MALDI-TOF mass spectrometry of Cyanobacteria: a global approach to the discovery of novel secondary metabolites

Cyanobacteria (blue-green algae) are a group of ancient prokaryotic organisms dating back between three and four billion years.¹ They have been attributed with oxygenating the earth’s atmosphere² but, since the anthropogenic euthrophication of lakes, ponds and oceans, they have become synonymous with water hygiene issues.³ This is due to the alteration of the nutrient composition of their habitat to one which is optimal for growth (or blooms). Cyanobacterial blooms may simply cause foul tastes and odours,⁴ but they can also lead to the production of toxic secondary metabolites poisonous to humans and animals upon ingestion.⁵ NZ has yet to suffer a human fatality, but the deaths of several dogs in Wellington was attributed to homoanatoxin-a 1 (Chart 1) from a Phormidium species.

Spectroscopic Investigations of Oligopeptides from Aquatic Cyanobacteria: Characterisation of New Oligopeptides, Development of Microcystin Quantification Tools and Investigations into Microcystin Production

Cyanobacteria (blue-green algae) are a group of ancient prokaryotic organisms which have become synonymous with water quality deterioration. An array of compounds is produced by aquatic cyanobacteria, the largest group being the oligopeptides. A major class of cyanobacterial oligopeptides are the microcystins; cyclic heptapeptides which contain the unique amino acid Adda (3 amino-9-methoxy-2,6,8-trimethyl-10-phenyldeca-4,6-dienoic acid). Due to their ability to inhibit protein phosphatases 1 and 2A and as they are concentrated in the liver, microcystins can be highly toxic to animals. Anabaenopeptins are cyclic hexapeptides which contain a carbonyl-linked sidechain amino acid and a ring which is cyclised through the sidechain amine of the position two D-lysine. Matrix-assisted laser desorption/ionisation-time of flight mass spectrometry (MALDI TOF MS) analysis of a cyanobacterial bloom sample from Lake Wiritoa (Manawatu, New Zealand) led to the identification of a new anabaenopeptin. The putative structure for anabaenopeptin 906 was constructed using tandem MS (MS/MS) data, in conjunction with the sequences of the known anabaenopeptins. A Microcystis species (CYN06) isolated from Lake Hakanoa (Huntly, New Zealand) was investigated as it produced a large number of microcystin congeners. Liquid chromatography-MS/MS analysis was used to identify seventeen known microcystin congeners and to characterise ten new variants. Two of the new microcystins, MC-FA and MC-WA, were structurally characterised using amino acid analysis and nuclear magnetic resonance (NMR) spectroscopy. The other new congeners included MC FAba, MC WAba, MC FL, MC-WL as well as [Asp³] analogues of MC FA, MC WA, and [Asp³] analogues of two known congeners; MC-RA and MC-RAba. A review of the number of microcystin congeners produced by reported cyanobacterial strains placed CYN06 in the upper ten percentile. However, CYN06 differed from the other strains as it showed relaxed substrate specificity at position two and four and simultaneously produced microcystin congeners which contained no arginine residues, one arginine residue and two arginine residues. Microcystis CYN06 also contained an unidentified microcystin with a mass of 1014.5 Da. Structural characterisation by NMR spectroscopy indicated that it was an analogue of MC-WA which contained N-formylkynurenine at position two. As N-formylkynurenine is a known oxidation product of tryptophan it was proposed that further unidentified microcystins from CYN06 contained two other oxidation products of tryptophan; kynurenine and oxindolyalanine. This is the first report of the presence of oxidised tryptophan residues in microcystins. Analysis of a cyanobacterial hydroterrestrial mat sample collected from Miers Valley in Eastern Antarctica indicated the presence of fourteen new oligopeptides. A combination of MS/MS and amino acid analysis was used to characterise eight new microcystins which each contained a position one glycine. A recently-developed thiol derivatisation technique indicated that the position seven amino acid in each of the microcystins was very likely dehydrobutyrine. Different combinations of variable modifications at positions two, four and five resulted in eight unique structures. These included four microcystins with Adda moieties which possessed an O-acetyl group (ADMAdda) instead of the conventional C9 methoxy. As well as being the first report of microcystins containing a position one glycine, this is the first report of ADMAdda-containing microcystins in the southern hemisphere. The putative structures of six new Antarctic linear peptides were determined through a combination of mass spectrometric techniques. The compounds contained an N terminus with the molecular formula C₉H₁₄NO₂ linked to isoleucine, two aromatic amino acids and an ester linked hydroxyphenyllactic acid. The hydroxyphenyllactic acid C terminus and the unidentified N terminus suggest that these new compounds are a novel class of cyanobacterial oligopeptide. Seven different sample preparation techniques for the quantitative analysis of microcystins by MALDI-TOF MS were assessed for signal reproducibility and sensitivity using a cost-effective internal standard (angiotensin I). The sensitivity of six of the preparations was acceptable, as was the reproducibility for two thin layer preparations performed on a polished steel target. Both thin layer preparations could be used with a MALDI-TOF mass spectrometer which automatically acquires data. The thin-layer-spot preparation could also be used in an automated sample preparation work-flow. Further investigation using this preparation demonstrated that linear quantification of three different microcystin congeners ([Dha⁷] MC-LR, MC-RR and MC-YR) was possible. Use of this MALDI sample preparation will allow large numbers of microcystin-containing samples to be analysed rapidly and at low cost. A batch culture experiment using Microcystis CYN06 exhibited a decreased abundance of arginine-containing microcystins as nitrate concentrations decreased. Linear regression of the relationship between log10 microcystin content and nitrate concentration revealed slopes which were dependent upon the number of arginine residues present in the compound. During this experiment the abundance of congeners with a single arginine residue at position two did not change (p > 0.05), whilst the abundance of the congeners with a position four arginine decreased significantly (p ≤ 0.001). This suggests that there could be an element of selectivity in regards to which arginine in the microcystin structure is modulated and could explain why congener modulation in response to nitrogen concentration has not been observed previously. Whilst it was not proven that nitrogen supply was the causative factor for the congener modulation, the results from this experiment warrant further study in this area. This research has significantly expanded our knowledge of oligopeptide diversity, improved an existing method of quantifying microcystins and shed new light on the regulation of the abundance of microcystin congeners. The identification of eighteen new microcystins is a 16% increase upon the 111 presently characterised. Also reported was the identification of nine oxidised analogues of tryptophan-containing microcystins from Microcystis CYN06. The presence of oxidised Trp residues in microcystins has not been reported previously and will allow researchers working with samples of Trp-containing microcystins to now assign the oxidised analogues. Seven new cyanobacterial oligopeptides were characterised during this study, six of which may belong to a novel class of linear peptides. A sample preparation designed for the quantification of microcystins by MALDI TOF MS gave comparative performance to the previously reported method but was compatible with automated high-throughput sample preparation and data acquisition. For the first time, a culturing experiment showed a relationship between the abundance of arginine-containing microcystins and nitrogen supply

