High throughput quantification of N-glycans using one-pot sialic acid modification and matrix assisted laser desorption ionization time of flight mass spectrometry

Appropriate glycosylation of recombinant therapeutic glycoproteins has been emphasized in biopharmaceutical industries because the carbohydrate component can affect safety, efficacy, and consistency of the glycoproteins. Reliable quantification methods are essential to ensure consistency of their products with respect to glycosylation, particularly sialylation. Mass spectrometry (MS) has become a popular tool to analyze glycan profiles and structures, showing high resolution and sensitivity with structure identification ability. However, quantification of sialylated glycans using MS is not as reliable because of the different ionization efficiency between neutral and acidic glycans. We report here that amidation in mild acidic conditions can be used to neutralize acidic N-glycans still attached to the protein. The resulting amidated N-glycans can then released from the protein using PNGase F, and labeled with permanent charges on the reducing end to avoid any modification and the formation of metal adducts during MS analysis. The N-glycan modification, digestion, and desalting steps were performed using a single-pot method that can be done in microcentrifuge tubes or 96-well microfilter plates, enabling high throughput glycan analysis. Using this method we were able to perform quantitative MALDI-TOF MS of a recombinant human glycoprotein to determine changes in fucosylation and changes in sialylation that were in very good agreement with a normal phase HPLC oligosaccharide mapping method

High throughput quantification of N-glycans using one-pot sialic acid modification and matrix assisted laser desorption ionization time of flight mass spectrometry

Appropriate glycosylation of recombinant therapeutic glycoproteins has been emphasized in biopharmaceutical industries because the carbohydrate component can affect safety, efficacy, and consistency of the glycoproteins. Reliable quantification methods are essential to ensure consistency of their products with respect to glycosylation, particularly sialylation. Mass spectrometry (MS) has become a popular tool to analyze glycan profiles and structures, showing high resolution and sensitivity with structure identification ability. However, quantification of sialylated glycans using MS is not as reliable because of the different ionization efficiency between neutral and acidic glycans. We report here that amidation in mild acidic conditions can be used to neutralize acidic N-glycans still attached to the protein. The resulting amidated N-glycans can then released from the protein using PNGase F, and labeled with permanent charges on the reducing end to avoid any modification and the formation of metal adducts during MS analysis. The N-glycan modification, digestion, and desalting steps were performed using a single-pot method that can be done in microcentrifuge tubes or 96-well microfilter plates, enabling high throughput glycan analysis. Using this method we were able to perform quantitative MALDI-TOF MS of a recombinant human glycoprotein to determine changes in fucosylation and changes in sialylation that were in very good agreement with a normal phase HPLC oligosaccharide mapping method

Stranded and equilibrated assemblages of late feldspars in two granitic pegmatites in the Pampean Ranges, Argentina

