traditionally been analyzed by gas chromatography/mass spectrometry (GC/MS) after necessary derivatization reactions. However, GC/MS is unsuitable for the analysis of many conjugated steroids and those with unsuspected functional groups. Here we describe an alternative analytical method specifically designed for the analysis of oxosteroids and those with a 3beta-hydroxy-delta5 or 5alpha-hydrogen-3beta-hydroxy structure.
View Article and Find Full Text PDFNeutral steroids are difficult to analyse using desorption ionisation methods coupled with mass spectrometry (MS). However, steroids with an unhindered ketone group can readily be derivatised with the Girard P (GP) reagent to give GP hydrazones. Steroid GP hydrazones contain a quaternary nitrogen atom and are readily desorbed in the matrix-assisted laser desorption/ionisation (MALDI) process, giving an improvement in sensitivity of two orders of magnitude.
View Article and Find Full Text PDFAccurate mass measurements are often used in the structural determination of unknown compounds of low molecular mass (i.e., below approximately 500 Da).
View Article and Find Full Text PDFJ Pediatr Gastroenterol Nutr
April 2005
Background: Inborn errors of bile acid synthesis are rare but potentially treatable causes of neonatal cholestasis. We here present a cholestatic infant with an ongoing cytomegalovirus infection who despite intensive treatment died of severe liver disease at 4 months of age.
Methods: The urinary steroids were investigated by electrospray mass spectrometry and gas chromatography mass spectrometry.
A compact disk (CD)-based microfluidic method for selective detection of phosphopeptides by mass spectrometry is described. It combines immobilized metal affinity chromatography (IMAC) and enzymatic dephosphorylation. Phosphoproteins are digested with trypsin and processed on the CD using nanoliter scale IMAC with and without subsequent in situ alkaline phosphatase treatment.
View Article and Find Full Text PDFEur J Mass Spectrom (Chichester)
June 2004
Neutral oxosteroids have been derivatised with Girard T and P hydrazine reagents to give the corresponding Girard hydrazone quaternary ammonium salts. Both Girard T (GT) and Girard P (GP) hydrazones of oxosteroids give very intense [M](+) ion signals in electrospray (ES) mass spectra and fragment within the ES interface and collision cell to give characteristic fragment ions. GT and GP derivatives give informative high-energy collision-induced dissociation spectra, from which the structure of the precursor oxosteroid can be determined.
View Article and Find Full Text PDFWithin a mixture of proteins, minor polymorphic components are difficult to identify using a conventional proteomic approach. Their identification generally requires multi-dimensional separation steps, before or after proteolytic cleavage, followed by sequence analysis of the proteolytic products. In this study, we investigated the potential of tandem mass spectrometry for protein characterization by identifying the delta-beta hybrid human hemoglobin variant Lepore-Boston-Washington using electrospray ionization tandem mass spectrometry.
View Article and Find Full Text PDFMushroom alcohol dehydrogenase (ADH) from Agaricus bisporus (common mushroom, champignon) was purified to apparent homogeneity. One set of ADH isozymes was found, with specificity against formaldehyde/glutathione. It had two highly similar subunits arranged in a three-member isozyme set of dimers with indistinguishable activity.
View Article and Find Full Text PDFDander from the domestic cat (Felis domesticus) is one of the most common causes of IgE-mediated allergy. Attempts to produce tetrameric folded major allergen Fel d 1 by recombinant methods with structural features similar to the natural allergen have been only partially successful. In this study, a recombinant folded Fel d 1 with molecular and biological properties similar to the natural counterpart was produced.
View Article and Find Full Text PDF