A comparative proteomics approach to studying skeletal muscle mitochondria from myostatin knockout mice

Myostatin is a negative regulator of muscle growth. When it is not present or non-functional double-muscling occurs, the primary characteristic of this phenotype being an increase in muscle mass. Another characteristic of double-muscling is an increased proportion of type IIB muscle fibres, which rely on glycolysis as their primary energy source, as opposed to type IIA and type I fibres which rely on oxidative phosphorylation. This switch in muscle metabolism directly impacts on the mitochondria, as mitochondria from glycolytic muscle fibres have been shown to have differences in metabolic activity. The increased proportion of glycolytic muscle fibres present in myostatin knockout animals provides a unique model to investigate alterations in muscle fibre type metabolism. The mouse model of myostatin knockout utilised during this study was generated by genetic deletion of exon three of the myostatin gene. Verification of this knockout was attempted by western blot analysis, but only the latency associated protein (LAP) was detected. Interestingly, the LAP was barely detectable in the knockout muscle suggesting deletion of exon three affects binding of anti-myostatin antibodies to the LAP, as that part of the gene is not deleted. A comparison of the basal mitochondrial stress levels was made, also by western blot analysis. The knockout mitochondria showed no change in levels of heat shock protein 60 or superoxide dismutase 2, indicating that they are not being subjected to any increased stress due to the myostatin knockout phenotype. A comparative proteomics approach was used to detect changes in the mitochondrial proteome of myostatin knockout gastrocnemius muscle to gain clues to how mitochondria from glycolytic muscle fibres differ from those present in oxidative fibres. This was undertaken using two-dimensional electrophoresis (2-DE), in-gel tryptic digests and peptide mass fingerprinting by mass spectrometry. A 2-DE gel protein loading of 220 g was shown to give the best protein spot resolution and the most crucial step in the loading process was found to be the laying of the immobilized pH gradient, which had to be performed very carefully to obtain a consistent loading pattern. This study resolved only around 160 protein spots out of the estimated 1,000 to 2,000 proteins present in the mitochondria. Modulation of six proteins was seen at a plt0.1 level, but were unable to be identified using the current methodology. More abundant mitochondrial proteins were able to be identified, but showed no significant modulation. Malate dehydrogenase and 3-hydroxyacyl-CoA dehydrogenase, which were identified during this study, have been reported to have decreased activity in mitochondria from glycolytic muscle fibres. This study suggests that the change in activity observed by other researchers is due to inhibition of these enzymes in the glycolytic fibres or activation in the oxidative fibres

