Mass spectrometric profiling of glucosamine, glucosamine polymers and their catecholamine adducts. Model reactions and cuticular hydrolysates of Toxorhynchites amboinensis (Culicidae) pupae.

Glucosamine (Gln), glucosamine polymers, and their catecholamine adducts were characterized using positive ion electrospray mass spectrometry (ESMS) and tandem mass spectrometry (ESMS-MS). N-acetyldopamine (NADA), a catecholamine found in many insect cuticles, was oxidized using mushroom tyrosinase, and the resulting quinone derivatives were reacted with Gln, (Gln)3, and polymeric glucosamine (chitosan). Adducts of glucosamine and its trisaccharide with NADA were readily identified as [M + H]+ ions in ESMS spectra, and ESMS-MS of selected ions confirmed the condensation of 1-3 NADA residues with Gln. In addition to Gln modification by the quinone derivatives of NADA, other spectra were consistent with the formation of adducts with N-acetylnoradrenaline and moieties formed by intramolecular cyclization following oxidation. The primary amine of glucosamine was involved in initial adduct formation, but the sites for subsequent additions of oxidized NADA to glucosamine, presumably via hydroxyl groups, could not be identified by ESMS alone. The ESMS spectra of chitosan films infused into the spectrometer following solubilization in acidic methanol/water produced spectra similar to that of (Gln)3 up to m/z 502. Ions of gradually decreasing intensity consistent with (Gln)x, where x = 4-8, were observed. Modification of chitosan films following incubation with NADA plus tyrosinase rendered the films insoluble in dilute acid, simulating the cross-linking process proposed to occur during insect cuticle sclerotization. Acid hydrolysates of the pupal stage of the mosquito Toxorhynchites amboinensis, using only two pupal exuviae for the hydrolyses, were infused into the mass spectrometer without preliminary chromatography. Eight amino acids, glucosamine, N-acetylglucosamine, catecholamines, and a variety of polymers incorporating these compound classes were identified.

Mass spectrometric analysis of catechol-histidine adducts from insect cuticle.

Adducts of catechols and histidine, which are produced by reactions of 1,2-quinones and p-quinone methides with histidyl residues in proteins incorporated into the insect exoskeleton, were characterized using electrospray ionization mass spectrometry (ESMS), tandem electrospray mass spectrometry (ESMS-MS, collision-induced dissociation), and ion trap mass spectrometry (ITMS). Compounds examined included adducts obtained from acid hydrolysates of Manduca sexta (tobacco hornworm) pupal cuticle exuviae and products obtained from model reactions under defined conditions. The ESMS and ITMS spectra of 6-(N-3')-histidyldopamine [6-(N-3')-His-DA, pi isomer] isolated from M. sexta cuticle were dominated by a [M + H]+ ion at m/z 308, rather than the expected m/z 307. High-resolution fast atom bombardment MS yielded an empirical formula of C14H18N3O5, which was consistent with this compound being 6-(N-1')-histidyl-2-(3, 4-dihydroxyphenyl)ethanol [6-(N-1')-His-DOPET] instead of a DA adduct. Similar results were obtained when histidyl-catechol compounds linked at C-7 of the catechol were examined; the (N-1') isomer was confirmed as a DA adduct, and the (N-3') isomer identified as an (N-1')-DOPET derivative. Direct MS analysis of unfractionated cuticle hydrolysate revealed intense parent and product ions characteristic of 6- and 7-linked adducts of histidine and DOPET. Mass spectrometric analysis of model adducts synthesized by electrochemical oxidative coupling of N-acetyldopamine (NADA) quinone and N-acetylhistidine (NAcH) identified the point of attachment in the two isomers. A prominent product ion corresponding to loss of CO2 from [M + H]+ of 2-NAcH-NADA confirmed this as being the (N-3') isomer. Loss of (H2O + CO) from 6-NAcH-NADA suggested that this adduct was the (N-1') isomer. The results support the hypothesis that insect cuticle sclerotization involves the formation of C-N cross-links between histidine residues in cuticular proteins, and both ring and side-chain carbons of three catechols: NADA, N-beta-alanyldopamine, and DOPET.