Late primary K-feldspar in two granitic pegmatites from the Pampean pegmatite province in Argentina shows similar patterns of growth and exsolution, but different structural states and mineral associations. (i) In El Criollo, a beryl - columbite - phosphate pegmatite in the Punilla pegmatite field, a single miarolitic cavity in the blocky zone is lined with pale rusty crystals of veinperthitic K-feldspar and quartz. The Kfs host, Or89.7 Ab7.8 Rbf0.3 LEf(? 1.1 (□Si4O8)1.2 (∼88 vol.%), corresponds to optically and XRD-monoclinic, but highly (Al,Si -ordered orthoclase (2t1 = 0.82), with only a slight incipient conversion to intermediate grid-twinned microcline along contacts with exsolved low albite, Ab98.1 An1.2 Or0.9 (∼12 vol.%). The homogeneous precursor of the perthite contained ∼22 mole % Abss. The perthitic orthoclase is overgrown, mainly on the {010} faces, by nonperthitic, gemmy, pale grey to beige orthoclase, Or91.6 Ab6.1 Rbf0.5 Cno.2 Srf0.1 LEf(?)0.2 (□Si4O8)1.5, with 2t1 equal to 0.84. Small tabular crystals of white low albite, Ab99.5 Or0.7, elongate parallel to c, are situated on the {010} faces of the orthoclase in epitactic orientation. (ii) In San Luis II, a spodumene pegmatite in El Totoral pegmatite field, crystals of coarsely perthitic K-feldspar and quartz line cavities in the core-margin zone. The mottled-grey euhedral K-feldspar, Or92.3 Ab4.1 Rbf1.1 Csf0.1 Cn0.1 Srf0.1 LEf(?)1.5 (□Si4O8)0.8 (∼92 vol.%), locally altered to muscovite and clay minerals, is very close to end-member low microcline with vein-shaped exsolution lamellae of low albite. Ab99.1 An0.1 Or0.4 Cn0.1 Srf0.1 (∼8 vol.%). The homogeneous precursor of the perthite contained ∼13 mole % Abss. All faces of the perthite crystals have an even overgrowth of pale grey, nonperthitic low microcline, Or92.8 Ab4.8 Rbf0.2 Srf0.1 LEf(? 1.8(□Si4O8)0.3. The cavity space among the crystals of the K-feldspar and primary quartz is largely filled by microcrystalline secondary quartz, "chalcedony", opal and goethite. Different subsolidus histories of the two host vugs explain the conspicuous contrast between the structural states of their cavity-lining K-feldspar. In El Criollo pegmatite, the cavity probably quickly lost residual aqueous fluid, as the orthoclase of both the primary perthitic crystals and particularly of the gemmy overgrowth show only stranded, coherent domain-scale order and twinning, and very limited alteration. In the San Luis II pegmatite, the cavity-lining K-feldspar shows considerable alteration and overgrowth by low-temperature phases, indicative of substantial hydrothermal activity, which promoted (Al,Si)-ordering to give tartan-twinned low microcline in both the primary perthitic crystals and the norperthitic overgrowths.Le feldspath potassique primaire qui s’est formé tardivement dans deux pegmatites granitiques de la province pegmatitique de Pampea, en Argentine, montre des points communs en termes de croissance et d’exsolution, mais des différences marquées en termes du degré d’ordre et d’associations minérales. (i) A El Criollo, une pegmatite à béryl – columbite – phosphate du champ pegmatitique de Punilla, une seule cavité miarolitique de la zone à cristaux polyédriques est tapissée de cristaux pâles de couleur rouille de feldspath potassique, perthitique à veines, avec quartz. L’hôte dans cette perthite, Or89.7 Ab7.8 Rbf0.3 LEf(?)1.1 (Si4O8)1.2 (~88% en volume), correspond optiquement et en diffraction X à une phase monoclinique (orthose) fortement ordonnée, avec 2t1 = 0.82, et un début de conversion à la microcline intermédiaire avec maclage quadrillé le long des contacts avec l’albite ordonnée exsolvée, Ab98.1 An1.2 Or0.9 (~12% en volume). Le précurseur homogène de la perthite contenait ~22% Abss (base molaire). L’orthose perthitique est recouverte, surtout sur les faces {010}, d’une surcroissance d’orthose non perthitique, gemme, gris pâle à beige, Or91.6 Ab6.1 Rbf0.5 Cn0.2 Srf0.1 LEf(?)0.2 (Si4O8)1.5, avec 2t1 egal à 0.84. De petits cristaux tabulaires blancs d’albite ordonnée, Ab99.5 Or0.7, allongés parallèles à c, recouvrent les faces {110} de l’orthose, en relation épitactique. (ii) A San Luis II, une pegmatite à spodumène du champ de El Totoral, des cristaux grossiers et perthitiques de feldspath potassique et de quartz tapissent les cavités de la zone externe du coeur du massif. Les cristaux idiomorphes de feldspath potassique sont gris nuagé, Or92.3 Ab4.1 Rbf1.1 Csf0.1 Cn0.1 Srf0.1 LEf(?)1.5 (Si4O8)0.8 (~92% en volume), localement transformés en muscovite et en argiles; ils sont très près du pôle microcline ordonné, avec des lamelles exsolvées en veines d’albite ordonnée, Ab99.1 An0.1 Or0.4 Cn0.1 Srf0.1 (~8% en volume). Le précurseur homogène de la perthite contenait ~13% d’albite Abss (base molaire). Toutes les faces des cristaux de perthite sont recouvertes d’une surcroissance uniforme gris pâle de microline ordonné non perthitique, Or92.8 Ab4.8 Rbf0.2 Srf0.1 LEf(?)1.8(Si4O8)0.3. Les espaces entre les cristaux de feldspath potassique et de quartz sont surtout remplis de quartz microcristallin secondaire, “calcédoine”, opale et goethite. Des différences dans l’évolution subsolidus des deux cavités hôtes expliquent le contraste frappant en termes du degré d’ordre du feldspath potassique sur les parois. Dans la pegmatite de El Criollo, la cavité a probablement perdu la phase fluide résiduelle rapidement; l’orthose des cristaux primaires devenus perthitiques et de la surcroissance gemme ne contient que des domaines ordonnés cohérents et maclés, conservés de façon métastable, et très peu d’altération. Dans la pegmatite de San Luis II, les cristaux de feldspath potassique le long des parois montrent des signes importants d’altération et de surcroissances de phases de basse température, indications d’une activité hydrothermale substantielle, qui a favorisé la mise en ordre (Al,Si) du feldpath pour mener à la forme à maclage quadrillé du microcline ordonné, aussi bien dans les cristaux perthitiques que dans les surcroissances non perthitiques.Fil: Cerny, Petr. University of Manitoba; CanadáFil: Galliski, Miguel Angel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto Argentino de Nivología, Glaciología y Ciencias Ambientales. Provincia de Mendoza. Instituto Argentino de Nivología, Glaciología y Ciencias Ambientales. Universidad Nacional de Cuyo. Instituto Argentino de Nivología, Glaciología y Ciencias Ambientales; Argentina. Ministerio de Relaciones Exteriores, Comercio Internacional y Culto. Direccion Nacional del Antártico. Instituto Antártico Argentino. Instituto Antártico Argentino - Sede Cricyt (Mendoza); ArgentinaFil: Oyarzabal, Julio C.. Universidad Nacional de San Luis; ArgentinaFil: Teertstra, David K.. University of Manitoba; CanadáFil: Chapman, Ron. University of Manitoba; CanadáFil: MacBride, Lyndsey. University of Manitoba; CanadáFil: Ferreira, Karen. University of Manitoba; Canad

