M. Rosário M. Domingues

Mass spectrometry analysis of oxidized phospholipids

The evidence that oxidized phospholipids play a role in signaling, apoptotic events and in age-related diseases is responsible for the increasing interest for the study of this subject. Phospholipid changes induced by oxidative reactions yield a huge number of structurally different oxidation products which difficult their isolation and characterization. Mass spectrometry (MS), and tandem mass spectrometry (MS/MS) using the soft ionization methods (electrospray and matrix-assisted laser desorption ionization) is one of the finest approaches for the study of oxidized phospholipids. Product ions in tandem mass spectra of oxidized phospholipids, allow identifying changes in the fatty acyl chain and specific features such as presence of new functional groups in the molecule and their location along the fatty acyl chain. This review describes the work published on the use of mass spectrometry in identifying oxidized phospholipids from the different classes.

Characterization of Phenolic Components in Polar Extracts of Eucalyptus globulus Labill. Bark by High-Performance Liquid Chromatography–Mass Spectrometry

High-performance liquid chromatography-electrospray ionization mass spectrometry (HPLC-ESI-MS) and tandem mass spectrometry (MS(n)) were used to investigate the phenolic constituents in methanol, water, and methanol/water extracts of Eucalyptus globulus Labill. bark. Twenty-nine phenolic compounds were identified, 16 of them referenced for the first time as constituents of E. globulus bark, namely, quinic, dihydroxyphenylacetic, and caffeic acids, bis(hexahydroxydiphenoyl (HHDP))-glucose, galloyl-bis(HHDP)-glucose, galloyl-HHDP-glucose, isorhamentin-hexoside, quercetin-hexoside, methylellagic acid (EA)-pentose conjugate, myricetin-rhamnoside, isorhamnetin-rhamnoside, mearnsetin, phloridzin, mearnsetin-hexoside, luteolin, and a proanthocyanidin B-type dimer. Digalloylglucose was identified as the major compound in the methanol and methanol/water extracts, followed by isorhamnetin-rhamnoside in the methanol extract and by catechin in the methanol/water extract, whereas in the water extract catechin and galloyl- HHDP-glucose were identified as the predominant components. The methanol/water extract was shown be the most efficient to isolate phenolic compounds identified in E. globulus bark.

Direct photodegradation of carbamazepine followed by micellar electrokinetic chromatography and mass spectrometry

Carbamazepine, a widely consumed psychotropic pharmaceutical, is one of the most commonly detected drugs in the environment. To better assess the environmental persistence of carbamazepine in aqueous matrices, the effect of pH and dissolved oxygen on the direct photodegradation rate of this pharmaceutical was evaluated in this study, using simulated solar irradiation. In order to follow the degradation and the emergence of photoproducts, a micellar electrokinetic chromatography based method was developed, consisting on the use of a dynamically coated capillary column. The developed methodology showed good repeatability and efficiency in the separation of carbamazepine and photoirradiation products. Also, seven photodegradation products were identified by electrospray mass spectrometry (ESI-MS), including the known carcinogenic acridine that was produced under all the pH and oxygenation levels studied and one newly identified photoproduct. This paper gives new insights into the role of dissolved oxygen on the photodegradation rate of carbamazepine. The results indicate that acidic pH, combined with the absence of dissolved oxygen in the aqueous matrix, results in very high direct photodegradation rates. At basic pH, dissolved oxygen does not interfere with the process and very low rates were observed. At environmentally relevant conditions, carbamazepine was shown to persist in the environment from 4.5 to 25 days.

Photodegradation of psychiatric pharmaceuticals in aquatic environments--kinetics and photodegradation products.

Benzodiazepines are widely consumed psychiatric pharmaceuticals which are frequently detected in the environment. The environmental persistence and fate of these pharmaceuticals as well as their degradation products is of high relevance and it is, yet, scarcely elucidated. In this study, the relevance of photodegradation processes on the environmental persistence of four benzodiazepines (oxazepam, diazepam, lorazepam and alprazolam) was investigated. Benzodiazepines were irradiated under simulated solar irradiation and direct and indirect (together with three different fractions of humic substances) photodegradation kinetics were determined. Lorazepam was shown to be quickly photodegradated by direct solar radiation, with a half-life time lower than one summer sunny day. On the contrary, oxazepam, diazepam and alprazolam showed to be highly resistant to photodegradation with half-life times of 4, 7 and 228 summer sunny days, respectively. Apparent indirect and direct photodegradation rates are of the same order of magnitude. However, humic acids were consistently responsible for a decrease in the photodegradation rates while fulvic acids and XAD4 fraction caused an enhancement of the photodegradation. Overall, the results highlight that photodegradation might not be an efficient pathway to prevent the aquatic environmental accumulation of oxazepam, diazepam and alprazolam. Also, nineteen direct photodegradation products were identified by electrospray mass spectrometry, the majority of which are newly identified photoproducts. This identification is crucial to a more complete understanding of the environmental impact of benzodiazepines in aquatic systems.

