Laura Molin

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.

Protein Glycation in Diabetes as Determined by Mass Spectrometry

Diabetes is a common endocrine disorder characterized by hyperglycemia leading to nonenzymatic glycation of proteins, responsible for chronic complications. The development of mass spectrometric techniques able to give highly specific and reliable results in proteome field is of wide interest for physicians, giving them new tools to monitor the disease progression and the possible complications related to diabetes, as well as the effectiveness of therapeutic treatments. This paper reports and discusses some of the data pertaining protein glycation in diabetic subjects obtained by matrix-assisted laser desorption ionization (MALDI) mass spectrometry (MS). The preliminary studies carried out by in vitro protein glycation experiments show clear differences in molecular weight of glycated and unglycated proteins. Then, the attention was focused on plasma proteins human serum albumin (HSA) and immunoglobulin G (IgG). Enzymatic degradation products of in vitro glycated HSA were studied in order to simulate the in vivo enzymatic digestion of glycated species by the immunological system leading to the highly reactive advanced glycation end-products (AGEs) peptides. Further studies led to the evaluation of glycated Apo A-I and glycated haemoglobin levels. A different MALDI approach was employed for the identification of markers of disease in urine samples of healthy, diabetic, nephropathic, and diabetic-nephropathic subjects.

A further investigation on a MALDI-based method for evaluation of markers of renal damage.

The validity of the urinary protein profile to characterize the pathological states of diabetic, nephropathic and diabetic-nephropathic patients was considered on the basis of previously obtained results by MALDI/MS, showing a different abundance ratio of the collagen alpha1 and alpha5 chain precursor fragments at m/z 1219 and 2049 and of the uromodulin precursor fragment at m/z 1912 observed in healthy subjects and patients; a larger number of subjects was examined and the obtained results were statistically evaluated. The p values related to the observed differences indicate that they are statistically significant when comparing all patients versus healthy controls, diabetic with normo or microalbuminuria versus nephropathic with advanced renal disease patients and diabetic with normo or microalbuminuria versus diabetic with advanced nephropathy patients. The scatter plot matrix gives evidence of the strict inverse relationship between the abundances of ions at m/z 1912 and 1219, the correlation coefficient being particularly high (r = 0.921, p < 0.001). The relationship between the true positive rate (sensitivity) and false positive rate (1-specificity) for every possible cutoff value in abundance of the considered ionic species was investigated through the receiver-operating characteristic (ROC) curve. The obtained data indicate that a good differentiation of nephropathic patients with advanced renal disease and diabetic patients with advanced nephropathy versus healthy subjects can be easily obtained by this approach.

The double nature of 1,5-diaminonaphthalene as matrix-assisted laser desorption/ionization matrix: some experimental evidence of the protonation and reduction mechanisms.

1,5-Diaminonaphthalene (DAN) has been described as an interesting and effective matrix for matrix-assisted laser desorption/ionization (MALDI) experiments in positive ion mode, being able to activate in-source decomposition phenomena and, when employed for the analysis of proteins containing disulphide bridge(s), being able to activate reduction processes, resulting in disulphide bridge cleavage. The mechanisms of the DAN reactivity have been studied in detail, and the results indicate that the reduction properties of the matrix are of a radical nature. In the present study the structure of the reactive species produced by DAN, responsible for its reductive properties, has been investigated by accurate mass measurements and tandem mass spectrometry (MS/MS) experiments. Contrary to what is usually observed by laser irradiation of other MALDI matrices (with the sole formation of the MH(+) ion of the matrix), DAN leads to the formation of odd-electron molecular ions M(+•) . This can be rationalized by the occurrence of two photon pooling processes, due to the low ionization energy of DAN. Thus the M(+•) ion of DAN can be considered responsible for both analyte protonation and disulphide bond reduction and some mechanisms are proposed for this behaviour.

