Size-exclusion chromatography as a stand-alone methodology identifies novel markers in mass spectrometry analyses of plasma-derived vesicles from healthy individuals.

Plasma-derived vesicles hold a promising potential for use in biomedical applications. Two major challenges, however, hinder their implementation into translational tools: (a) the incomplete characterization of the protein composition of plasma-derived vesicles, in the size range of exosomes, as mass spectrometric analysis of plasma sub-components is recognizably troublesome and (b) the limited reach of vesicle-based studies in settings where the infrastructural demand of ultracentrifugation, the most widely used isolation/purification methodology, is not available. In this study, we have addressed both challenges by carrying-out mass spectrometry (MS) analyses of plasma-derived vesicles, in the size range of exosomes, from healthy donors obtained by 2 alternative methodologies: size-exclusion chromatography (SEC) on sepharose columns and Exo-Spin™. No exosome markers, as opposed to the most abundant plasma proteins, were detected by Exo-Spin™. In contrast, exosomal markers were present in the early fractions of SEC where the most abundant plasma proteins have been largely excluded. Noticeably, after a cross-comparative analysis of all published studies using MS to characterize plasma-derived exosomes from healthy individuals, we also observed a paucity of "classical exosome markers." Independent of the isolation method, however, we consistently identified 2 proteins, CD5 antigen-like (CD5L) and galectin-3-binding protein (LGALS3BP), whose presence was validated by a bead-exosome FACS assay. Altogether, our results support the use of SEC as a stand-alone methodology to obtain preparations of extracellular vesicles, in the size range of exosomes, from plasma and suggest the use of CD5L and LGALS3BP as more suitable markers of plasma-derived vesicles in MS.

Studies of polyether toxins in the marine phytoplankton, Dinophysis acuta, in Ireland using multiple tandem mass spectrometry.

Diarretic shellfish poisoning (DSP) is a toxic syndrome associated with the consumption of bivalve molluscs. The DSP toxins are polyether compounds, which include okadaic acid (OA), dinophysistoxins (DTXs), pectenotoxins (PTXs) and pectenotoxin seco acids (PTX2SAs). These toxins originate in marine dinoflagellates, including Dinophysis spp. Phytoplankton samples were collected from the southwest coast of Ireland and D. acuta was the predominant species. Monocultures of D. acuta cells were prepared by hand picking from microscope slides in order to confirm their toxin profiles. There was a remarkable consistency in the toxin profiles in all of the phytoplankton samples collected during the summer months, irrespective of location, depth or mesh size. Analysis using liquid chromatography-multiple tandem mass spectrometry (LC-MS/MS) revealed that DTX2 and OA were the predominant toxins at a consistent ratio. The average toxin composition was: DTX2 (53+/-5%), OA (26.5+/-2.3%) and total pectenotoxins (20.8+/-4.7%). Toxin profiles in D. acuta from Europe were distinctly different from those found in New Zealand, where PTX2 was the predominant toxin and DTX2 was absent.

Geographical, temporal, and species variation of the polyether toxins, azaspiracids, in shellfish.

Azaspiracid Poisoning (AZP) is a new toxic syndrome that has caused human intoxications throughout Europe following the consumption of mussels (Mytilus edulis), harvested in Ireland. Shellfish intoxication is a consequence of toxin-bearing microalgae in the shellfish food chain, and these studies demonstrated a wide geographic distribution of toxic mussels along the entire western coastal region of Ireland. The first identification of azaspiracids in other bivalve mollusks including oysters (Crassostrea gigas), scallops (Pecten maximus), clams (Tapes phillipinarium), and cockles (Cardium edule) is reported. Importantly, oysters were the only shellfish that accumulated azaspiracids at levels that were comparable with mussels. The highest levels of total azaspiracids (microg/g) recorded to-date were mussels (4.2), oysters (2.45), scallops (0.40), cockles (0.20), and clams (0.61). An examination of the temporal variation of azaspiracid contamination of mussels in a major shellfish production area revealed that, although maximum toxin levels were recorded during the late summer period, significant intoxications were observed at periods when marine dinoflagellate populations were low. Although human intoxications have so far only been associated with mussel consumption, the discovery of significant azaspiracid accumulation in other bivalve mollusks could pose a threat to human health.