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Advanced glycation end products/peptides (AGE/peptides) originate by in vivo enzymatic digestion of nonenzymatically glycated proteins, which are produced by reaction of glucose with primary amino groups present in the protein chain following the Maillard pattern. AGE/peptides are highly reactive species and can interact with tissue and circulating proteins, leading to tissue modification and impaired protein functionality. Serum levels of AGE/peptides are reported to be particularly high in diabetes (in terms of higher production) or in end-stage renal disease (in terms of accumulation). For these reasons, their structural identification is of high interest, giving information on their relationship with the pathological state and allowing the design of possible therapeutic interventions. We report here some preliminary results obtained by liquid chromatography/electrospray ionization/mass spectrometry (LC/ESI/MS) and matrix-assisted laser desorption ionization MS (MALDI-MS) investigations carried out on the low-molecular-weight serum peptide fraction from 10 healthy subjects, 10 patients with poorly controlled diabetes, and 10 patients with end-stage nephropathy.

Cadmium bis(O-alkylxanthates) are potential single-source molecular precursors for the chemical vapor deposition (CVD) of Cd(II) sulfide thin films. In this work, a multi-technique characterization of Cd(O-RXan)2 compounds [where O-RXan is CH3CH2OCS2 (O-EtXan) or (CH3)2CHOCS2 (O-iPrXan)] is performed by means of several analytical methods (extended x-ray absorption fine structure, Raman, Fourier transform infrared and optical absorption, spectroscopics 1H and 13C NMR, thermal analysis and mass spectrometry) for a thorough investigation of their structure and chemical–physical properties. The most important results concerning the chemical behavior under different experimental conditions, with particular attention to relevant properties for CVD applications, are presented and discussed. Copyright © 2005 John Wiley & Sons, Ltd.

Theutilizationofsingle-sourcemolecularprecursorsinchemicalvapordeposition(CVD)experiments requires a deep knowledge of their chemico-physical properties, with particular regard to thermal stability and fragmentation pattern. This study describes the synthesis and characterization of zinc bis(O-isopropylxanthate), Zn(O- i PrXan)2 ,[ O- i PrXan = (CH3)2CHOCS2], a single-source precursor for the CVD of zinc(II) sulfide thin films and nanorods. Several analytical methods yielding complementary information (extended X-ray absorptionfine structure, Raman, FT-IR, UV‐Vis optical absorption, 1 Ha nd 13 C NMR, thermogravimetric analysis, differential scanning calorimetry as well as mass spectrometry techniques, i.e. electrospray and electron ionization, mass-analyzed ion kinetic energy) are adopted for a comprehensive investigation of purity, structure, thermal behavior and decomposition pathways of the molecule. The most significant results are discussed critically and the properties useful for CVD applications are highlighted. Copyright  2005 John Wiley & Sons, Ltd.

Advanced glycation end products (AGEs) accumulate in serum and tissues of patients with chronic renal failure, even in the absence of diabetes, and a different clearance of these species has been observed by hemodialysis and peritoneal dialysis (CAPD). Furthermore, it has been shown that not only AGE but also 1,2-dicarbonyl compounds are formed during heat sterilization of glucose-based peritoneal dialysis fluids. Therefore, we investigated the level of some AGEs (pentosidine and free pentosidine) and dicarbonyl compounds (glyoxal and methylglyoxal) in end-stage renal disease patients subjected to peritoneal dialysis. Samples (20 from healthy subjects, 16 from uremic patients before and after 12 h of peritoneal dialysis) were analyzed, and the plasma and dialysate levels of glyoxal, methylglyoxal, pentosidine, and free pentosidine were determined. In plasma of uremic patients, mean values of pentosidine showed a small decrease after dialysis and were always higher than those of healthy control subjects. An analogous trend was observed for free pentosidine. In the case of peritoneal dialysate, no pentosidine and free pentosidine were found at time zero, whereas both compounds were detected after 12 h of dialysis. Glyoxal and methylglyoxal mean levels showed a decrease in plasma after dialysis even if their values were always higher than those of healthy control subjects. Surprisingly, an analogous trend was observed also in dialysate. These results might indicate that glyoxal and methylglyoxal already present in the dialysis fluid react with the peritoneal matrix proteins, accounting for the gradual loss of peritoneal membrane function that is often observed in patients subjected to CAPD for a long time.

New pyridylthioether-based dendrons bearing a thiol moiety at their focal point have been prepared by a convergent synthetic approach. These dendrons were readily attached to a Fe 2 (CO) 6 core to generate two-directional dendritic molecules incorporating an iron-carbonyl cluster.

The reaction products of RNase with glucose and fructose, at different concentrations and incubation times were studied by Matrix Assisted Laser Desorption/Ionization Mass Spectrometry. Clear differences in the reactivity of the two sugars were found. Different spectra were obtained analyzing diabetic and healthy serum samples by the same analytical technique.