A first update on mapping the human genetic architecture of COVID-19

peer reviewe

Combining 2-DE immunoblots and mass spectrometry to identify putative soybean ( Glycine max ) allergens

Soybean is recognized as a commonly allergenic food, but the identity of important allergens is not well studied. Recently, some global regulatory agencies started requiring quantitative analysis of individual allergens, including unproven allergens, as part of the risk assessment for genetically engineered (GE) soybeans. We sought to identify soybean proteins that bind IgE from any of 10 individual soybean-sensitized subjects. Soybean IgE binding proteins were identified by 2-DE immunoblots using sera from four soy-allergic and plasma from six soy-sensitized human subjects. Corresponding spots were excised from stained gels, digested, and analyzed using a quadrupole TOF Synapt G2-S tandem mass spectrometer. Results showed the major IgE binding proteins were subunits of either β-conglycinin (Gly m 5) or glycinin (Gly m 6). Soybean Kunitz trypsin inhibitor (SKTI) was a significant IgE binding protein for four subjects. Soybean agglutinin, seed biotinylated protein (SBP) of 65 kDa, late embryogenesis protein (LEP), and sucrose-binding protein were identified as IgE binding only for soy-sensitized subjects. We conclude that the major soybean allergens are isoforms of Gly m 5, Gly m 6, and possibly SKTI and that requirements for quantitative measurement of proteins that are not clear allergens is not relevant to safety

Virtue ethics and social psychology

Virtue ethics has emerged as an alternative to deontological and utilitarian theory in recent moral philosophy. The basic notion of virtue ethics is to reassert the importance of virtuous character in ethical judgement in contrast to the emphasis on principles and consequences. Since questions of virtue have been largely neglected in modern moral theory, there has been a return to Aristotle’s account of virtue as character. This in turn has been questioned as the basis of virtue ethics and there has been a search for alternative accounts of moral agency. One aspect of this critical reflection on virtue ethics is an engagement with social psychology as a source of criticism of the Aristotelian conception of character and as a more plausible alternative foundation for a theory of moral character with contemporary relevance. This paper aims to introduce this area of moral theory to a psychological audience and reflect on the interpretation of social psychological theory and evidence in criticisms of virtuous character, focusing on the use of Milgram’s (1974) experiments on obedience to authority as an argument for situationism. A number of questions emerge concerning the interpretation and use of social psychological theory and evidence in debates within moral philosophy