The Determination of a Threshold Level of Exogenous Methyl Jasmonate Effecting an Increase in Peroxidase Activity in Protoplasts of Avena Sativa

Methyl jasmonate plays a dual role in plant systems by participating in both developmental and defense mechanisms. Recent studies have shown that methyl jasmonate can specifically alter gene expression, while wounding and specific elicitors can cause a buildup of methyl jasmonate itself. Peroxidase enzymes are ubiquitous to all plant systems and have long been associated with plant stress and defense responses. The objective of this research was to establish a threshold concentration of methyl jasmonate effecting a maximum increase in peroxidase activity less than twenty-four hours after treatment. While previous experiments have explored the activity of methyl jasmonate on peroxidase activity using whole leaf tissue, we developed a system for testing peroxidase activity in oat protoplasts after isolation and subsequent treatment and incubation in a suitable culture media

But my peaks are not Gaussian! Part 3: Physicochemical causes of peak tailing

Although symmetric peaks with Gaussian shapes are predicted by models of the chromatographic process, "perfect peaks" are not observed often outside of textbooks. Several physico-chemical phenomena can lead to asymmetric peak shapes, including analyte adsorption to different types of sites within the stationary phase, and overload tailing, which may involve a variety of factors. Understanding these phenomena can help identify whether the cause of asymmetry is most likely to have a physical or chemical origin, which in turn dictates which troubleshooting steps to start with when dealing with poor peak shapes

The Effects of Wounding and Ethylene on Cellular Fatty Acid Composition of Avena Sativa

Plant defense responses to wounding, pathogen attack, and environmental stresses often involve the production of various intermediates of the octadecanoid pathway, which are derived from the fatty acid linolenic acid. Ethylene is a plant hormone that has also been implicated in defense responses. The goal of this project has been to determine if wounding causes changes in the fatty acid composition of plants and to determine whether ethylene plays a role in this response. Controlled and wounded nine-day old Avena sativa (oats) plants were treated with ethylene, norbomadiene or silver thiosulfate. Norbomadiene and silver thiosulfate inhibit the action of endogenous ethylene. The oat leaves were harvested at 0, 1,4, 8, and 22 hours following wounding and treatment. Lipids were then extracted from leaves and the lipids were fractionated using solid phase extraction. The methyl esters of the fatty acids in these fractions were prepared by transesterification with sulfuric acid in methanol. The resulting fatty acid methyl esters will be analyzed using gas chromatography with methyl heptadecanoate as an internal standard

Smart Templates for peak pattern matching with comprehensive two-dimensional liquid chromatography

Comprehensive two-dimensional liquid chromatography (LC × LC) generates information-rich but complex peak patterns that require automated processing for rapid chemical identification and classification. This paper describes a powerful approach and specific methods for peak pattern matching to identify and classify constituent peaks in data from LC × LC and other multidimensional chemical separations. The approach records a prototypical pattern of peaks with retention times and associated metadata, such as chemical identities and classes, in a template. Then, the template pattern is matched to the detected peaks in subsequent data and the metadata are copied from the template to identify and classify the matched peaks. Smart Templates employ rule-based constraints (e.g., multispectral matching) to increase matching accuracy. Experimental results demonstrate Smart Templates, with the combination of retention-time pattern matching and multispectral constraints, are accurate and robust with respect to changes in peak patterns associated with variable chromatographic conditions

Tips, Tricks, and Troubleshooting for Separations of Biomolecules, Part I: Contemporary Reversed-Phase Protein Separations

Several new materials and columns have been introduced in recent years for reversed-phase separations of proteins. How do I know which one to choose, and which separation conditions will be best for my protein separation

Tips, Tricks, and Troubleshooting for Separations of Biomolecules, Part 1: Contemporary Reversed-Phase Protein Separations

Several new materials and columns have been introduced in recent years for reversed-phase separations of proteins. How do I know which one to choose, and which separation conditions will be best for my protein separation

A Strategy for Assessing Peak Purity of Pharmaceutical Peptides in Reversed-Phase Chromatography Methods using Two-Dimensional Liquid Chromatography Coupled to Mass Spectrometry. Part I: Selection of Columns and Mobile Phases

The current study describes the development of a 2D-LC-MS-based strategy for assessing main peak purity in the analysis of pharmaceutical peptides. The focus is on 2D-LC using reversed-phase (RP) separations in both dimensions, and particularly peptide isomer selectivity, since compounds with the same mass to charge ratio are not readily differentiated by mass spectrometry and therefore must be separated chromatographically. Initially, 30 column / mobile phase combinations were evaluated for both general separation performance (i.e., selectivity and peak shape) and isomer selectivity using forcibly degraded peptide samples and mixtures of synthetic diastereomers. A ranking of more than 300 UV and MS chromatograms suggests that when developing a new method, screening a set of four columns and four volatile mobile phases with differing characteristics should be adequate to both cover the selectivity space, and yield good separation performance. When 2D-LC-MS is to be used to evaluate peak purity for a new method, our results show that a second-dimension separation comprising a C8/C18 column possessing no ionic functionality, and an acetic acid / ammonium acetate mobile phase buffered at pH 5, provides good selectivity at 25 °C for peptide isomers with a MW Retention data for 29 diverse peptides (1 i.e., 30 in total) are consistent with the classification of these various chromatographic conditions using the previously reported Peptide RPC Column Characterisation Protocol. For the investigated peptides trifluoroacetic acid was found to reduce selectivity differences between columns of diverse properties, probably due to its potential to form ion-pairs with peptides. Trifluoroacetic acid often improves peak shape for very large peptides (i.e. MW > 10 kDa). In the current dataset which also contain smaller peptides it received the highest ranking for 40% of the column and mobile phase combinations due to better selectivity and/or peak shape. The reported work here constitutes part one of a series of two papers. The second paper focuses on the use of retention modelling for rapid and accurate selection of the shallow gradients (i.e., << 1% ACN/min) required to obtain sufficient peptide isomer retention and separation in the second dimension. The overall results presented in this series of papers provides the guidance needed to develop a 2D-LC-MS method from start to finish for the analysis of main peak purity of therapeutic peptides