Ceramide profiling of complex lipid mixtures by electrospray ionization mass spectrometry.

Ceramides and sphingoid bases are important intracellular second messengers that play a role in the regulation of cell growth, differentiation, and programmed cell death. Until now, quantitative and qualitative analysis of ceramide second messengers has been limited by a lack of analytical methods capable of detecting endogenous levels of, and differentiating between, individual ceramide species. Here we report the use of electrospray ionization tandem mass spectrometry for the qualitative and quantitative analysis of ceramides. Collision-induced fragmentation resulted in characteristic product ions for the sphingosine and dihydrosphingosine (sphinganine) head groups at m/z 264 and 282 and m/z 266 and 284, respectively, regardless of the length of the fatty acyl chains, with spectra being reproducible at concentrations as low as 25 nM (25 fmol/microliter). These reporter ions were used to detect both sphingosine- and sphinganine-based ceramides in complex mixtures using precursor ion scan analysis. We demonstrated the application of this method for profiling the composition of ceramides in a commercial preparation of bovine brain ceramides and, following minimal chromatographic separation, a lipid extract of cultured T cells. Furthermore, we easily detected relative differences in individual ceramide species levels by comparing the profiles of three related lymphocyte cell lines, Jurkat, U937, and WEHI 231. Finally, by the addition of a nonnaturally occurring internal standard, we show that the technique can be used to measure quantitative changes in ceramide levels in such biologically derived lipid preparations.

Profiling peptide adducts of oxidized N-acetyldopamine by electrospray mass spectrometry.

Phenolic and catecholamine polymers are common constituents of many biological systems. Cross-linking of polyphenols with other phenols, peptides, proteins and carbohydrates results in the synthesis of complex natural products which are not easily characterized. Electrospray mass spectrometry (ES-MS) and tandem mass spectrometry (ES-MS/MS) were used to identify polymers of oxidized N-acetyldopamine (NADA) and peptide adducts with oxidized NADA. Following incubation with mushroom tyrosinase, NADA adducts of trityrosine were identified. It was not possible to locate the site of NADA binding to this tripeptide. Compounds formed by incubation of N-acetylhistidine and N-acetyllysine with oxidized NADA, previously characterized using classical chemical techniques, were confirmed using ES-MS/MS. The peptide angiotensin (DRVYIHPFHL) was used as a model substrate to determine whether the site(s) to which oxidized NADA bound could be determined. The lot of angiotensin used was contaminated with a peptide of mass 14 u greater than angiotensin, and it was found that the H in position 9 of the contaminant peptide was modified. ES-MS/MS of the angiotensin and the contaminant peptide following incubation with oxidized NADA revealed that the C-terminal aspartic acid was the primary amino acid to which NADA adducts were covalently bound, but other residues were also modified. Femto-molar sensitivity for analysis of complex mixtures of catecholamine-peptide adducts will facilitate structural elucidation of natural products not amenable to characterization using other spectroscopic techniques.

Negative ion electrospray mass spectrometry of polyphenols, catecholamines and their oxidation products.

Polyphenols, catecholamines and their oxidation products have a variety of physiological effects and are key components of insect cuticle and other biological structures. Three dihydroxybenzene isomers were easily differentiated using negative ion electrospray mass spectrometry (ESMS) or tandem mass spectrometry (MS/MS). Ions at m/z 107-109 were diagnostic for more complex polyphenols and catecholamines with a dihydroxybenzene moiety. The product ion spectra of other compounds including 3,4-dihydroxyphenylacetic acid, 3,4-dihydroxyphenylalanine (DOPA) and N-acetyldopamine (NADA) had product ions at m/z 121-123 corresponding to methylene homologues of dioxygenated benzene. Oxidation of DOPA using tyrosinase, Ag2O or NaIO4 could be followed using ESMS and several proposed intermediates proceeding from DOPA through a quinone methide intermediate, 5,6-dihydroxyindole and dihydroxyindole quinone were confirmed using negative ion ESMS/MS. Spectra of the NaIO4 oxidation products of 1,2,3-trihydroxybenzene (pyrogallol) were consistent with tetrahydroxybenzene, two non-cyclic tetraoxygenated derivatives and a bicyclic polymerization product, purpurogallin. ESMS can be used to analyse complex polyphenolic conjugates commonly encountered in biological specimens.