Quantification of Soyasaponins I and βg in Italian Lentil Seeds by Solid Phase Extraction (SPE) and High Performance Liquid Chromatography-Mass Spectrometry (HPLC-MS)

Lentil saponins are triterpene glycosides, mainly soyasaponins I and betag (also known as VIota), with multiple health-promoting properties. This paper reports the isolation of soyasaponins I and betag from soybeans as analytical standards and the development of a new analytical procedure for quantification of their content in various cultivars of Italian lentils, by SPE-HPLC-MS. Soyasaponins I and betag were isolated from soybeans at a purity of >90% and characterized by MS/MS (ion trap) experiments. The determination of soyasaponins in lentils was performed by extraction, SPE purification, and HPLC-MS (single quadrupole) analysis; results were confirmed by MALDI-TOF experiments. Calibration curves for soyasaponin I and betag showed correlation coefficients of 0.998 and 0.997, respectively. LOD and LOQ values were 0.02 and 0.2 mg kg(-1) for soyasaponins I and 0.1 and 1 mg kg(-1) for soyasaponin betag. Recoveries calculated at a 100 mg kg(-1) fortification level ranged from 85 to 97%, with n = 10 and RSDs of <12%. In the 32 lentil samples, contents of soyasaponin I ranged from 28 to 407 mg kg(-1), whereas that of soyasaponin betag ranged from 110 to 1242 mg kg(-1).

Seed oil triglyceride profiling of thirty-two hybrid grape varieties.

Triglyceride profile of seed oil samples from 32 hybrid grape varieties not studied before was investigated. A new method for the analysis of triacylglycerols (TAGs) has been developed based on the direct infusion in the electrospray ionization (ESI) source and employing tetrahydrofuran/methanol/water (85:10:5 v|v|v) as solvent; the formation of [M + Na](+) ions in high yield has been observed. TAGs were identified by ESI-tandem mass spectrometry analysis, and the matrix-assisted-laser-desorption-ionization and time-of-flight profile of samples was determined. Six were the principal TAGs identified in seed oil: trilinolein (LLL) was the most abundant (43%), followed by dilinoleoyl-oleoylglycerol (LOL, 23%), and dilinoleoyl-palmitoylglycerol (LPL, 15%). Compounds present in lower concentration were LSL and LOO (11%), LOP (6%), and LSP (2%). Compared with seed oils produced from V. Vinifera grapes, some significant differences in the relative abundances of TAGs were found, in particular hybrid grape seed oils showed higher LOL and lower LPL content, respectively. Among the samples studied, a particularly high content of LLL (rich in unsaturated fatty acids) was found in seed oils from two red varieties.

Cocaine adulterants used as marker compounds

Cocaine has become a widely employed drug of abuse. In Italy its use may be estimated in the order of 9000 kg/year. In fact, considering that seizures in the last year in Italy were 3900 kg (Italian Minister of the Interior)[1] and that the worldwide interception rate has been estimated in the order of 42%,[2] it follows that the circulating cocaine is in the order of magnitude mentioned earlier. Large amounts of seized drug samples have been analyzed in order to identify, on the basis of the adulterants contained therein, their possible production area and the mechanisms by which they have been placed on the market. In fact the determination of cocaine content and adulterants in street samples is important not only from the clinical and toxicological points of view, but also for forensic purposes, related to the determination of geographical origin, comparison of various street samples for intelligence purposes and route of synthesis of the drugs. Nowadays illicit cocaine powders contain purposely a myriad of ‘new’ adulterants, having or not having any pharmacological effect, with the main aim to increase the amount of product available for sale with profit maximization. Although the risks to human health from cocaine use are known, the consequences from assumption of cocaine adulterants are not fully explored.[3] The cocaine adulterants most frequently employed are listed in Table 1.[4] A great number of adulterants have significant toxicological effects that may, in particular at high concentrations, amplify those of cocaine. The main categories of adulterants with pharmacological effects are: stimulants and local anesthetics (which are absorbed across mucosal surface and simulate the assumption of Cocaine) and sedatives (which are often assumed with cocaine to reduce the intensity of its unpleasant side effects).[5] The presence of adulterants could reflect on another aspect. In fact, as usually observed in pharmaceutical preparation, the impurities present at trace level (usually intermediates of the active product synthesis) are an effective marker of the chemical process used for production and covered by the related patent. Their presence is an unequivocal proof of the production process. Nowadays the street drug production can be considered due to, unfortunately, highly active industrial structures which necessarily, at marketing level, must define their sale regions. An effective method to control the presence of drugs from concurrent producers in the reserved area could be monitored on the basis of specific adulterants. In the Padova area (North-East of Italy) in the period March, 2006 – September, 2007 among the seized cocaine samples (259, for a

