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Intracellular Maturation of Acute Phase Proteins
Published in Andrzej Mackiewicz, Irving Kushner, Heinz Baumann, Acute Phase Proteins, 2020
Erik Fries, E. Mathilda Sjöberg
The carbohydrate processing and intracellular transport of secretory proteins can to some extent be monitored by pulse-chase experiments followed by SDS/PAGE. For many proteins, the increase in size due to the addition of galactose and sialic acid residues in the trans-Golgi results in an easily detectable decrease in electrophoretic mobility.65-67 Alternatively, the immunoprecipitated protein is treated with the enzyme endoglycosidase H before electrophoresis. This enzyme will remove all early (high-mannose) forms and thus produce a clear shift in the mobility of the protein;65-67 resistance to the enzyme marks the arrival of the pulse-labled protein in the medial Golgi.12
Water Permeability of Amphibian Urinary Bladder
Published in Gheorghe Benga, Water Transport in Biological Membranes, 1989
Jacques Bourguet, Jacques Chevalier, Mario Parisi, Pierre Ripoche
Glucosidase to remove the cell coat and proteases to identify some external fragment of protein was also investigated by several authors. In this way, it was difficult to identify some peptides extracted specifically when the preparation was stimulated by vasopressin.216 Endoglycosidase H was very effective as determined by the absence of wheat germ agglutinin (WGA) binding sites in the apical surface after this action. Peptides extracted by papain, protease V8, and proteinase K have been analyzed. Some bands in SDS PAGE were particularly important; a 76-kdalton protein was clearly identified.
Gas Phase Sequence Analysis of Proteins/Peptides
Published in Ajit S. Bhown, Protein/Peptide Sequence Analysis: Current Methodologies, 1988
Asparaginyl residues N-linked to complex carbohydrate moieties produce ATZ derivatives that are insoluble in Applied Biosystems Sequencer Solvent S3 (1-chlorobutane). However, removal of all but the directly linked N-acetylgalactosamine (AGA), by treatment of the protein or peptide prior to sequencing with endoglycosidase H, results in formation of an S3-soluble ATZ. PTH-AGAAsn elutes between oxidized DTT and PTH-Asp.16
GlycoVHH: optimal sites for introducing N-glycans on the camelid VHH antibody scaffold and use for macrophage delivery
Published in mAbs, 2023
Loes van Schie, Wander Van Breedam, Charlotte Roels, Bert Schepens, Martin Frank, Ahmad Reza Mehdipour, Bram Laukens, Wim Nerinckx, Francis Santens, Simon Devos, Iebe Rossey, Karel Thooft, Sandrine Vanmarcke, Annelies Van Hecke, Xavier Saelens, Nico Callewaert
To assess the presence of N-linked glycans, samples of P. pastoris supernatant or purified protein were heat-denatured in buffer containing 5% SDS and 400 mM dithiothreitol, treated with PNGaseF, H. jecorina endoT,41S. plicatus endoglycosidase H (endoH; #P0702, New England Biolabs) or mock treated, and assayed via Coomassie Brilliant Blue-stained SDSPAGE and His-tag-specific western blot. Selected glycovariants were further characterized by intact protein MS. LC-MS was performed on an Ultimate 3000 HPLC (Thermo Fisher Scientific, Bremen, Germany) equipped with a Poroshell 300SB-C8 column (Thermo Scientific, 1.0 mm of I.D. × 150 mm), in-line connected with an ESI source to an LTQ XL mass spectrometer (Thermo Fischer Scientific). Mobile phases were 0.1% formic acid and 0.05% trifluoroacetic acid (TFA) in H2O (solvent A) and 0.1% formic acid and 0.05% TFA in acetonitrile (solvent B). The proteins (as indicated in the Results section: in 10 µl of spent P. pastoris supernatant or purified at 1 mg/ml) were separated using a gradient ranging from 10% to 80% solvent B at a flow rate of 100 µl/min for 15 min. The mass spectrometer was operated in MS1 profile mode in the orbitrap analyzer at a resolution of 60,000 (at m/z 400) and a mass range of 600–4000 m/z. The following ESI parameters were used: a surface-induced dissociation of 30 V, a spray voltage of 5.0 kV, capillary temperature of 325°C, capillary voltage of 35 V and a sheath gas (N2) flow rate setting of 7. The summed MS1 spectra of the eluting peak (2 min range) were deconvoluted to molecular mass in Xcalibur Qual Browser (Thermo Scientific).