Enantiomeric purity analysis of synthetic peptide therapeutics by direct chiral high-performance liquid chromatography-electrospray ionization tandem mass spectrometry.

The determination of chiral purity is critical to the evaluation of the quality of peptide pharmaceutical products. For synthetic peptides, the undesirable d-isomers can be introduced as impurities in amino acid starting materials and can also be formed during peptide synthesis and in some cases during product shelf life. A chiral high-performance liquid chromatography-electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS) method is described that facilitates rapid and accurate determination of amino acid chiral purity of a peptide.

Development, Validation, and Interlaboratory Evaluation of a Quantitative Multiplexing Method To Assess Levels of Ten Endogenous Allergens in Soybean Seed and Its Application to Field Trials Spanning Three Growing Seasons.

As part of the regulatory approval process in Europe, comparison of endogenous soybean allergen levels between genetically engineered (GE) and non-GE plants has been requested. A quantitative multiplex analytical method using tandem mass spectrometry was developed and validated to measure 10 potential soybean allergens from soybean seed. The analytical method was implemented at six laboratories to demonstrate the robustness of the method and further applied to three soybean field studies across multiple growing seasons (including 21 non-GE soybean varieties) to assess the natural variation of allergen levels.

Development and Validation of a Multiplexed Protein Quantitation Assay for the Determination of Three Recombinant Proteins in Soybean Tissues by Liquid Chromatography with Tandem Mass Spectrometry.

Currently, traditional immunochemistry technologies such as enzyme-linked immunosorbent assays (ELISA) are the predominant analytical tool used to measure levels of recombinant proteins expressed in genetically engineered (GE) plants. Recent advances in agricultural biotechnology have created a need to develop methods capable of selectively detecting and quantifying multiple proteins in complex matrices because of increasing numbers of transgenic proteins being coexpressed or "stacked" to achieve tolerance to multiple herbicides or to provide multiple modes of action for insect control. A multiplexing analytical method utilizing liquid chromatography with tandem mass spectrometry (LC-MS/MS) has been developed and validated to quantify three herbicide-tolerant proteins in soybean tissues: aryloxyalkanoate dioxygenase (AAD-12), 5-enol-pyruvylshikimate-3-phosphate synthase (2mEPSPS), and phosphinothricin acetyltransferase (PAT).

NMR structure of the S-linked glycopeptide sublancin 168.

Sublancin 168 is a member of a small group of glycosylated antimicrobial peptides known as glycocins. The solution structure of sublancin 168, a 37-amino-acid peptide produced by Bacillus subtilis 168, has been solved by nuclear magnetic resonance (NMR) spectroscopy. Sublancin comprises two α-helices and a well-defined interhelical loop.

The glycosyltransferase involved in thurandacin biosynthesis catalyzes both O- and S-glycosylation.

The S-glycosyltransferase SunS is a recently discovered enzyme that selectively catalyzes the conjugation of carbohydrates to the cysteine thiol of proteins. This study reports the discovery of a second S-glycosyltransferase, ThuS, and shows that ThuS catalyzes both S-glycosylation of the thiol of cysteine and O-glycosylation of the hydroxyl group of serine in peptide substrates. ThuS-catalyzed S-glycosylation is more efficient than O-glycosylation, and the enzyme demonstrates high tolerance with respect to both nucleotide sugars and peptide substrates.

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.

Non-proteinogenic amino acids in lacticin 481 analogues result in more potent inhibition of peptidoglycan transglycosylation.

Lantibiotics are ribosomally synthesized and post-translationally modified peptide natural products that contain the thioether structures lanthionine and methyllanthionine and exert potent antimicrobial activity against Gram-positive bacteria. At present, detailed modes-of-action are only known for a small subset of family members. Lacticin 481, a tricyclic lantibiotic, contains a lipid II binding motif present in related compounds such as mersacidin and nukacin ISK-1.

An engineered lantibiotic synthetase that does not require a leader peptide on its substrate.

Ribosomally synthesized and post-translationally modified peptides are a rapidly expanding class of natural products. They are typically biosynthesized by modification of a C-terminal segment of the precursor peptide (the core peptide). The precursor peptide also contains an N-terminal leader peptide that is required to guide the biosynthetic enzymes.

Haloduracin α binds the peptidoglycan precursor lipid II with 2:1 stoichiometry.

The two-peptide lantibiotic haloduracin is composed of two post-translationally modified polycyclic peptides that synergistically act on gram-positive bacteria. We show here that Halα inhibits the transglycosylation reaction catalyzed by PBP1b by binding in a 2:1 stoichiometry to its substrate lipid II. Halβ and the mutant Halα-E22Q were not able to inhibit this step in peptidoglycan biosynthesis, but Halα with its leader peptide still attached was a potent inhibitor.

Sublancin is not a lantibiotic but an S-linked glycopeptide.

Sublancin is shown to be an S-linked glycopeptide containing a glucose attached to a cysteine residue, establishing a new post-translational modification. The activity of the S-glycosyl transferase was reconstituted in vitro, and the enzyme is shown to have relaxed substrate specificity, allowing the preparation of analogs of sublancin. Glycosylation is essential for its antimicrobial activity.

Follow the leader: the use of leader peptides to guide natural product biosynthesis.

The avalanche of genomic information in the past decade has revealed that natural product biosynthesis using the ribosomal machinery is much more widespread than originally anticipated. Nearly all of these compounds are crafted through post-translational modifications of a larger precursor peptide that often contains the marching orders for the biosynthetic enzymes. We review here the available information for how the peptide sequences in the precursors govern the post-translational tailoring processes for several classes of natural products.

Insights into the mode of action of the two-peptide lantibiotic haloduracin.

Haloduracin, a recently discovered two-peptide lantibiotic composed of the post-translationally modified peptides Halalpha and Halbeta, is shown to have high potency against a range of Gram-positive bacteria and to inhibit spore outgrowth of Bacillus anthracis. The two peptides display optimal activity in a 1:1 stoichiometry and have efficacy similar to that of the commercially used lantibiotic nisin. However, haloduracin is more stable at pH 7 than nisin.

In vitro mutasynthesis of lantibiotic analogues containing nonproteinogenic amino acids.

Lantibiotics are ribosomally synthesized and post-translationally modified peptide antibiotics containing the characteristic thioether cross-links lanthionine and methyllanthionine. To date, no analogues of lantibiotics that contain nonproteinogenic amino acids have been reported. In this study, in vitro-reconstituted lacticin 481 synthetase was used in conjunction with synthetic peptide substrates containing nonproteinogenic amino acids to generate 11 analogues of lacticin 481.