Proteomics
| Researcher Contact: Brian Gregson |
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| Figure 1: Active sites on the Pro-teinChip Arrays selectively bind whole classes of proteins in crude samples for detection by the Pro-teinChip mass-spec reader. Arrays are available with chromatographic (e.g. ion-exchange, hydrophobic-interaction, etc) or biologically reac-tive surfaces (e.g. antibodies, nucleic acids, etc). Image courtesy of Ci-phergen Biosystems, Inc. |
Issues related to harmful algal blooms (HABs) are of concern to Florida marine scientists, as well as ecologists and public health officials worldwide. Since HABs often result in fish die-offs and human health problems, there is a strong demand for the development of improved technology to identify and characterize HAB organisms and their toxins. We’ve been using a novel proteomics technology known as surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-MS, Ciphergen Biosystems; and ref.) to (1) analyze the protein characteristics of HAB-forming cyanobacteria as well as develop new assays to directly detect cyanotoxins in water supplies. SELDI-MS is a powerful proteomics tool used in medical and ecology research to identify unique protein biomarkers of organisms in “diseased” states (2). Proteins in solution are separated on ProteinChip arrays coated with chromatographic or biologically reactive surfaces (Figure 1). Subsequent ionization and desorption by TOF-MS allows further differentiation of separated proteins according to differences in molecular size |
| In Florida, the red tide cyanobacterium Karenia brevis is of particular concern because its toxin, brevetoxin, is responsible for fish kills and respiratory ailments throughout the coastal regions. We have used SELDI-MS to analyze patterns of K. brevis protein expression, in order to better understand the molecular behavior of the organism. Samples of total protein from lysed cells cultured in either a normal light:dark cycle or 24 hours constant dark were captured on ionexchange ProteinChip surfaces and analyzed by the mass-spec reader. Of 42 proteins exhibiting significantly different levels of expression in normal compared to dark conditions, 31 demonstrated a rhythmic “circadian” pattern (Figure 2). While the proteins have not yet been identified, many are suspected to be involved in pathways regulating the organism’s metabolism as well as toxin production. Similar “profiling” experiments are underway with other samples, including seagrasses from polluted versus clean locations and freely dissolved organic matter. |
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| Figure 2: Circadian expression of a representative pro-tein peak identified by SELDI-MS. From a K. brevis culture maintained in conditions of 12 hours light, 12 hours dark for approximately 3 weeks. As light intensity changes throughout the day (time 0-24 hours), the level of protein expression exhibits a “biorhythmic” pattern. |
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Since many cyanobacterial toxins consist of protein-like peptide structures, direct detection in contaminated waters is possible by SELDI-MS. We are currently in the process of developing a promising newmethod for detecting and identifying microcystins, acutely toxic freshwater cyanotoxins known to target mammalian and fish livers. A highly specific antibody that captures all the known microcystins is first bound to special reactive surfaces on ProteinChip arrays. Samples containing microcystin mixes are then enriched and purified on the immobilized antibody surfaces through a series of washes. SubsequentTOF-MS analysis allows us to differentiate between related toxins with small size differences (Figure 3). Since our method enables identification and quantification of individual microcystins from mixed samples, it is a substantial improvement over current methods that are unable to discriminate between different toxin peptides. We hope to apply this method to the development of assays for other cyanotoxins and toxic peptide-like compounds.
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| Figure 3: SELDI-MS spectrum of three different microcystins captured by a microcystin antibody bound to ProteinChip array reactive surface. The SELDI-MS immunoassay allows for the enrichment and differentiation of similar peptide toxins with small size differences (here, ca. 7 Da between microcystin-RR and –YR). |
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