Roberta Seraglia

Analysis of plasma cholesterol oxidation products using gas- and high-performance liquid chromatography/mass spectrometry

The application of gas chromatography and high-pressure liquid chromatography/mass spectrometry techniques for analysis of plasma cholesterol oxidation products is described. Cholesterol oxides that are widely identified in biological samples were subjected to gas (GC) and high-pressure liquid chromatographic (HPLC) separations, and their detection and characterization by mass spectrometry (MS) were compared. Analysis of cholesterol oxides from plasma samples revealed distinct advantages for each method according to the specific cholesterol oxide in question. Whereas HPLC/MS analysis of cholesterol oxides provided less resolution and lower sensitivity as compared to GC/MS, a distinct advantage was evident for direct measurements of cholesterol-7-hydroperoxides and 7-ketocholesterol. These two cholesterol oxides are particularly sensitive to storage in solvents, derivatization procedures, and analytical conditions used for GC analysis, which are minimized or avoided using the HPLC/MS conditions described. Analysis of human and rabbit plasma samples identified cholest-5-ene-3 beta, 7 beta-diol (7 beta-hydroxycholesterol); 5,6 alpha-epoxy-5 alpha-cholestan-3 beta-ol (cholesterol-5 alpha, 6 alpha-epoxide); 5 alpha-cholestane-3 beta, 5,6 beta-triol (cholestanetriol); 3 beta-hydroxycholest-5-ene-7-one (7-ketocholesterol); and 5,6 beta-epoxy-5 beta-cholestan-3 beta-ol (cholesterol-5 beta,6 beta-epoxide) as commonly occurring components (trivial names indicated in parentheses). The latter two compounds were dramatically increased in hypercholesterolemic samples and were found in approximately equal amounts in the free cholesterol and cholesteryl ester fractions. Although most of the plasma cholesterol oxides are found in the dietary cholesterol, others are not, particularly cholesterol-5 beta,6 beta-epoxide, suggesting that at least some of these compounds are formed by in vivo oxidation of cholesterol. Despite the readily measurable levels of the above cholesterol oxides, as well as other less prominent oxides, there was no evidence of cholesterol-7-hydroperoxides associated with plasma free cholesterol. Although several of the plasma cholesterol oxides may derive from cholesterol-7-hydroperoxides, it appears that the latter are either unstable and decompose in plasma, are metabolized to other cholesterol oxidation products, or break down during their isolation.

Matrix-assisted laser desorption/ionization mass spectrometry, enzymatic digestion, and molecular modeling in the study of nonenzymatic glycation of IgG

The glycation-induced functional change of immunoglobulins is of particular interest. The glycation levels of IgG in 10 healthy subjects and 20 diabetic patients with different degrees of metabolic control were studied by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. It reveals the number of glucose molecules that have condensed on the protein, which range from 1 to 5 for healthy subjects, from 5 to 9 for well controlled diabetic patients, and from 10 to 25 for poorly controlled ones. The identification of the most favored glycation sites has been obtained by MALDI analysis of standard and in vitro glycated IgG and plasma protein fraction of a healthy subject after digestion with papain, releasing Fab and Fc fragments of the molecule. Both experiments, as well as molecular modeling of the whole protein, confirm that the most of glucose molecules have condensed on the Fab fragment of IgG, suggesting that the immune deficiency observed in diabetic patients may be explained at the molecular level by a more effective glycation of the Fab fragment, thus inhibiting the process of molecular recognition between antibody and antigen.

Identification of Partially Degraded Oligomers of 5,6-Dihydroxyindole-2-carboxylic Acid inSepia Melanin by Matrix-assisted Laser Desorption/Ionization Mass Spectrometry

Despite extensive efforts over more than half a century, the direct structural investigation of natural melanins has, so far, been largely unsuccessful, because of the adverse physical and chemical properties of these pigments. In the present study, we applied matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) to the direct analysis of fresh melanin samples from the cuttlefish Sepia officinalis, and succeeded in identifying clearly distinct patterns of low molecular weight ionic species, ranging from 450 to 1200 Da. Detailed analysis of the molecular weights and mass differences between the major species, aided by comparison with MALDI-MS spectra of synthetic melanins, allowed formulation of the major components as oligomers of 5,6-dihydroxyindole-2-carboxylic acid partially degraded by peroxidative fission of the o-diphenol moieties with concomitant decarboxylation and oxygenation reactions. These results provide the first direct insight into the structure of Sepia melanin and confirm the unique value of MALDI-MS techniques for the investigation of highly complex and heterogeneous polymers such as melanins.

Low molecular weight proteins in urines from healthy subjects as well as diabetic, nephropathic and diabetic-nephropathic patients : a MALDI study

Urine samples from healthy subjects as well as diabetic, nephropathic and diabetic-nephropathic patients were analyzed by matrix assisted laser desorption/ionization (MALDI) mass spectrometry in order to establish evidence of some possible differences in the peptide profile related to the pathological states. Multivariate analysis suggested the possibility of a distinction among the considered groups of patients. Some differences have been found, in particular, in the relative abundances of three ions at m/z 1912, 1219 and 2049. For these reasons, further investigation was carried out by MALDI/TOF/TOF to determine the sequence of these peptides and, consequently, to individuate their possible origin. By this approach, the peptide at m/z 1912 was found to originate from uromodulin, and its lower expression in the case of nephropathy can be well related to the pathological condition. Ions at m/z 2049 and 1219 originate from the collagen alpha-1(I) chain precursor and from the collagen alpha-5 (IV) chain precursor, respectively, and, also in this case, their different expressions can be related to the pathologies under investigation. The obtained data seem to indicate that urine is an interesting biological fluid to investigate on the peptide profile and to obtain, consequently, information on the dismetabolism activated by specific pathologies.

