Elisabete da Costa

Differentiation of isomeric pentose disaccharides by electrospray ionization tandem mass spectrometry and discriminant analysis

RATIONALE The structural characterization of unknown oligosaccharides remains a big challenge since a large number of isomeric structures are possible even for disaccharides. In this work, electrospray ionization collision-induced dissociation tandem mass spectrometry (ESI-CID-MS/MS) was used for the differentiation of isomeric pentose disaccharides, α-(1 → 5)-L-arabinobiose (Ara(2)) and β-(1 → 4)-D-xylobiose (Xyl(2)). METHODS ESI-MS/MS spectra of [M + Li](+) and [M + Na](+) ions of Ara(2) and Xyl(2), as well as these precursor ions of (18)O-labelled disaccharides, were acquired using two mass spectrometers equipped with different analyzers: LIT (linear ion trap) and Q-TOF (quadrupole time-of-flight). RESULTS Product ions observed in MS/MS spectra arise from the cleavage at the nonreducing side of the glycosidic bond (Y(1)(+)) and from cross-ring cleavages (0,1)A(2)(+), (0,2)A(2)(+), and (0,3)A(2)(+) at the reducing residue. Statistically significant differences were observed between the relative abundance of specific product ions, when comparing both disaccharides. These differences allowed discriminant models to be built and to propose a criterion using the relative abundances of selected ions capable of discriminating between the isomers for both adduct ions and spectrometers. CONCLUSIONS Isomeric pentose disaccharides can be distinguished based on the fragmentation of both [M + Li](+) and [M + Na](+) ions and using different mass spectrometers. However, LIT instrument has a better discriminant power.

Lipidomic Approaches towards Deciphering Glycolipids from Microalgae as a Reservoir of Bioactive Lipids.

In recent years, noteworthy research has been performed around lipids from microalgae. Among lipids, glycolipids (GLs) are quite abundant in microalgae and are considered an important source of fatty acids (FAs). GLs are rich in 16- and 18-carbon saturated and unsaturated fatty acids and often contain polyunsaturated fatty acids (PUFAs) like n-3 α-linolenic (ALA 18:3), eicosapentaenoic (EPA, 20:5) and docosahexaenoic (DHA, 22:6). GLs comprise three major classes: monogalactosyldiacyl glycerolipids (MGDGs), digalactosyl diacylglycerolipids (DGDGs) and sulfoquinovosyl diacylglycerolipids (SQDGs), whose composition in FA directly depends on the growth conditions. Some of these lipids are high value-added compounds with antitumoral, antimicrobial and anti-inflammatory activities and also with important nutritional significance. To fully explore GLs’ bioactive properties it is necessary to fully characterize their structure and to understand the relation between the structure and their biological properties, which can be addressed using modern mass spectrometry (MS)-based lipidomic approaches. This review will focus on the up-to-date FA composition of GLs identified by MS-based lipidomics and their potential as phytochemicals.

Oxidation of amylose and amylopectin by hydroxyl radicals assessed by electrospray ionisation mass spectrometry.

The hydroxyl radicals (HO) are one of the most reactive oxygen species (ROS) involved in the oxidative damage of biological molecules, including carbohydrates. During the industrial processing of food, ROS can be formed. In order to identify the structural changes induced in starch by oxidation, amylose, amylopectin, and maltotriose, an oligosaccharide structurally related to these polysaccharides, were subjected to oxidation with HO generated under Fenton reaction conditions (Fe(2+)/H2O2). The oxidised polysaccharides were hydrolysed by α-amylase and the obtained oligosaccharides were fractionated by ligand-exchange/size-exclusion chromatography. Both acidic and neutral α-amylase resistant oligosaccharides were characterized by mass spectrometry. In oxidised neutral products, new keto, hydroxyl, and hydroperoxy moieties, and oxidative ring scission were observed at the reducing end of the oligosaccharides. The acid sugar residues occurred at the reducing end and included gluconic and glucuronic acid derivatives, and acids formed by oxidative ring scission, namely, arabinonic, erythronic, glyceric and glycolic acids.

Neutral and acidic products derived from hydroxyl radical-induced oxidation of arabinotriose assessed by electrospray ionisation mass spectrometry.