Sterol and steryl ester regulation of phospholipase A2 from the mosquito parasite Lagenidium giganteum.

Lagenidium giganteum, a facultative parasite of mosquito larvae, cannot synthesize sterols, and requires an exogenous source of these lipids in order to enter its reproductive cycle. This parasite grows vegetatively in the absence of sterols, but requires cholesterol or structurally related compounds to produce motile zoospores, which are the only stage capable of infecting mosquitoes. Sterols structurally related to cholesterol and some steryl esters inhibited the activity of L. giganteum phospholipase A2 (PLA2), an enzyme that hydrolyzes fatty acids from the sn-2 position of glycerophospholipids. Sterols that induce reproduction inhibited L. giganteum PLA2 activity, while sterols and steroids that do not support sporulation had minimal effect. Most steryl esters had no effect on enzyme activity, but cholesteryl arachidonate (CA) was a potent inhibitor of parasite PLA2. Not all enzymes partly purified using a DEAE-Sephacel column were affected by these lipids, demonstrating selective inhibition of specific enzymes. Potency was enhanced by up to several orders of magnitude if epoxy fatty acids were esterified to the cholesterol nucleus. The steryl ester pool was dynamic during morphogenesis, and the fatty acid composition of the steryl esters did not mimic total cell or membrane (glycerophospholipid) fatty acid composition as L. giganteum proceeded through its growth cycle. Synthesis of CA and monoepoxy CA by the parasite was confirmed using electrospray mass spectrometry and collision-induced dissociation. Steryl derivatives selectively inhibited PLA2 enzymes from bovine pancreas, snake venom, and human cytoplasmic 85-kDa PLA2.

Identification of monohydroxy fatty acids by electrospray mass spectrometry and tandem mass spectrometry.

Negative-ion electrospray mass spectrometry (ESI-MS) and tandem mass spectrometry (ESI-MS/MS) were used to characterize saturated and unsaturated monohydroxy fatty acids and fatty acid metabolites formed following incubation with soybean lipoxygenase. Ions corresponding to [M-H]- of eicosanoids were readily observed using ESI-MS, but double bond migration precluded the use of MS to localize double bonds or the position of hydroxyl moieties; however, by following MS analysis with negative-ion ESI-MS/MS of precursor ions, the position of oxygenation could be determined for picogram quantities of underivatized monohydroxy fatty acids. Loss of 46 mass units from the precursor ion of saturated monohydroxy compounds was explained in some cases by resonance stabilization of enolate ions, but this product ion was found in spectra of compounds where resonance was not possible. Spectra of deuterated analogs supported charge-driven vinylic processes as the most common mechanism of fragmentation. The utility of low-collision-energy ESI-MS/MS to examine biological samples was shown by examining the products formed by the metabolism of linoleic (18:2omega6) and arachidonic (20:4omega6) acids by soybean lipoxygenase using aerobic and anaerobic incubation conditions that generated increasingly complex mixes of metabolites.

Identification of fatty acids by electrospray mass spectrometry and tandem mass spectrometry.

