Pedro 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.

Analysis of the human saliva proteome

Interest in the characterization of the salivary proteome has increased in the last few years. This review discusses the different techniques and methodologies applied to the separation and identification of salivary proteins. Nowadays, proteomic techniques are the state of the art for the analysis of biologic materials and saliva is no exception. 2D electrophoresis and tryptic digest analysis by mass spectrometry are the typical methodology, but new approaches using 2D liquid chromatography/mass spectrometry methods have already been introduced for saliva analysis. Due to their important physiologic role in the oral cavity, low-molecular-weight proteins and peptides are also included in this article and the methodologies discussed.

Keggin-type polyoxotungstates as catalysts in the oxidation of cyclohexane by dilute aqueous hydrogen peroxide

Abstract Oxidation of cyclohexane by hydrogen peroxide in the presence of catalytic amounts of the Keggin-type heteropolytungstates [PW 11 O 39 ] 7− and [PW 11 M(L)O 39 ] (7− m )− , M m + =first row transition metal cation, L=H 2 O or CH 3 CN, was found to produce cyclohexanol, cyclohexanone and, in certain cases, cyclohexyl hydroperoxide. The presence of the latter was demonstrated by negative chemical ionization GC-MS. The reactions were carried out in acetonitrile, using tetra n -butylammonium salts of the catalysts and aqueous 30% hydrogen peroxide as oxidant. The polyanions [PW 11 O 39 ] 7− and [PW 11 Fe(H 2 O)O 39 ] 4− showed higher catalytic activity and different selectivity for the oxidation of cyclohexane than did the corresponding Cu-, Co-, Mn- and Ni-substituted complexes.

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.

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.

Effect of the pH of growth on the survival of Lactobacillus delbrueckii subsp. bulgaricus to stress conditions during spray-drying

Aims:  The aim of this study was to optimize survival of Lactobacillus delbrueckii subsp. bulgaricus during spray‐drying and subsequent storage through optimizing the pH of growth conditions.

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.

Novel biocompatible and self-buffering ionic liquids for biopharmaceutical applications.

Antibodies obtained from egg yolk of immunized hens, immunoglobulin Y (IgY), are an alternative to the most focused mammal antibodies, because they can be obtained in higher titers by less invasive approaches. However, the production cost of high-quality IgY for large-scale applications remains higher than that of other drug therapies due to the lack of efficient purification methods. The search for new purification platforms is thus vital. The solution could be liquid-liquid extraction by using aqueous biphasic systems (ABS). Herein, we report the extraction and attempted purification of IgY from chicken egg yolk by using a new ABS composed of polymers and Goods buffer ionic liquids (GB-ILs). New self-buffering and biocompatible ILs based on the cholinium cation and anions derived from Goods buffers were synthesized and the self-buffering characteristics and toxicity were characterized. Moreover, when these GB-ILs are combined with PPG 400 (poly(propylene) glycol with a molecular weight of 400 g mol(-1)) to form ABS, extraction efficiencies, of the water-soluble fraction of proteins, ranging between 79 and 94% were achieved in a single step. Based on computational investigations, we also demonstrate that the preferential partitioning of IgY for the GB-IL-rich phase is dominated by hydrogen-bonding and van der Waals interactions.

Lipoxidation adducts with peptides and proteins: deleterious modifications or signaling mechanisms?

Protein lipoxidation refers to the modification by electrophilic lipid oxidation products to form covalent adducts, which for many years has been considered as a deleterious consequence of oxidative stress. Oxidized lipids or phospholipids containing carbonyl moieties react readily with lysine to form Schiff bases; alternatively, oxidation products containing α,β-unsaturated moieties are susceptible to nucleophilic attack by cysteine, histidine or lysine residues to yield Michael adducts, overall corresponding to a large number of possible protein adducts. The most common detection methods for lipoxidized proteins take advantage of the presence of reactive carbonyl groups to add labels, or use antibodies. These methods have limitations in terms of specificity and identification of the modification site. The latter question is satisfactorily addressed by mass spectrometry, which enables the characterization of the adduct structure. This has allowed the identification of lipoxidized proteins in physiological and pathological situations. While in many cases lipoxidation interferes with protein function, causing inhibition of enzymatic activity and increased immunogenicity, there are a small number of cases where lipoxidation results in gain of function or activity. For certain proteins lipoxidation may represent a form of redox signaling, although more work is required to confirm the physiological relevance and mechanisms of such processes. This article is part of a Special Issue entitled: Posttranslational Protein modifications in biology and Medicine.