Spatial and Depth Profiling of Agricultural Formulations in Leaf Tissue Using LAESI Mass Spectrometry.

Formulating agrochemical products involves combining several chemical components, including the active ingredient(s), to obtain a final product with desirable efficacy. A formulated product incorporates additional components to modulate properties that enhance the efficacy of the active(s) by modifying physical properties such as viscosity, hydrophobicity, miscibility, and others. In plants, understanding the formulation's ability to spread on tissues and penetrate through the outer layer is critical in evaluating the efficacy of the final product. We have previously demonstrated the use of mass spectrometry imaging to determine spreadability. In this study, we show that laser ablation electrospray mass spectrometry (LAESI-MS) can be a valuable tool to assess the penetrability of formulations into the leaf tissues by selectively sampling various layers of leaf tissue by manipulating the laser intensity and analyzing the ablated material using a mass spectrometer. Using this technique, we were able to identify a formulation composition that can improve the penetration and uptake of active ingredients.

Modification of a Quadrupole/Orbitrap/Linear Quadrupole Ion Trap Tribrid Mass Spectrometer for Diagnostic Gas-Phase Ion-Molecule Reactions.

Tandem mass spectrometry based on diagnostic gas-phase ion-molecule reactions represents a robust method for functional group identification in unknown compounds. To date, most of these reactions have been studied using unit-resolution instruments, such as linear quadrupole ion traps and triple quadrupoles, which cannot be used to obtain elemental composition information for the species of interest. In this study, a high-resolution mass spectrometer, a quadrupole/orbitrap/linear quadrupole ion trap tribrid, was modified by installing a portable reagent inlet system to obtain high-resolution data for ion-molecule reactions. Examination of a previously published test system, the reaction between protonated 1,1'-sulfonyldiimizadole with 2-methoxypropene, demonstrated the ability to perform ion-molecule reactions on the modified tribrid mass spectrometer. High-resolution data were obtained for ion-molecule reactions of three isobaric ions (protonated glycylalanine, protonated glutamine, and protonated lysine) with diethylmethoxyborane. On the basis of these data, the isobaric ions can be differentiated based on both their measured accurate mass as well as the different product ions they generated upon the ion-molecule reactions. In a different experiment, analyte ions were subjected to collision-induced dissociation (CID), and the structures of the resulting fragment ions were examined via diagnostic ion-molecule reactions. This experiment allows for the functional group interrogation of fragment ions and can be used to improve the understanding of the structures of fragment ions generated in the gas phase.

Investigation of fragmentation pathways of protonated 2-methoxypyrimidine derivatives.

Several representative pyrimidine derivatives were selected to undergo electrospray ionization (ESI) followed by collision-induced dissociation tandem mass spectrometry (CID MS/MS) experiments. Two competitive pathways were found to govern the formation of major fragment ions from protonated species of these molecules. The pathways were largely affected by the 2-O-methyl group but not significantly influenced by the substitution on C-5 site of the pyrimidine ring. These findings were supported by both deuterium labeling CID MS/MS experiments and theoretical calculations. The deuterium labeled pyrimidine ion molecules were generated in-source in ESI from the fully deuterated hydrazinyl pyrimidines, which were readily obtained through hydrogen/deuterium (H/D) exchange when dissolved in deuterium oxide (D2 O).

Alveolarides: Antifungal Peptides from Microascus alveolaris Active against Phytopathogenic Fungi.

Three novel cyclodepsipeptides, alveolarides A (1), B (2), and C (3), each possessing the rare 2,3-dihydroxy-4-methyltetradecanoic acid unit and a ß-phenylalanine amino acid residue, along with the known peptide scopularide were isolated and identified from the culture broth of Microascus alveolaris strain PF1466. The pure compounds were evaluated for biological activity, and alveolaride A (1) provided strong in vitro activity against the plant pathogens Pyricularia oryzae, Zymoseptoria tritici, and Ustilago maydis. Moderate activity of alveolaride A was observed under in planta conditions against Z. tritici, Puccinia triticina, and Phakopsora pachyrhizi. Structures of 1, 2, and 3 were determined by detailed analysis of NMR (1D and 2D) and mass spectrometry data. The partial absolute configuration of alveolaride A (1) was established.

Fragmentation of Protonated N-(3-Aminophenyl)Benzamide and Its Derivatives in Gas Phase.

