63 research outputs found
Comparison of LC and LC/MS Methods for Quantifying N-Glycosylation in Recombinant IgGs
High-performance liquid chromatography (LC) and liquid chromatography/electrospray ionization time-of-flight mass spectrometry (LC/ESI-MS) methods with various sample preparation schemes were compared for their ability to identify and quantify glycoforms in two different production lots of a recombinant monoclonal IgG1 antibody. IgG1s contain a conserved N-glycosylation site in the fragment crystallizable (Fc) subunit. Six methods were compared: (1) LC/ESI-MS analysis of intact IgG, (2) LC/ESI-MS analysis of the Fc fragment produced by limited proteolysis with Lys-C, (3) LC/ESI-MS analysis of the IgG heavy chain produced by reduction, (4) LC/ESI-MS analysis of Fc/2 fragment produced by limited proteolysis and reduction, (5) LC/MS analysis of the glycosylated tryptic fragment (293EEQYNSTYR301) using extracted ion chromatograms, and (6) normal phase HPLC analysis of N-glycans cleaved from the IgG using PNGase F. The results suggest that MS quantitation based on the analysis of Fc/2 (4) is accurate and gives results that are comparable to normal phase HPLC analysis of N-glycans (6)
Accurate determination of key surface properties that determine the efficient separation of bovine milk BSA and LF proteins
The aim of this work is to accurately measure fundamental surface properties, i.e., zeta potential, isoelectric point and protein size that determine the optimal separation conditions of Bovine serum albumin and lactoferrin, two high added value food proteins whose similarity in weight makes their separation a scientific and technical challenge. The systematic study of these proteins’ surface properties was performed under different conditions: (i) 3.0 < pH < 10.0, (ii) electrolyte type: KCl, NaCl and CaCl2 and concentration (0.01–0.1 M KCl) and (iii) protein concentration in the range of 0.04–4.0 g L-1 for BSA and 0.01–1.0 g L-1 for LF with the objective of establishing the optimal separation conditions. Finally, the comparison of the experimental and theoretically calculated values revealed significant deviations under specific conditions, highlighting the simplicity of the theoretical assumptions and leading to the conclusion that the use of experimental surface properties is still needed for the correct design of food protein separation processes.Financial support from the Projects CTQ2011-25262, CTM2011- 23912 and CTQ2012- 31639 (Ministerio de EconomÃa y Competitividad-MINECO/SPAIN and Fondo Europeo de Desarrollo Regional-FEDER) is gratefully acknowledged
The feeding tube of cyst nematodes: characterisation of protein exclusion
Plant parasitic nematodes comprise several groups; the most economically damaging of these are the sedentary endoparasites. Sedentary endoparasitic nematodes are obligate biotrophs and modify host root tissue, using a suite of effector proteins, to create a feeding site that is their sole source of nutrition. They feed by withdrawing host cell assimilate from the feeding site though a structure known as the feeding tube. The function, composition and molecular characteristics of feeding tubes are poorly characterised. It is hypothesised that the feeding tube facilitates uptake of host cell assimilate by acting as a molecular sieve. Several studies, using molecular mass as the sole indicator of protein size, have given contradictory results about the exclusion limits of the cyst nematode feeding tube. In this study we propose a method to predict protein size, based on protein database coordinates in silico. We tested the validity of these predictions using travelling wave ion mobility spectrometry--mass spectrometry, where predictions and measured values were within approximately 6%. We used the predictions, coupled with mass spectrometry, analytical ultracentrifugation and protein electrophoresis, to resolve previous conflicts and define the exclusion characteristics of the cyst nematode feeding tube. Heterogeneity was tested in the liquid, solid and gas phase to provide a comprehensive evaluation of three proteins of particular interest to feeding tube size exclusion, GFP, mRFP and Dual PI. The data and procedures described here could be applied to the design of plant expressed defence compounds intended for uptake into cyst nematodes. We also highlight the need to assess protein heterogeneity when creating novel fusion proteins
The glycan patterns at the individual glycosylation sites in orosomucoid from allergic reaction patients
Purification of the Alpha and Beta Subunits of (Na,K)-ATPase by Continuous Elution Electrophoresis
Membrane Interactions, Ligand-Dependent Dynamics, and Stability of Cytochrome P4503A4 in Lipid Nanodiscs
Membrane-bound
cytochrome P4503A4 (CYP3A4) is the major source of enzymatic drug
metabolism. Although several structural models of CYP3A4 in various
ligand complexes are available, none includes a lipid bilayer. Details
of the effects of the membrane on protein dynamics and solvation,
and access channels for ligands, remain uncertain. H/D exchange mass
spectrometry (H/DXMS) with ligand free CYP3A4 containing a deletion
of residues 3–12, compared to that of the full length wild
type, in lipid nanodiscs afforded 91% sequence coverage. Deuterium
exchange was fast in the F- and G-helices, HI loop, and C-terminal
loop. In contrast, there is very low exchange in the F′- and <i>G</i>′-helices. The results are consistent with the overall
membrane orientation of CYP3A4 suggested by published MD simulations
and spectroscopic results, and the solvent accessibility of the F/G
loop suggests that it is not deeply membrane-embedded. Addition of
ketoconazole results in only modest, but global, changes in solvent
accessibility. Interestingly, with ketoconazole bound some peptides
become less solvent accessible or dynamic, including the F- and G-helices,
but several peptides demonstrate modestly increased accessibility.
Differential scanning calorimetry (DSC) of CYP3A4-nanodiscs suggests
membrane-induced stabilization compared to that of aggregated CYP3A4
in buffer, and this stabilization is enhanced upon addition of the
ligand ketoconazole. This ligand-induced stabilization is accompanied
by a very large increase in Δ<i>H</i> for CYP3A4 denaturation
in nanodiscs, possibly due to increased CYP3A4–membrane interactions.
Together, the results suggest a distinct orientation of CYP3A4 on
the lipid membrane, and they highlight likely solvent access channels,
which are consistent with several MD simulations
Purification of the glycosylated beta subunit of (Na,K)-ATPase by lectin affinity chromatography
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