Low-energy negative-ion electrospray mass spectrometry (ESI-MS) and ESI-MS/MS were used to characterize saturated and unsaturated fatty acids. The carbon number and degree of unsaturation of fatty acids were determined using ESI-MS, and MS/MS was used to localize some double bond positions of mono-and polyunsaturated fatty acids. For compounds with up to two unsaturated bonds, fragmentation was dominated by loss of H2O from the carboxyl moiety and very low-intensity peaks generated from bonds cleaved at carbons alpha and/or beta to sites of unsaturation. Fragmentation of monounsaturated fatty acids was minimal using this soft method of mass spectrometric analysis, but increased with progressively greater degrees of fatty acid unsaturation. There was extensive hydride migration during ESI-MS/MS of compounds with three or more double bonds. Although this behavior complicated localization of double and triple bonds, the spectra were reproducible. Many peaks could not be definitively assigned to specific product ions, but the spectra of standards and complementary natural products were similar and isobaric compounds could be differentiated. The utility of this technique to examine biological samples was shown by analysis of the fatty acid composition of cod liver oil. Detection limits for negative-ion ESI-MS/MS were at or below 1 pg.

Lagenidium giganteum in Culex tarsalis larvae: production of infective propagules.

Zoospore and oospore production of the entomopathogenic fungus, Lagenidium giganteum, was quantified from Culex tarsalis larvae in rice irrigation water. For the first through fourth instars, respectively, the average zoospore production (+/- SE) was 521 +/- 106, 2458 +/- 365, 10,546 +/- 582, and 14,360 +/- 1076, and the average oospore production was 17 +/- 1.8, 33 +/- 3.5, 44 +/- 2.6, and 34 +/- 3.1. Zoosporogenesis varied with the water sample. The ionic composition of the water samples was significantly correlated with zoospore production. The correlations, however, changed as different subsets of data were analyzed, suggesting that other factors also affected zoosporogenesis. The extent to which a cadaver was colonized by other microorganisms had a clear effect on the resulting Lagenidium reproduction. The interval of peak zoosporogenesis occurred between 24 and 36 hr postmortem for the first instar and 12 to 24 hr postmortem for other instars. Measurable production was observed up to 96 hr postmortem from fourth instar larvae.

Isoprenoid-mediated changes in the glycerophospholipid molecular species of the sterol auxotrophic fungus Lagenidium giganteum.

The mosquito pathogenic fungus Lagenidium giganteum (Oomycetes: Lagenidiales) is a sterol auxotroph that can grow vegetatively in the absence of these compounds, but requires an exogenous source of sterols to enter its sexual and asexual reproductive cycles. Electrospray mass spectrometry (MS) and electrospray MS/MS were used to examine three major glycerophospholipid molecular species--glycerophosphocholine (GPC), glycerophosphoethanolamine (GPE) and glycerophosphoinositol (GPI)--from fungal mycelium and nuclei grown in defined medium with and without isoprenoids which induce (cholesterol and ergosterol) or do not induce (squalene, cholestane) reproduction. Testosterone supplementation of defined media inhibited growth of L. giganteum, so the effect of this steroid on phospholipid metabolism could not be assessed. Mycelium grown in defined media supplemented with these isoprenoids produced significantly different quantities of total phospholipid relative to unsupplemented media and to each other, ranging from a mean of 292 micrograms phosphate per g wet weight for cholesterol-supplemented media to 56 micrograms phosphate per g wet weight for mycelium grown in the presence of squalene. A very large percentage of the GPC (69-80 mol%) and GPI (74-79 mol%) molecular species from mycelia and nuclei contained ether linkages. GPE molecular species had 13-20 mol% ether-containing moieties. The elevated levels of ether lipids may be related to the sterol auxotrophic nature of the fungus. Isoprenoid supplementation of defined growth media resulted in many significant changes in molecular species for all three lipid classes. Significant differences (P < 0.05) in the percentage of total cell ether lipids in GPC and GPE were generated by isoprenoid supplements to culture media. Mycelium grown in the presence of the two sterols which induce asexual and sexual reproduction in L. giganteum, cholesterol and ergosterol, had a significantly greater percentage of ether-containing GPE moieties. The glycerolipid species from nuclei isolated from cultures grown with cholesterol and ergosterol were similar to the composition of nuclei isolated from fungus cultured in defined medium without any supplement or supplemented with squalene. The nuclear membrane from mycelia grown in cholestane-supplemented media, however, had a very different glycerophospholipid composition relative to either whole cells or nuclei from cells grown on other media. It appears that one of the reasons that cyclic isoprenoids such as cholestane do not induce fungal reproduction is that they drastically alter the nuclear membrane glycerophospholipid composition.