Doxorubicin coupled to lactosaminated albumin: effect of heterogeneity in drug load on conjugate disposition and hepatocellular carcinoma uptake in rats.

Coupling to lactosaminated human albumin (L-HSA) makes doxorubicin (DOXO) an effective drug against chemically induced rat hepatocellular carcinomas (HCCs). In the conjugate there is a large heterogeneity in the number of DOXO molecules bound to one L-HSA molecule. After lyophilization, the molecules with the higher DOXO load form large complexes (C-DOXOL), whereas those with low drug load (C-DOXOS) have the size of the carrier L-HSA. In the present experiments, we demonstrated that in C-DOXOL the molecules are not linked by covalent bonds, but are strongly aggregated probably because of mutual drug-drug interaction between the DOXO residues. In healthy rats and in animals with HCCs which received the same dose (1 microg/g) of DOXO injected in C-DOXOL or in C-DOXOS forms, penetration of the drug in tumors and in tissues was more rapid after administration of the former complex. Three hours after injection of both conjugate forms the intracellular release of DOXO from the carrier was completed. The AUCs from 0.5 to 4h of the levels of the released DOXO in HCCs, surrounding liver and bone marrow of animals injected with C-DOXOL were similar to those calculated in animals given C-DOXOS. This suggests that after administration of the dose of DOXO used in the present experiments the conjugate molecules with lower or higher drug load can exert comparable pharmacological and toxic effects.

A preliminary matrix-assisted laser desorption/ionization time-of-flight approach for the characterization of Italian lentil varieties.

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) was used in this study to obtain protein fingerprints of seven different lentil varieties, to characterize their differences and similarities. Two different matrices have been tested in order to obtain reproducible and significant mass spectra. Extraction with water containing 0.1% of trifluoroacetic acid has been used as preparative step to obtain hydrophilic protein samples of lentil seeds. The obtained MALDI protein profiles identified clear differences between the seven studied lentil varieties. Moreover, considering the high complexity of the obtained MALDI spectra, multivariate techniques of data analysis were employed to find further classification details. These multivariate analyses confirmed the possibility of a clear classification of the seven lentil varieties, indicating that the proposed procedure can be a valid taxonomic tool, and a method to certify the origin of lentils, useful for high added value lentils (Italian lentils).

On the Primary Ionization Mechanism(s) in Matrix-Assisted Laser Desorption Ionization

A mechanism is proposed for the first step of ionization occurring in matrix-assisted laser desorption ionization, leading to protonated and deprotonated matrix (Ma) molecules ([Ma + H]+ and [Ma − H]− ions). It is based on observation that in solid state, for carboxyl-containing MALDI matrices, the molecules form strong hydrogen bonds and their carboxylic groups can act as both donors and acceptors. This behavior leads to stable dimeric structures. The laser irradiation leads to the cleavage of these hydrogen bonds, and theoretical calculations show that both [Ma + H]+ and [Ma − H]− ions can be formed through a two-photon absorption process. Alternatively, by the absorption of one photon only, a heterodissociation of one of the O–H bonds can lead to a stable structure containing both cationic and anionic sites. This structure could be considered an intermediate that, through the absorption of a further photon, leads to the formation of matrix ions. Some experiments have been performed to evaluate the role of thermal ionization and indicate that its effect is negligible. Some differences have been observed for different matrices in the formation of analyte molecule (M) ion [M + H]+, [M − H]−, M+•, and [M − 2H]-•, and they have been explained in terms of ionization energies, pKa values, and thermodynamic stability.