Structure of the asparagine-linked sugar chains of porcine kidney and human urine cerebroside sulfate activator protein

The specific sugar residues and their linkages in the oligosaccharides from pig kidney and human urine cerebroside sulfate activator proteins (saposin B), although previously hypothesized, have been unambiguously characterized. Exhaustive sequential exoglycosidase digestion of the trimethyl-p-aminophenyl derivatives, followed by either matrix-assisted laser desorption/ionization and/or mass spectrometry, was used to define the residues and their linkages. The oligosaccharides were enzymatically released from the proteins by treatment with peptidyl-N-glycosidase F and separated from the proteins by reversed-phase high-performance liquid chromatography (HPLC). Reducing termini were converted to the trimethyl-p-aminophenyl derivative and the samples were further purified by normal-phase HPLC. The derivatized carbohydrates were then treated sequentially with a series of exoglycosidases of defined specificity, and the products of each digestion were examined by mass spectrometry. The pentasaccharides from pig kidney and human urine protein were shown to be of the asparagine-linked complex type composed of mannose-alpha 1-6-mannose-beta 1-4-N-acetylglucosamine-N-acetylglucosamine(alpha 1-6-fucose). This highly degraded structure probably represents the final product of intra-lysosomal exoglycosidase digestion. Oligosaccharide sequencing by specific exoglycosidase degradation coupled with mass spectrometry is more rapid than conventional oligosaccharide sequencing. The procedures developed will be useful for sequencing other oligosaccharides including those from other members of the lipid-binding protein class to which cerebroside sulfate activator belongs. (c) 2000 John Wiley & Sons, Ltd.     

Isotope ratio monitoring gas chromatography/Mass spectrometry of D/H by high temperature conversion isotope ratio mass spectrometry

Of all the elements, hydrogen has the largest naturally occurring variations in the ratio of its stable isotopes (D/H). It is for this reason that there has been a strong desire to add hydrogen to the list of elements amenable to isotope ratio monitoring gas chromatography/mass spectrometry (irm-GC/MS). In irm-GC/MS the sample is entrained in helium as the carrier gas, which is also ionized and separated in the isotope ratio mass spectrometer (IRMS). Because of the low abundance of deuterium in nature, precise and accurate on-line monitoring of D/H ratios with an IRMS requires that low energy helium ions be kept out of the m/z 3 collector, which requires the use of an energy filter. A clean mass 3 (HD(+.)) signal which is independent of a large helium load in the electron impact ion source is essential in order to reach the sensitivity required for D/H analysis of capillary GC peaks. A new IRMS system, the DELTA(plus)XL(trade mark), has been designed for high precision, high accuracy measurements of transient signals of hydrogen gas. It incorporates a retardation lens integrated into the m/z 3 Faraday cup collector. Following GC separation, the hydrogen bound in organic compounds must be quantitatively converted into H(2) gas prior to analysis in the IRMS. Quantitative conversion is achieved by high temperature conversion (TC) at temperatures >1400 degrees C. Measurements of D/H ratios of individual organic compounds in complicated natural mixtures can now be made to a precision of 2 per thousand (delta notation) or, better, with typical sample amounts of approximately 200 ng per compound. Initial applications have focused on compounds of interest to petroleum research (biomarkers and natural gas components), food and flavor control (vanillin and ethanol), and metabolic studies (fatty acids and steroids). Copyright 1999 John Wiley & Sons, Ltd.     

Cerebroside sulfate activator protein (Saposin B): chromatographic and electrospray mass spectrometric properties.

Cerebroside sulfate activator protein is a small, heat-stable protein that is exceptionally resistant to proteolytic attack. This protein is essential for the catabolism of cerebroside sulfate and several other glycosphingolipids. Protein purified from pig kidney and human urine was extensively characterized by reversed-phase liquid chromatography and electrospray mass spectrometry. These two sources revealed 20 and 18 different molecular isoforms of the protein, respectively. Plausible explanations of the structures of the majority of these isoforms can be made on the basis of accurate molecular mass assignments. The reversed-phase chromatographic and electrospray mass spectrometric properties of enzymatically deglycosylated and disulfide-reduced protein were also compared. In addition to a demonstration of the power of electrospray ionization mass spectrometry for revealing a wealth of information on protein microheterogeneity and structural detail, the results also demonstrate the utility of this technique for monitoring spontaneous chemical and enzymatically mediated changes that occur as a result of metabolic processing and protein purification.     

How does cold constrain life cycles of terrestrial plants and animals?

Short summer seasons with low thermal energy input characterise polar ecosystems. Climatic variables and physical isolation of terrestrial habitats act as selective filters which must be passed to allow colonisation, establishment and survival in these extreme environments. Life history studies of the terrestrial biota of such ecosystems give little evidence of adaptive responses to low temperatures having evolved in situ, even though behavioural, ecophysiological and biochemical features allowing tolerance of the likely extremes are well-developed. Observed life history strategies are often consistent with the general predictions of adversity (A) or stress (S) selection. However, biota successful in these extreme environments may be better-grouped by the lack of particular life history features, rather than common possession.     

Hydrogen-deuterium exchange signature of porcine cerebroside sulfate activator protein

Hydrogen-deuterium exchange can be a sensitive indicator of protein structural integrity. Comparisons were made between cerebroside sulfate activator protein (CSAct) in the native state and after treatment with guanidine hydrochloride plus dithiothreitol. Native protein has three internal disulfide bonds and treated protein has no internal disulfide bonds. The comparisons were made using hydrogen-deuterium exchange measured by electrospray ionization mass spectrometry, percentage alpha-helical content measured by circular dichroism and biological activity measured by the ability to support arylsulfatase A-catalyzed sulfate hydrolysis from cerebroside sulfate. In acidic solvent native protein has 59 exchange refractory protons and treated protein has 20 exchange refractory protons (44 and 14% of the exchangeable proton populations, respectively). In native protein the size of the exchange refractory proton population is sensitive to changes in pH, temperature and the presence of a ligand. It is uninfluenced by the presence or absence of glycosyl groups attached to Asn21. Helical content is virtually identical in native and treated protein. Biological activity is significantly reduced but not obliterated in treated protein. The hydrogen-deuterium exchange profile appears to be a sensitive signature of the correctly folded protein, and reflects a dimension of the protein structure that is not apparent in circular dichroic spectra or in the ability of the protein to support arylsulfatase A-catalyzed sulfate hydrolysis from sulfatide. The hydrogen-deuterium exchange profile will be a valuable criterion for characterizing mutant forms of CSAct produced by recombinant and synthetic paradigms and also the native and mutant forms of related proteins.