Pilot scale production and application in wildlife ponds of Lagenidium giganteum (Oomycetes: Lagenidiales).

Lagenidium giganteum, a facultative parasite of mosquito larvae, has recently been registered by the U.S. Environmental Protection Agency for operational mosquito control. We report here the first pilot scale production of the mycelium formulation. Scale-up from 10 to 650 liters was accomplished by a proportionate increase of medium components and volume of water. Foaming of the culture medium had not been encountered previously, but was a serious problem in pilot scale production due to the very rapid growth of a large volume of L. giganteum. Addition of an antifoaming agent did not adversely affect growth, but reduced the ability of the fungus to sporulate. Despite what was effectively a 100-fold reduction in the desired application rate due to reduced sporulation, L. giganteum infected sentinel mosquito larvae and reduced field populations for more than 2 months following application.

Identification of molecular species of glycerophospholipids and sphingomyelin using electrospray mass spectrometry.

This paper describes the use of positive and negative ion electrospray mass spectrometry (MS) and MS/MS (tandem mass spectrometry) to identify glycerophospholipid and ceramide headgroups and their alkyl, alkenyl and acyl constituents. Molecular ion adducts were the primary products formed by positive ionization, occurring as [M+H]+, [M+Na]+, [M+K]+, [M+formate]+, or [M+acetate]+, depending upon the class of glycerophospholipid and the presence or absence of these ionization-promoting species. Similar (negatively charged) ions corresponding to the loss of the groups listed above were formed in negative ion MS. Positive ion electrospray MS/MS provides information on the nature of the headgroup, with the formation of an ion corresponding to the headgroup itself, or the loss of the headgroup from the molecular ion H+ or Na+ adduct. Acyl constituents are identified during negative ion MS/MS from the formation of their RCOO- ions. The nature of alkyl or alkenyl substituents in glycerophosphoethanolamine (PE) molecular species can be identified from residual ions following the loss of ethanolamine plus loss of the acyl moiety in the sn-2 position, and cyclization of a phosphate oxygen with C-2 of glycerol. In glycerophosphoinositol (PI) species, it appears that an RCO- ion is formed during negative ion MS/MS, possibly to steric interference from the bulky phosphoinositol headgroup that prevents cyclization (and subsequent stabilization) of the ion described for PE species. Positive and negative ion electrospray MS spectra for molecular species of commercial preparations of PE, PI, phosphatidylserine (PS), glycerophosphocholine (PC) and sphingomyelin (SM) produced similar profiles. For phospholipids occurring as Na+ adducts, concentrations above ca. 1 ng/microliter produced significant quantities of both [M+H]+ and [M+Na]+ ions for those molecular species present in the largest quantities, complicating interpretation of the spectra. Complete profiles of molecular species were obtained from as little as 10 picograms of material. Major components of PE were identified from 0.1 picogram total lipid. Using single ion monitoring of the Na+ adduct of beta-acetyl-gamma-O-hexadecyl L-alpha-phosphatidylcholine, 10 femtograms of material was detected. A mixture of 1 nanogram each of PE, PI, PS, and PC was readily resolved into individual molecular species, with little apparent loss of resolution or preferential ionization. Electrospray MS did not provide information on the position (sn-1 or sn-2) of fatty acids, and was not capable of differentiating in all instances between alkyl-acyl and alkenyl-acyl substituents without prior separation of these lipid subclasses.(ABSTRACT TRUNCATED AT 400 WORDS)

Effects of exogenous phospholipids on lipid composition and sporulation by three strains of Lagenidium giganteum.