Identification of oxidation products and free radicals of tryptophan by mass spectrometry

New oxidation products and free radicals derived from tryptophan (Trp) oxidation under Fenton reaction conditions were identified using mass spectrometry. After the oxidation of tryptophan using hydrogen peroxide and iron (II) system (Fenton reaction), mono- and dihydoxy tryptophans and N-formylkynurenine were identified using electrospray mass spectrometry (ES-MS) and ES-MS/MS. Besides these products, new products resulting from the reaction of tryptophan and oxidized tryptophan and 3-methyl indole derivatives were also identified. The 3-methyl indole derivatives resulted, most probably, from the oxidation process and not from in-source processes. A dimer formed by cross-linking between two Trp radicals (Trp-Trp), similar to the previously described tyrosine dimer was observed, as well as the corresponding monohydroxy-dimer (Trp-Trp-OH). Tandem mass spectrometry was used to identify the structures of these new oxidation products. Free radicals derived from tryptophan oxidation under Fenton reaction were detected using as spin trap the DMPO. The free radical species originated during the oxidation reaction formed stable adducts with the spin trap, and these adducts were identified by ES-MS. New adducts of oxidized tryptophan radicals, namely monohydroxy-tryptophan and dihydroxy-Trp dimer radicals, with one and two DMPO spin trap molecules where identified. Tandem mass spectrometry was used to confirm the proposed structure of the observed adducts.

Rhamnoarabinosyl and rhamnoarabinoarabinosyl side chains as structural features of coffee arabinogalactans.

The hot water soluble green coffee arabinogalactans, representing nearly 7% of total coffee bean arabinogalactans, were characterized by (1)H and (13)C NMR and, after partial acid hydrolysis, by ESI-MS/MS. Data obtained showed that these are highly branched type II arabinogalactans covalently linked to proteins (AGP), with a protein moiety containing 10% of 4-hydroxyproline residues. They possess a beta-(1-->3)-Galp/beta-(1-->3,6)-Galp ratio of 0.80, with a sugars composition of Rha:Ara:Gal of 0.25:1.0:1.5, and containing 2mol% of glucuronic acid residues. Beyond the occurrence of single alpha-L-Araf residues and [alpha-L-Araf-(1-->5)-alpha-L-Araf-(1-->] disaccharide residues as side chains, these AGPs contain unusual side chains at O-3 position of the beta-(1-->6)-linked galactopyranosyl residues composed by [alpha-L-Rhap-(1-->5)-alpha-L-Araf-(1-->] and [alpha-L-Rhap-(1-->5)-alpha-L-Araf-(1-->5)-alpha-L-Araf-(1-->] oligosaccharides. Rhamnoarabinosyl and rhamnoarabinoarabinosyl side chains are reported for the first time as structural features of plant arabinogalactan-proteins.

Lipidomic approach to identify patterns in phospholipid profiles and define class differences in mammary epithelial and breast cancer cells.

Breast cancer is the leading cause of cancer-related deaths in women. Altered cellular functions of cancer cells lead to uncontrolled cellular growth and morphological changes. Cellular biomembranes are intimately involved in the regulation of cell signaling; however, they remain largely understudied. Phospholipids (PLs) are the main constituents of biological membranes and play important functional, structural and metabolic roles. The aim of this study was to establish if patterns in the PL profiles of mammary epithelial cells and breast cancer cells differ in relation to degree of differentiation and metastatic potential. For this purpose, PLs were analyzed using a lipidomic approach. In brief, PLs were extracted using Bligh and Dyer method, followed by a separation of PL classes by thin layer chromatography, and subsequent analysis by mass spectrometry (MS). Differences and similarities were found in the relative levels of PL content between mammary epithelial and breast cancer cells and between breast cancer cells with different levels of aggressiveness. When compared to the total PL content, phosphatidylcholine levels were reduced and lysophosphatydilcholines increased in the more aggressive cancer cells; while phosphatidylserine levels remained unchanged. MS analysis showed alterations in the classes of phosphatidylcholine, lysophosphatidylcholine, sphingomyelin, and phosphatidylinositides. In particular, the phosphatidylinositides, which are signaling molecules that affect proliferation, survival, and migration, showed dramatic alterations in their profile, where an increase of phosphatdylinositides saturated fatty acids chains and a decrease in C20 fatty acids in cancer cells compared with mammary epithelial cells was observed. At present, information about PL changes in cancer progression is lacking. Therefore, these data will be useful as a starting point to define possible PLs with prospective as biomarkers and disclose metabolic pathways with potential for therapy.