Atmospheric pressure photoionization mechanisms - 1. The case of acetonitrile

Abstract Experimental data show that acetonitrile, even though it has an ionization energy higher than the photon energy employed in atmospheric pressure photoionization (APPI) conditions, participates in the protonation of furocumarins. In order to clarify this unexpected behaviour, the processes activated by APPI on acetonitrile have been studied in detail. The formation of protonated molecules (C2H4N+ cations), and of a complex between acetonitrile and water (C2H6NO+ cations), is observed. To clarify the nature of this species, 13 C and 2 H labelling experiments and product ion spectra have been employed. The data suggest that photon irradiation leads to a first isomerization of acetonitrile molecules that leads to species that exhibit an IE

Some thoughts on electrospray ionization mechanisms.

Electrospray ionization (ESI) mechanisms are highly complex, due to a series of physical and chemical phenomena taking place on a complex system, as a solution is. In fact, even if the solution of an analyte in a protic medium can be considered at first sight to be a two-component system, the presence of solvent dissociation equilibria and the possible interactions solvent–solvent dissociation products, solvent dissociation products–analyte make this system highly complex, also for the presence of possible ionic compounds (for example, Na+, K+) which strongly affect the above equilibria. A high number of research articles have been published, mainly devoted to charged droplet production and to gas-phase ion generation. They all show the high complexity of the processes affecting electrospray measurements related to either the chemical equilibria present in the condensed phase and to electrolysis processes at the emitter tip or to the processes occurring in the sprayed droplets. As a result, the chemical composition inside the small droplets from which the analyte ions are generated can be significantly different from those in sprayed solution. In this review, after a short survey of the proposed ESI mechanisms, some experiments are described. They were performed to examine if ion mobility in solution, before the formation of the sprayed charged droplets, can affect the ESI results. The data, obtained by studying both inorganic and organic analytes, indicate that the ESI spectra are dependent on the analyte dimension and charge state which, as a consequence, affect their ion mobility in solution.

Structural Analysis of Synthetic Melanins from 5,6-Dihydroxyindole by Matrix-assisted Laser Desorption/Ionization Mass Spectrometry

Despite extensive investigation, the structure of melanins, the major determinants of skin colour differences in man and other mammals, is still poorly defined, mainly because of the unfavourable properties of the materials. In this study, analysis of model pigments prepared by enzymically- or chemically-induced oxidative polymerization of 5,6-dihydroxyindole (DHI), the ultimate biosynthetic precursor, was investigated by means of matrix-assisted laser desorption/ionization mass spectrometry. All DHI melanins exhibited quite distinct pattern of ionic species of low m/z ranging from 500 to 1500, none of which corresponded to intact DHI oligomers. Analysis of the molecular weights and mass differences between the oligomer species provided evidence for a significant breakdown of the pigment backbone by peroxidative fission of the indole units with concomitant decarboxylation and oxygenation reactions. These processes, which take place to a different extent depending on the preparation conditions, are possibly initiated by hydrogen peroxide, either added as the oxidant or slowly generated in the reaction medium. In support of this, melanin samples prepared by tyrosinase oxidation in the presence of catalase comprised, as major components, intact DHI oligomers up to hexamers. Overall, these results offer a new picture of the structure of DHI melanins which may significantly contribute to the understanding of the high degree of molecular heterogeneity of the natural pigments.

Evaluation of IgG glycation levels by matrix-assisted laser desorption/ionization mass spectrometry†

Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry has been employed for the evaluation of the glycation level of IgG from healthy subjects and also from well- and badly-controlled diabetic patients. The measurements have been performed on untreated plasma protein fractions. The data obtained have shown that a clear mass increase, originating from non-enzymatic glycation processes, is observed in the case of diabetic patients: for well-controlled ones it is in the range 512-1565 Da, while it becomes 827-4270 Da for badly-controlled diabetic patients. This approach indicates that MALDI mass spectrometry is a highly specific tool that can be employed in the metabolic control of diabetic patients and in studies relating the IgG glycation level to possible immunological impairment.

The potential of matrix-assisted laser desorption/ionization mass spectrometry in the quality control of water buffalo mozzarella cheese.

Adulteration by addition of bovine milk to water buffalo milk employed for mozzarella cheese production is often observed. Water buffalo milk and mozzarella cheese were analysed by matrix-assisted laser desorption/ionization mass spectrometry in order to achieve their rapid and accurate characterization and to evaluate possible fraudulence in mozzarella cheese production.

Protein profiles in sera of patients with malignant cutaneous melanoma

Seventeen samples of sera from patients with malignant cutaneous melanoma at various stages and 14 samples from healthy subjects were analysed by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. Results highlighted the presence of several protein species at molecular weights lower than 30000 Da, presumably originating from proteolysis, in the sera of the patients with melanoma. These species were completely absent in healthy subjects. In particular, the presence and abundance of species with molecular weights in the range 2500-3500 Da exhibit significant variations related to the different clinical stages of the disease.