Further characterization of glycine-containing microcystins from the McMurdo Dry Valleys of Antarctica

Microcystins are hepatotoxic cyclic peptides produced by several cyanobacterial genera worldwide. In 2008, our research group identified eight new glycine-containing microcystin congeners in two hydro-terrestrial mat samples from the McMurdo Dry Valleys of Eastern Antarctica. During the present study, high-resolution mass spectrometry, amino acid analysis and micro-scale thiol derivatization were used to further elucidate their structures. The Antarctic microcystin congeners contained the rare substitution of the position-1 D-alanine for glycine, as well as the acetyl desmethyl modification of the position-5 Adda moiety (3S-amino-9S-methoxy-2S,6,8S-trimethyl-10-phenyldeca-4E,6E-dienoic acid). Amino acid analysis was used to determine the stereochemistry of several of the amino acids and conclusively demonstrated the presence of glycine in the microcystins. A recently developed thiol derivatization technique showed that each microcystin contained dehydrobutyrine in position-7 instead of the commonly observed N-methyl dehydroalanine

Simultaneous Detection of 14 Microcystin Congeners from Tissue Samples Using UPLC- ESI-MS/MS and Two Different Deuterated Synthetic Microcystins as Internal Standards

International audienceCyanobacterial microcystins (MCs), potent serine/threonine-phosphatase inhibitors, pose an increasing threat to humans. Current detection methods are optimised for water matrices with only a few MC congeners simultaneously detected. However, as MC congeners are known to differ in their toxicity, methods are needed that simultaneously quantify the congeners present, thus allowing for summary hazard and risk assessment. Moreover, detection of MCs should be expanded to complex matrices, e.g., blood and tissue samples, to verify in situ MC concentrations, thus providing for improved exposure assessment and hazard interpretation. To achieve this, we applied two synthetic deuterated MC standards and optimised the tissue extraction protocol for the simultaneous detection of 14 MC congeners in a single ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) run. This procedure was validated using plasma and liver homogenates of mice (male and female) spiked with deuterated MC standards. For proof of concept, tissue and plasma samples from mice i.p. injected with MC-LR and MC-LF were analysed. While MC-LF was detected in all tissue samples of both sexes, detection of MC-LR was restricted to liver samples of male mice, suggesting different toxicokinetics in males, e.g., transport, conjugation or protein binding. Thus, deconjugation/-proteinisation steps should be employed to improve detection of bound MC. Key Contribution: The use of deuterated microcystin standards and an improved extraction procedure using UPLC-MS/MS analytics, provides for the simultaneous detection of fourteen microcystin congeners. Thus, it allows more accurate quantitation of total microcystin load of a given sample in complex matrices like blood or tissue, and therefore better hazard interpretation

Toxic cyanobacteria in Svalbard: chemical diversity of microcystins detected using a liquid chromatography mass spectrometry precursor ion screening method

Cyanobacteria synthesize a large variety of secondary metabolites including toxins. Microcystins (MCs) with hepato- and neurotoxic potential are well studied in bloom-forming planktonic species of temperate and tropical regions. Cyanobacterial biofilms thriving in the polar regions have recently emerged as a rich source for cyanobacterial secondary metabolites including previously undescribed congeners of microcystin. However, detection and detailed identification of these compounds is difficult due to unusual sample matrices and structural congeners produced. We here report a time-efficient liquid chromatography-mass spectrometry (LC-MS) precursor ion screening method that facilitates microcystin detection and identification. We applied this method to detect six different MC congeners in 8 out of 26 microbial mat samples of the Svalbard Archipelago in the Arctic. The congeners, of which [Asp3, ADMAdda5, Dhb7] MC-LR was most abundant, were similar to those reported in other polar habitats. Microcystins were also determined using an Adda-specific enzyme-linked immunosorbent assay (Adda-ELISA). Nostoc sp. was identified as a putative toxin producer using molecular methods that targeted 16S rRNA genes and genes involved in microcystin production. The mcy genes detected showed highest similarities to other Arctic or Antarctic sequences. The LC-MS precursor ion screening method could be useful for microcystin detection in unusual matrices such as benthic biofilms or liche

Fine-scale cryogenic sampling of planktonic microbial communities: Application to toxic cyanobacterial blooms