Bioconversion of agro-industrial by-products in rhamnolipids toward applications in enhanced oil recovery and bioremediation.

In this work, biosurfactant production by a Pseudomonas aeruginosa strain was optimized using low-cost substrates. The highest biosurfactant production (3.2 g/l) was obtained using a culture medium containing corn steep liquor (10% (v/v)) and molasses (10% (w/v)). The biosurfactant reduced the surface tension of water up to 30 mN/m, and exhibited a high emulsifying activity (E24=60%), with a critical micelle concentration as low as 50 mg/l. The biosurfactant produced in this alternative medium was characterized as a mixture of eight different rhamnolipid congeners, being the most abundant the mono-rhamnolipid Rha-C10-C10. However, using LB medium, nine different rhamnolipid congeners were identified, being the most abundant the di-rhamnolipid Rha-Rha-C10-C10. The rhamnolipid mixture produced in the alternative medium exhibited a better performance in removing oil from contaminated sand when compared with two chemical surfactants, suggesting its potential use as an alternative to traditional chemical surfactants in enhanced oil recovery or bioremediation.

Lipidomic analysis of phospholipids from human mammary epithelial and breast cancer cell lines.

Alterations of phospholipid (PL) profiles have been associated to disease and specific lipids may be involved in the onset and evolution of cancer; yet, analysis of PL profiles using mass spectrometry (MS) in breast cancer cells is a novel approach. Previously, we reported a lipidomic analysis of PLs from mouse mammary epithelial and breast cancer cells using off‐line thin layer chromatography (TLC)‐MS, where several changes in PL profile were found to be associated with the degree of malignancy of cells. In the present study, lipidomic analysis has been extended to human mammary epithelial cells and breast cancer cell lines (MCF10A, T47‐D, and MDA‐MB‐231), using TLC‐MS, validated by hydrophilic interaction liquid chromatography‐MS. Differences in phosphatidylethanolamine (PE) content relative to total amount of PLs was highest in non‐malignant cells while phosphatidic acid was present with highest relative abundance in metastatic cells. In addition, the following differences in PL molecular species associated to cancer phenotype, metastatic potential, and cell morphology were found: higher levels of alkylacyl PCs and phosphatidylinositol (PI; 22:5/18:0) were detected in migratory cells, epithelial cells had less unsaturated fatty acyl chains and shorter aliphatic tails in PE and sphingomyelin classes, while PI (18:0/18:1) was lowest in non‐malignant cells compared to cancer cells. To date, information about PL changes in cancer progression is scarce, therefore results presented in this work will be useful as a starting point to define possible PLs with prospective as biomarkers and disclose metabolic pathways with potential for cancer therapy. J. Cell. Physiol. 228: 457–468, 2013.

Post-translational modifications and mass spectrometry detection

In this review, we provide a comprehensive bibliographic overview of the role of mass spectrometry and the recent technical developments in the detection of post-translational modifications (PTMs). We briefly describe the principles of mass spectrometry for detecting PTMs and the protein and peptide enrichment strategies for PTM analysis, including phosphorylation, acetylation and oxidation. This review presents a bibliographic overview of the scientific achievements and the recent technical development in the detection of PTMs is provided. In order to ascertain the state of the art in mass spectrometry and proteomics methodologies for the study of PTMs, we analyzed all the PTM data introduced in the Universal Protein Resource (UniProt) and the literature published in the last three years. The evolution of curated data in UniProt for proteins annotated as being post-translationally modified is also analyzed. Additionally, we have undertaken a careful analysis of the research articles published in the years 2010 to 2012 reporting the detection of PTMs in biological samples by mass spectrometry.