An ion of m/z 110.06036 (ion formula [C6H8NO](+); error: 0.32 mDa) was observed in the collision induced dissociation tandem mass spectrometry experiments of protonated N-(3-aminophenyl)benzamide, which is a rearrangement product ion purportedly through nitrogen-oxygen (N-O) exchange. The N-O exchange rearrangement was confirmed by the MS/MS spectrum of protonated N-(3-aminophenyl)-O (18) -benzamide, where the rearranged ion, [C6H8NO (18) ](+) of m/z 112 was available because of the presence of O (18) . Theoretical calculations using Density Functional Theory (DFT) at B3LYP/6-31 g(d) level suggest that an ion-neutral complex containing a water molecule and a nitrilium ion was formed via a transition state (TS-1), followed by the water molecule migrating to the anilide ring, eventually leading to the formation of the rearranged ion of m/z 110. The rearrangement can be generalized to other protonated amide compounds with electron-donating groups at the meta position, such as, -OH, -CH3, -OCH3, -NH(CH3)2, -NH-Ph, and -NHCOCH3, all of which show the corresponding rearranged ions in MS/MS spectra. However, the protonated amide compounds containing electron-withdrawing groups, including -Cl, -Br, -CN, -NO2, and -CF3, at the meta position did not display this type of rearrangement during dissociation. Additionally, effects of various acyl groups on the rearrangement were investigated. It was found that the rearrangement can be enhanced by substitution on the ring of the benzoyl with electron-withdrawing groups.

MALDI-MS Imaging Analysis of Fungicide Residue Distributions on Wheat Leaf Surfaces.

Improved retention and distribution of agrochemicals on plant surfaces is an important attribute in the biological activity of pesticide. Although retention of agrochemicals on plants after spray application can be quantified using traditional analytical techniques including LC or GC, the spatial distribution of agrochemicals on the plants surfaces has received little attention. Matrix assisted laser desorption/ionization (MALDI) imaging technology has been widely used to determine the distribution of proteins, peptides and metabolites in different tissue sections, but its application to environmental research has been limited. Herein, we probed the potential utility of MALDI imaging in characterizing the distribution of three commercial fungicides on wheat leaf surfaces. Using this MALDI imaging method, we were able to detect 500 ng of epoxiconazole, azoxystrobin, and pyraclostrobin applied in 1 µL drop on the leaf surfaces using MALDI-MS. Subsequent dilutions of pyraclostrobin revealed that the compound can be chemically imaged on the leaf surfaces at levels as low as 60 ng of total applied in the area of 1 µL droplet. After application of epoxiconazole, azoxystrobin, and pyraclostrobin at a field rate of 100 gai/ha in 200 L water using a track sprayer system, residues of these fungicides on the leaf surfaces were sufficiently visualized. These results suggest that MALDI imaging can be used to monitor spatial distribution of agrochemicals on leaf samples after pesticide application.

Quantification of the sulfated cholecystokinin CCK-8 in hamster plasma using immunoprecipitation liquid chromatography-mass spectrometry/mass spectrometry.

Cholecystokinin (CCK) and the different molecular forms of CCK are well established as biomarkers for satiety but accurate analysis has been limited by the multiple naturally occurring forms and extensive similarities to gastrin. Changes in levels of one form, CCK-8, a naturally occurring eight amino acid peptide of CCK, have been correlated with satiety responses. Endogenous CCK-8 has not been well characterized in Syrian Golden hamsters, an important model in the study of fat uptake and digestion. We have cloned and sequenced hamster CCK and identified and characterized endogenous CCK-8 from hamster plasma. Hamster CCK-8 is composed of eight amino acid residues which are highly conserved among other species. Following accurate identification and characterization of hamster CCK-8, we have developed a highly specific and sensitive immunoprecipitation based LC-MS/MS assay for its quantification. The present assay enables determination of active CCK-8 over a concentration range from 0.05 to 2.5 ng/mL in hamster plasma samples. This range covers both the basal and postprandial levels of CCK-8. Method performance validation samples were examined at three concentrations replicated over the course of 4 days. The assay accuracy (percent relative error, % RE) average was 11.3% with a precision (percent coefficient of variation, % CV) of 15.5% over all samples in this 4 day period. Additionally, the method was used to determine increases of endogenous plasma CCK-8 in hamsters challenged with a high-fat meal.

Discovery of the butenyl-spinosyn insecticides: novel macrolides from the new bacterial strain Saccharopolyspora pogona.

A new bacterium, Saccharopolyspora pogona (NRRL30141) was discovered which produced a series of very potent insecticidal compounds structurally related to the 'classical' (i.e., C-21-ethyl) spinosyns. A series of fermentations gave sufficient extract to allow the isolation and characterization of a total of 31 new metabolites. The majority of these compounds contained a but-1-enyl group at C-21 of the macrolide in place of the ethyl group in the 'classical' spinosyn series, corresponding to an additional acetate group incorporated during their biosynthesis. Additionally a variety of other new functionality was seen including hydroxylations, several novel forosamine sugar replacements, and a novel 14-membered macrolide ring analog.

Albucidin: a novel bleaching herbicide from Streptomyces albus subsp. chlorinus NRRL B-24108.

