2DLC-UV/MS assay for the simultaneous quantification of intact soybean allergens Gly m 4 and hydrophobic protein from soybean (HPS).

Top-down approaches for quantification of proteins based on separation and mass spectrometric assays hold promise due to their high specificity and avoidance of both proteolytic steps and need for generation of monoclonal antibodies. In this study, a 2DLC-UV/MS assay was developed for the simultaneous quantification of two intact soybean allergens, hydrophobic protein from soybean (HPS) and Gly m 4. Both of these allergens were purified from soybean seeds followed by complete characterization.

Characterization of aryloxyalkanoate dioxygenase-12, a nonheme Fe(II)/α-ketoglutarate-dependent dioxygenase, expressed in transgenic soybean and Pseudomonas fluorescens.

Aryloxyalkanoate dioxygenase-12 (AAD-12) was discovered from the soil bacterium Delftia acidovorans MC1 and is a nonheme Fe(II)/α-ketoglutarate-dependent dioxygenase, which can impart herbicide tolerance to transgenic plants by catalyzing the degradation of certain phenoxyacetate, pyridyloxyacetate, and aryloxyphenoxypropionate herbicides. (1) The development of commercial herbicide-tolerant crops, in particular AAD-12-containing soybean, has prompted the need for large quantities of the enzyme for safety testing. To accomplish this, the enzyme was produced in Pseudomonas fluorescens (Pf) and purified to near homogeneity.

Quantification of Gly m 4 protein, a major soybean allergen, by two-dimensional liquid chromatography with ultraviolet and mass spectrometry detection.

Soybean (Glycine max) is considered a major allergenic food. Gly m 4 is one of several soybean allergens that has been identified to cause an allergic reaction, typically the symptoms are localized effects including the skin, gastrointestinal tract, or respiratory tract. Soybean allergens are considered a complete food allergen in that they are capable of inducing specific IgE as well as eliciting a range of severity from mild rashes up to anaphylaxis.

Immunodepletion of high abundance proteins coupled on-line with reversed-phase liquid chromatography: a two-dimensional LC sample enrichment and fractionation technique for mammalian proteomics.

Proteomic analysis can be hampered by the large concentration distribution of proteins. Immunoaffinity techniques have been applied to selectively remove high abundant proteins (HAP's) from samples prior to analysis. Although immunodepletion of HAP's has been shown to enable greater detection of low abundance proteins, the resulting fractions are often diluted 5-10-fold during the process.

Sequence microheterogeneity of parvalbumin pI 5.0 of pike: a mass spectrometric study.

Parvalbumin (PA) is a muscle and neuronal calcium-binding protein, the major fish and frog allergen. Its characteristic feature is the presence of multiple isoforms with significantly different amino acid sequences. Here we show that the major isoform of northern pike muscle PA (pI 5.

A novel HPLC-UV-MS method for quantitative analysis of protein glycosylation.

Monoclonal antibody samples derived from transgenic plants (plantibodies) may often contain significant amounts of aglycosylated variants. Because glycosylated and non-/de-glycosylated proteins exhibit different functional and pharmacokinetic properties, accurate measurement of non- and de-glycosylated glycoprotein abundances is important. Glycosylation of plant-derived glycoproteins presents specific challenges.

An antibody produced in tobacco expressing a hybrid beta-1,4-galactosyltransferase is essentially devoid of plant carbohydrate epitopes.

N-glycosylation of a mAb may have a major impact on its therapeutic merits. Here, we demonstrate that expression of a hybrid enzyme (called xylGalT), consisting of the N-terminal domain of Arabidopsis thaliana xylosyltransferase and the catalytic domain of human beta-1,4-galactosyltransferase I (GalT), in tobacco causes a sharp reduction of N-glycans with potentially immunogenic core-bound xylose (Xyl) and fucose (Fuc) residues as shown by Western blot and MALDI-TOF MS analysis. A radioallergosorbent test inhibition assay with proteins purified from leaves of WT and these transgenic tobacco plants using sera from allergic patients suggests a significant reduction of potential immunogenicity of xylGalT proteins.

O-linked glycosylation in maize-expressed human IgA1.

O-Linked glycans vary between eukaryotic cell types and play an important role in determining a glycoprotein's properties, including stability, target recognition, and potentially immunogenicity. We describe O-linked glycan structures of a recombinant human IgA1 (hIgA1) expressed in transgenic maize. Up to six proline/hydroxyproline conversions and variable amounts of arabinosylation (Pro/Hyp + Ara) were found in the hinge region of maize-expressed hIgA1 heavy chain (HC) by using a combination of matrix-assisted laser-desorption ionization mass spectrometry (MALDI MS), chromatography, and amino acid analysis.

Dihedral psi angle dependence of the amide III vibration: a uniquely sensitive UV resonance Raman secondary structural probe.

UV resonance Raman studies of peptide and protein secondary structure demonstrate an extraordinary sensitivity of the amide III (Am III) vibration and the C(alpha)H bending vibration to the amide backbone conformation. We demonstrate that this sensitivity results from a Ramachandran dihedral psi angle dependent coupling of the amide N-H motion to (C)C(alpha)H motion, which results in a psi dependent mixing of the Am III and the (C)C(alpha)H bending motions. The vibrations are intimately mixed at psi approximately 120 degrees, which is associated with both the beta-sheet conformation and random coil conformations.

Transient UV Raman spectroscopy finds no crossing barrier between the peptide alpha-helix and fully random coil conformation.

Transient UV resonance Raman measurements excited within the amide pi --> pi transitions of a 21 unit alpha-helical peptide has for the first time determined a lower bound for the unfolding rate of the last alpha-helical turn to form a fully random coil peptide. A 3 ns T-jump is generated with 1.9 microm laser pulses, which are absorbed by water.

Association of partially-folded intermediates of staphylococcal nuclease induces structure and stability.

Staphylococcal nuclease forms three different partially-folded intermediates at low pH in the presence of low to moderate concentration of anions, differing in the amount of secondary structure, globularity, stability, and compactness. Although these intermediates are monomeric at low protein concentration (< or =0.25 mg/mL), increasing concentrations of protein result in the formation of dimers and soluble oligomers, ultimately leading to larger insoluble aggregates.

Anion-induced folding of Staphylococcal nuclease: characterization of multiple equilibrium partially folded intermediates.

The refolding of acid-unfolded staphylococcal nuclease (SNase) induced by anions was characterized, and revealed the existence of three different partially folded intermediates (A states). The three intermediates lack the rigid tertiary structure characteristic of native states, and differ in their degree of folding as measured by probes of secondary structure, size, stability and globularity. The least structured conformation, A1, is stabilized by chloride (600 mM) or sulfate (100 mM).

Size-exclusion chromatography of protected synthetic polypeptide tandems in an organic solvent.

A novel method for analytical and preparative size exclusion chromatography of large water-insoluble protected peptides in an organic solvent was developed. This method was applied to analysis and separation of protected synthetic peptide tandem repeats and to a control of the reaction of peptide fragment coupling. Columns containing Toyopearl HW-40, HW-50; HW-55 and HW-60 gels of Fine grade were used, and the selectivity of each sorbent, as well as the chromatographic behaviour of the peptides on them, were examined.