The oxidation of α-(1 → 5)-L-arabinotriose (Ara3), an oligosaccharide structurally related to side chains of coffee arabinogalactans, was studied in reaction with hydroxyl radicals generated under conditions of Fenton reaction (Fe(2+)/H2O2). The acidic and neutral oxidation products were separated by ligand exchange/size-exclusion chromatography, subsequently identified by electrospray ionisation mass spectrometry (ESI-MS) and structurally characterised by tandem MS (ESI-MS/MS). In acidic fraction were identified several oxidation products containing an acidic residue at the corresponding reducing end of Ara3, namely arabinonic acid, and erythronic, glyceric and glycolic acids formed by oxidative scission of the furanose ring. In neutral fractions were identified derivatives containing keto, hydroxy and hydroperoxy moieties, as well as derivatives resulting from the ring scission at the reducing end of Ara3. In both acidic and neutral fractions, beyond the trisaccharide derivatives, the corresponding di- and monosaccharide derivatives were identified indicating the occurrence of oxidative depolymerisation. The structural characterisation of these oxidation products by ESI-MS/MS allowed the differentiation of isobaric and isomeric species of acidic and neutral character. The species identified in this study may help in detection of roasting products associated with the free radical-mediated oxidation of coffee arabinogalactans.

Valorization of Lipids from Gracilaria sp. through Lipidomics and Decoding of Antiproliferative and Anti-Inflammatory Activity

The lipidome of the red seaweed Gracilaria sp., cultivated on land-based integrated multitrophic aquaculture (IMTA) system, was assessed for the first time using hydrophilic interaction liquid chromatography-mass spectrometry and tandem mass spectrometry (HILIC–MS and MS/MS). One hundred and forty-seven molecular species were identified in the lipidome of the Gracilaria genus and distributed between the glycolipids classes monogalactosyl diacylglyceride (MGDG), digalactosyl diacylglyceride (DGDG), sulfoquinovosyl monoacylglyceride (SQMG), sulfoquinovosyl diacylglyceride (SQDG), the phospholipids phosphatidylcholine (PC), lyso-PC, phosphatidylglycerol (PG), lyso-PG, phosphatidylinositol (PI), phosphatidylethanolamine (PE), phosphatic acid (PA), inositolphosphoceramide (IPC), and betaine lipids monoacylglyceryl- and diacylglyceryl-N,N,N-trimethyl homoserine (MGTS and DGTS). Antiproliferative and anti-inflammatory effects promoted by lipid extract of Gracilaria sp. were evaluated by monitoring cell viability in human cancer lines and by using murine macrophages, respectively. The lipid extract decreased cell viability of human T-47D breast cancer cells and of 5637 human bladder cancer cells (estimated half-maximal inhibitory concentration (IC50) of 12.2 μg/mL and 12.9 μg/mL, respectively) and inhibited the production of nitric oxide (NO) evoked by the Toll-like receptor 4 agonist lipopolysaccharide (LPS) on the macrophage cell line RAW 264.7 (35% inhibition at a concentration of 100 μg/mL). These findings contribute to increase the ranking in the value-chain of Gracilaria sp. biomass cultivated under controlled conditions on IMTA systems.

Effect of urea on cellulose degradation under conditions of alkaline pulping

The effect of urea on the cellulose degradation under conditions of alkaline pulping has been studied using purified cellulose powder. The increased cellulose yield in the presence of urea was assigned essentially to the carbamation reactions which were confirmed by elemental analysis, UV-resonance Raman and solid-state 13C nuclear magnetic resonance spectroscopy. The stabilizing effect of urea on the cellulose peeling reactions during heating up period of pulping process was suggested based on kinetic studies and additionally confirmed in model reactions using cellobiose. The reaction products formed in alkaline urea solutions were analysed by tandem electrospray ionization mass spectrometry and the occurrence of Maillard type reactions between reducing end groups of cellobiose and urea were evidenced. Both Maillard type reactions and carbamation of reducing end groups were proposed to be a part of cellulose protection mechanism against peeling under the conditions of alkaline pulping.

High-Resolution Lipidomics of the Early Life Stages of the Red Seaweed Porphyra dioica