The California (LGCA) and Butte Sink (LGBS) strains of the sterol auxotrophic fungus Lagenidium giganteum (Oomycetes: Lagenidiales) enter the sexual cycle on media supplemented with sterols. A third isolate of this mosquito pathogen, the North Carolina strain (LGNC), requires sterols plus phospholipids to produce oospores in vitro. Enrichment of the polar and neutral lipid fractions of the LGCA and LGBS strains with unsaturated fatty acids promoted oospore induction, and increased oospore viability. With the exception of the LGCA strain, there was no consistent relationship between phospholipid supplementation in growth media and mycelial phospholipid content.

Confirmation of the safety of Lagenidium giganteum (Oomycetes: Lagenidiales) to mammals.

Lagenidium giganteum Couch, a fungal parasite of mosquito larvae, is considered a promising agent for operational control. Quantitative reisolation of the fungus from rodent blood and tissue is easily accomplished because the cell walls are much more persistent than those of mammalian cells. The mycelium forms discrete, septate cells very early in its development, in marked contrast to filamentous fungi. The fungus is rapidly cleared from mice following intraperitoneal injection of large quantities of mycelium and oospores. More than 95% of viable cells are cleared within 24 h. Mortality following intravenous injection of mycelium in tail veins of mice is caused solely by embolism of heart and lung tissue. Recently revised U.S. Environmental Protection Agency protocols for testing of microbial pest control agents may have to be altered further to accommodate physically large microbial agents such as L. giganteum.

Carbohydrate-mediated Recognition of Larval Mosquito Hosts by Romanomermis culicivorax.

Proteases, glycosidases, and lectins were tested and the results supported a role in host recognition for glycoproteins containing beta-glucose and alpha-mannose on the cuticular surface of host and parasite. Carbohydrates containing alpha-glucose, galactose, fucose, or N-acetylglucosamine residues apparently are not involved in nematode attachment. Chitin or a related N-acetylglucosamine polymer was found in R. culicivorax preparasites. Treatment of preparasites with neuraminidase, which hydrolyzes sialic acids, increased nematode attachment to Anopheles freeborni larvae.

Reassessment of the role of phospholipids in sexual reproduction by sterol-auxotrophic fungi.

Several genera of oomycete fungi which are incapable of de novo sterol synthesis do not require these compounds for vegetative growth. The requirement for an exogenous source of sterols for sexual reproduction by several members of the Pythiaceae has been questioned by reports of apparent induction and maturation of oospores on defined media supplemented with phospholipids in the absence of sterols. A more detailed examination of this phenomenon suggested that trace levels of sterols in the inoculum of some pythiaceous fungi act synergistically with phospholipid medium supplements containing unsaturated fatty acid moieties to induce oosporogenesis. Phospholipid analysis of one species, Pythium ultimum, suggested that only the fatty acid portion of the exogenous phospholipid is taken up by the fungus. Enrichment of the phospholipid fraction of total cell lipid of P. ultimum with unsaturated fatty acids promoted oospore induction, and enhanced levels of unsaturated fatty acids in the neutral lipid fraction increased oospore viability. For some pythiaceous fungi, the levels of sterols required for the maturation of oospores with appropriate phospholipid medium supplementation suggest that these compounds are necessary only for the sparking and critical domain roles previously described in other fungi.

Ground and aerial application of the asexual stage of Lagenidium giganteum for control of mosquitoes associated with rice culture in the Central Valley of California.

A series of ground and aerial applications of Lagenidium giganteum, a facultative fungal parasite of mosquito larvae, was made in rice fields and associated habitats in the Sacramento Valley, CA. Initial trials using ground applications of the fungus in 400 m2 plots indicated that asexually competent mycelium from 30 liters of fermentation beer per hectare was sufficient to control Culex tarsalis in rice field habitats. Two multi-hectare applications using a Micronair Atomizer were made at rates of mycelium from either 20 or 30 liters of fermentation beer per hectare. The lower application rate resulted in 40% confirmed infection of Cx. tarsalis and Anopheles freeborni sentinel larvae, while the higher application rate resulted in greater than 90% initial mortality of sentinel Cx. tarsalis and An. freeborni and 65% Aedes melanimon sentinel mortality. This was accompanied by a 10-fold decrease in indigenous populations of the 2 former species.