A novel nucleoside phytotoxin, albucidin (1), was isolated from the culture broth of Streptomyces albus subsp. chlorinus NRRL B-24108 using bioassay directed fractionation. The structure of the new natural product, albucidin, was determined by NMR and MS; however, the compound has been reported earlier in the literature following synthetic modification of oxetanocin. This is the first report of herbicidal activity for compounds of this structural type. Albucidin shows high levels of broad spectrum activity following post-emergence applications as well as moderate levels of pre-emergence activity. Accordingly, albucidin could be an important new lead for herbicide discovery.

The ansacarbamitocins: polar ansamitocin derivatives.

The ansacarbamitocins are a new family of maytansinoids that are unusually substituted with a glucose subunit and two carbamate functional groups and exhibit modest activity against some agricultural fungal disease organisms. Ansacarbamitocins A-F ( 1- 6) all consist of the same macrocyclic core as the ansamitocins, with variation occurring on the glucose unit, while ansacarbamitocins A1 and B1 ( 7, 8) additionally lack the epoxide group on C-4 and C-5.

Discovery, synthesis, and insecticidal activity of cycloaspeptide E.

Several Penicillia and one Tricothecium strain produced a new, insecticidally active member of the cycloaspeptide family, with the proposed name cycloaspeptide E (1). The structure, which was determined on the basis of spectroscopic (NMR, UV, MS) data and Marfey amino acid analysis, was the tyrosine desoxy version of cycloaspeptide A (2). Two synthetic routes to compound 1 were developed: one a partial synthesis from 2 and the other a total synthesis from methyl alaninate hydrochloride. Cycloaspeptide E, the first member of this series not to contain a tyrosine moiety, is also the first to be reported with insecticidal activity.

Purification and characterization of a chimeric Cry1F delta-endotoxin expressed in transgenic cotton plants.

Cotton plants were genetically modified through the introduction of a synthetic gene that encodes a Bacillus thuringiensis insecticidal protoxin referred to as Cry1F(synpro). This protoxin is a chimeric, full-length delta-endotoxin of 130 kDa, comprised of the core toxin of Cry1Fa2 protein and parts of the nontoxic portions of Cry1Ca3 and Cry1Ab1 proteins, all of which originated from Bacillus thuringiensis. The Cry1F(synpro) expressed in cotton plants confers resistance to lepidopteran pests. The current study was conducted to characterize the Cry1F(synpro) protein expressed in the transgenic cotton event 281-24-236. Results showed that the full-length Cry1F(synpro) produced in the transgenic cotton plants was sensitive to the host cell protease cleavage, resulting in a truncated, biologically active form (core toxin) with an apparent molecular mass of 65 kDa. This truncated toxin was purified by immunoaffinity chromatography from the cotton leaf extract. N-terminal sequencing, peptide mass fingerprinting by MALDI-TOF MS, and internal peptide sequencing by MS/MS confirmed the identity of the truncated core toxin of Cry1F. The mechanism of truncation was explored with Cry1F(synpro) derived from a recombinant Pseudomonas fluorescens. The transgenic cotton-produced Cry1F showed equivalent insecticidal activity to that of Pseudomonas fluorescens-derived Cry1F.

Characterization of Cry34Ab1 and Cry35Ab1 insecticidal crystal proteins expressed in transgenic corn plants and Pseudomonas fluorescens.

Cry34Ab1 and Cry35Ab1 proteins, identified from Bacillus thuringiensis strain PS149B1, act together to control corn rootworms. Transgenic corn lines coexpressing the two proteins were developed to protect corn against rootworm damage. Large quantities of the two proteins were needed to conduct studies required for assessing the safety of this transgenic corn crop. Because it was technically infeasible to obtain sufficient quantities of high purity Cry34Ab1 and Cry35Ab1 proteins from the transgenic corn plants, the proteins were produced using a recombinant Pseudomonas fluorescens (Pf) production system. The two proteins from both the transgenic corn and the Pf were purified and characterized. The proteins from each host had the expected molecular mass and were immunoreactive to specific antibodies in enzyme-linked immunosorbent assay and Western blot analysis. Data from N-terminal sequencing, tryptic peptide mass fingerprinting, internal peptide sequencing, and biological activity provided direct evidence that the Cry34Ab1 and Cry35Ab1 proteins produced in Pf and transgenic corn were, respectively, comparable or equivalent molecules. In addition, neither protein had detectable glycosylation regardless of the host.

Tartrolone C: a novel insecticidal macrodiolide produced by Streptomyces sp. CP1130.

A new member of the tartrolone series of macrodiolides, tartrolone C (1), was isolated from a Streptomyces species on the basis of its insecticidal activity. Metacycloprodigiosin (2) and undecylprodigiosin (3) were also isolated on the same basis. The structure of all compounds was established by spectroscopic data (NMR, MS, and UV).