A lack of fine-scale methods for sampling planktonic microbial populations hinders advancement in understanding the responses of these communities to environmental conditions. Current methods provide resolution at scales of centimeters to meters, but not at the millimeter-scale required to understand highly stratified communities. To address this we developed two cryogenic sampling tools to collect spatially-precise samples from aquatic environments while simultaneously preserving the microbial communities. The application of these samplers was examined over a 5.5 h period using a cyanobacterial scum (Microcystis) formed in experimental mesocosms. A cryogenic “surface snatcher” collected a discrete layer (ca. 1 mm) of surface water. Compared to conventional surface sampling methods, the surface snatcher samples contained up to 22-times more microcystin, indicating that less underlying water was incorporated into the sample. A cryogenic “cold finger” sampler was used to collect vertical profiles of the upper 40 mm of the water column. This profiler provided new insights into the fine-scale structure of Microcystis scums, demonstrating that more microcystin-producing Microcystis was contained in the surface 5 mm than the 35 mm below. The results also showed that upregulation of microcystin production was highly localized in the top 2.5 mm of the Microcystis scum. Our results demonstrate that extreme changes in cyanobacterial communities can occur over small distances, and indicate that sampling resolution is of great importance for improving knowledge on cyanobacterial blooms and toxin production. While this study focused on microcystin-producing Microcystis, the cryogenic sampling tools described here could be applied to any planktonic microbial community

The role of 44-methylgambierone in ciguatera fish poisoning: Acute toxicity, production by marine microalgae and its potential as a biomarker for Gambierdiscus spp.

Ciguatera fish poisoning (CFP) is prevalent around the tropical and sub-tropical latitudes of the world and impacts many Pacific island communities intrinsically linked to the reef system for sustenance and trade. While the genus Gambierdiscus has been linked with CFP, it is commonly found on tropical reef systems in microalgal assemblages with other genera of toxin-producing, epiphytic and/or benthic dinoflagellates - Amphidinium, Coolia, Fukuyoa, Ostreopsis and Prorocentrum. Identifying a biomarker compound that can be used for the early detection of Gambierdiscus blooms, specifically in a mixed microalgal community, is paramount in enabling the development of management and mitigation strategies. Following on from the recent structural elucidation of 44-methylgambierone, its potential to contribute to CFP intoxication events and applicability as a biomarker compound for Gambierdiscus spp. was investigated. The acute toxicity of this secondary metabolite was determined by intraperitoneal injection using mice, which showed it to be of low toxicity, with an LD50 between 20 and 38 mg kg-1. The production of 44-methylgambierone by 252 marine microalgal isolates consisting of 90 species from 32 genera across seven classes, was assessed by liquid chromatography-tandem mass spectrometry. It was discovered that the production of this secondary metabolite was ubiquitous to the eight Gambierdiscus species tested, however not all isolates of G. carpenteri, and some species/isolates of Coolia and Fukuyoa

In situ collection and preservation of intact Microcystis colonies to assess population diversity and microcystin quotas

Understanding of colony specific properties of cyanobacteria in the natural environment has been challenging because sampling methods disaggregate colonies and there are often delays before they can be isolated and preserved. Microcystis is a ubiquitous cyanobacteria that forms large colonies in situ and often produces microcystins, a potent hepatotoxin. In the present study a new cryo-sampling technique was used to collect intact Microcystis colonies in situ by embedding them in a sheet of ice. Thirty-two of these Microcystis colonies were investigated with image analysis, liquid chromatography-mass spectrometry, quantitative polymerase chain reaction and high-throughput sequencing to assess their volume, microcystin quota and internal transcribed spacer (ITS) genotype diversity. Microcystin quotas were positively correlated to colony volume (R² = 0.32; p = 0.004). Individual colonies had low Microcystis ITS genotype diversity and one ITS operational taxonomic unit predominated in all samples. This study demonstrates the utility of the cryo-sampling method to enhance the understanding of colony-specific properties of cyanobacteria with higher precision than previously possible

The effect of cyanobacterial biomass enrichment by centrifugation and GF/C filtration on subsequent microcystin measurement

Microcystins are cyclic peptides produced by multiple cyanobacterial genera. After accumulation in the liver of animals they inhibit eukaryotic serine/threonine protein phosphatases, causing liver disease or death. Accurate detection/quantification of microcystins is essential to ensure safe water resources and to enable research on this toxin. Previous methodological comparisons have focused on detection and extraction techniques, but have not investigated the commonly used biomass enrichment steps. These enrichment steps could modulate toxin production as recent studies have demonstrated that high cyanobacterial cell densities cause increased microcystin levels. In this study, three microcystin-producing strains were processed using no cell enrichment steps (by direct freezing at three temperatures) and with biomass enrichment (by centrifugation or GF/C filtration). After extraction, microcystins were analyzed using liquid chromatography-tandem mass spectrometry. All processing methods tested, except GF/C filtration, resulted in comparable microcystin quotas for all strains. The low yields observed for the filtration samples were caused by adsorption of arginine-containing microcystins to the GF/C filters. Whilst biomass enrichment did not affect microcystin metabolism over the time-frame of normal sample processing, problems associated with GF/C filtration were identified. The most widely applicable processing method was direct freezing of samples as it could be utilized in both field and laboratory environments