Porphyra dioica is a commercial seaweed consumed all over the world, mostly in the shape of nori sheets used for “sushi” preparation. It is a well-known part of the Asian diet with health benefits, which have been associated, among others, to the high levels of n-3 and n-6 fatty acids in this red alga. However, other highly valued lipids of Porphyra are polar lipids that remain largely undescribed and can have both nutritional value and bioactivity, thus could contribute to the valorization of this seaweed. In this context, the present work aims to identify the lipidome of two life cycle stages of the Atlantic species Porphyra dioica: the early life stage conchocelis produced in an indoor-nursery, and young blades produced outdoors using an integrated multitrophic aquaculture (IMTA) framework. Both the blades (gametophyte) and conchocelis (sporophyte) are commercialized in the food and cosmetics sectors. Liquid chromatography coupled to Q–Exactive high resolution-mass spectrometry (MS) platform was used to gain insight into the lipidome of these species. Our results allowed the identification of 110 and 100 lipid molecular species in the lipidome of the blade and conchocelis, respectively. These lipid molecular species were distributed as follows (blade/conchocelis): 14/15 glycolipids (GLs), 93/79 phospholipids (PLs), and 3/6 betaine lipids. Both life stages displayed a similar profile of GLs and comprised 20:4(n-6) and 20:5(n-3) fatty acids that contribute to n-3 and n-6 fatty acid pool recorded and rank among the molecular species with higher potential bioactivity. PLs’ profile was different between the two life stages surveyed, mainly due to the number and relative abundance of molecular species. This finding suggests that differences between both life stages were more likely related with shifts in the lipids of extraplastidial membranes rather than in plastidial membranes. PLs contained n-6 and n-3 precursors and in both life stages of Porphyra dioica the n-6/n-3 ratio recorded was less than 2, highlighting the potential benefits of using these life stages in human diet to prevent chronic diseases. Atherogenic and thrombogenic indexes of blades (0.85 and 0.49, respectively) and conchocelis (0.34 and 0.30, respectively) are much lower than those reported for other Rhodophyta, which highlights their potential application as food or as functional ingredients. Overall, MS-based platforms represent a powerful tool to characterize lipid metabolism and target lipids along different life stages of algal species displaying complex life cycles (such as Porphyra dioica), contributing to their biotechnological application.

Kleptoplasty does not promote major shifts in the lipidome of macroalgal chloroplasts sequestered by the sacoglossan sea slug Elysia viridis

Sacoglossan sea slugs, also known as crawling leaves due to their photosynthetic activity, are highly selective feeders that incorporate chloroplasts from specific macroalgae. These “stolen” plastids - kleptoplasts - are kept functional inside animal cells and likely provide an alternative source of energy to their host. The mechanisms supporting the retention and functionality of kleptoplasts remain unknown. A lipidomic mass spectrometry-based analysis was performed to study kleptoplasty of the sacoglossan sea slug Elysia viridis fed with Codium tomentosum. Total lipid extract of both organisms was fractionated. The fraction rich in glycolipids, exclusive lipids from chloroplasts, and the fraction rich in betaine lipids, characteristic of algae, were analysed using hydrophilic interaction liquid chromatography-mass spectrometry (HILIC-LC-MS). This approach allowed the identification of 81 molecular species, namely galactolipids (8 in both organisms), sulfolipids (17 in C. tomentosum and 13 in E. viridis) and betaine lipids (51 in C. tomentosum and 41 in E. viridis). These lipid classes presented similar lipidomic profiles in C. tomentosum and E. viridis, indicating that the necessary mechanisms to perform photosynthesis are preserved during the process of endosymbiosis. The present study shows that there are no major shifts in the lipidome of C. tomentosum chloroplasts sequestered by E. viridis.

Polar lipidome profiling of Salicornia ramosissima and Halimione portulacoides and the relevance of lipidomics for the valorization of halophytes

Some halophytes are currently used as gourmet plant ingredients for human consumption. The polar lipidome of the succulent organs of Salicornia ramosissima (fresh branch tips) and Halimione portulacoides (leaves) were characterized in-depth, with more than two hundred lipid species being identified in both halophytes. The lipid species identified were distributed over five classes of phospholipids, three classes of glycolipids and one class of glycosphingolipids. Despite the existence of some species-specific differences between the polar lipidome, phospholipids and glycolipids show a high content of n-3 fatty acids in both S. ramosissima and H. portulacoides. These results highlights the advantage of employing mass spectrometry based lipidomic platform towards the valorization of halophytes as a source of valuable nutrients and bioactives, fostering potential applications in the fields of healthy and functional food products, and for nutraceutical and pharmaceutical uses.

Lipidomic signature of the green macroalgae Ulva rigida farmed in a sustainable integrated multi-trophic aquaculture

Ulva species, green macroalgae, are widely distributed across the globe, being one of the most heavily traded edible seaweeds. Nonetheless, although this genus has been largely used in scientific studies, its lipidome remains rather unexplored. The present study sheds light over the lipid profile of Ulva rigida produced in a land-based integrated multi-trophic aquaculture (IMTA) system using liquid chromatography coupled to high-resolution mass spectrometry for molecular lipid species identification. The lipidome of U. rigida revealed the presence of distinct beneficial n-3 fatty acids for human health, namely alpha-linoleic acid (ALA) and docosapentaenoic acid (DPA). A total of 87 molecular species of glycolipids, 58 molecular species of betaine lipids, and 57 molecular species of phospholipids were identified in the lipidome of U. rigida including some species bearing PUFA and with described bioactive properties. Overall, the present study contributes to the valorization and quality validation of sustainably farmed U. rigida.