The synthesis, the spectroscopic and structural characterization of different thiophenolate-capped zinc clusters are reported and described. In particular, different reactions of 4-chlorobenzenethiol with zinc salts yield the clusters [Me4N][Et3NH][Zn4(μ-S-C6H4-Cl)6(S-C6H4-Cl)4] (2a), [Et3NH]2[Zn4(μ-S-C6H4-Cl)6(S-C6H4-Cl)4] (2b), and [Me4N]2[Zn4(μ-S-C6H4-Cl)6(S-C6H4-Cl)4] (2c), and also the thiophenolate derivative [Et3NH]2[Zn4(μ-S-C6H5)6(S-C6H5)4] (1b) was obtained. The nanosized thiophenolate-capped clusters were investigated by 1H and 13C NMR, elemental analysis, and electrospray ionization (ESI) mass spectrometry. NMR experiments provided insights into the dynamic behaviour of the clusters. The thermal decomposition patterns of 2c were analyzed in air as well as in nitrogen, indicating the formation of zinc oxide and metallic zinc, respectively. The X-ray structure of 2a revealed that the cluster core consists of an adamantane-like framework analogous to those realized in many other M4(SR)10 metal complexes.

Advanced glycation end products (AGE) and dicarbonyl compounds accumulate in serum and tissues of patients with diabetes and chronic renal failure. Pentosidine, free pentosidine, glyoxal and methylglyoxal have been evaluated in plasma of diabetic patients with poor metabolic control at baseline and after the improvement of glycemic levels, and in plasma and peritoneal dialysate of patients with renal failure before and after 12 h of peritoneal dialysis. In diabetic patients, acceptable metabolic control was unable to normalize levels of pentosidine (after 2 and 10 months), glyoxal and methylglyoxal (after 2 months). In patients with end-stage renal disease, mean values of pentosidine, free pentosidine, glyoxal and methylglyoxal decreased in plasma after dialysis. No pentosidine or free pentosidine were present in the peritoneal dialysate at time 0, but were found after 12 h of peritoneal dialysis; glyoxal and methylglyoxal decreased after 12 h of dialysis. So, glyoxal and methylglyoxal, already present in the dialysis fluid, can react with the peritoneal matrix protein, giving a reason for the gradual loss of peritoneal membrane function often observed in patients undergoing long-term peritoneal dialysis.

The di-nitrile complexes trans -[PtCl 2 (N CR) 2 ] (R = Me, Ph, CH 2 Ph) react with an excess of gaseous NH 3 in CH 2 Cl 2 at −10 °C to form, in high yield, the corresponding di-amidine complexes trans -[PtCl(NH 3 ){HN C(NH 2 )R} 2 ]Cl in which also one chlorine ligand has been displaced by NH 3 . The 1 H NMR spectra in DMSO showed the formation of different species which were characterized through NOESY, TOCSY and 1 H/ 13 C heteronuclear correlations as trans -[Pt(NH 3 ){HN C(NH 2 )R} 2 (DMSO)]Cl 2 and trans -[PtCl{HN C(NH 2 )R} 2 (DMSO)]Cl.

Matrix-assisted laser desorption/ionization (MALDI) has been employed for the determination of molecular weights of alpha- and beta-globins obtained from blood samples of healthy and diabetic subjects. Glycated species, originating from the reaction of glucose with the proteins, are easily evident. The MALDI measurements allowed one to distinguish between simple glycation and glyco-oxidation processes of haemoglobin. In fact, together with the products arising from simple glucose condensation on the two proteins, compounds at different oxidation levels were evident. Due to its high specificity, MALDI must be considered a valuable tool for diagnosis in the diabetes field.

Several lines of evidence indicate that perturbed cellular Ca2+ homeostasis may play a prominent role in synaptic dysfunction and neuronal death in Alzheimer's disease (AD), suggesting a potential benefit of drugs capable to stabilize Ca2+ homeostasis. We here investigated the effects of a panel of L-type Ca2+ channel antagonists on the secretion of the amyloid beta-peptide (Abeta), which abnormally accumulates in the senile plaques of the brain of AD patients. We found that, in primary and immortalized neuronal cells in culture, nimodipine robustly stimulated secretion (up to about four-fold at 30 microM) of the highly amyloidogenic 42-residue isoform of Abeta (Abeta42), while leaving largely unaffected total Abeta secretion. An analogous effect was also observed in vivo, as the administration of a single dose of nimodipine (10 mg/kg i.p.) induced a significant rise of Abeta42 levels in plasma of Tg2576 mice. The effect of nimodipine was independent of blockage of L-type Ca2+ channels and capacitative calcium entry. Accordingly, nimodipine effect was largely Ca2+-independent, as neither depletion nor rise of extracellular Ca2+ abolished it. Hence, by showing that the effect of nimodipine on Abeta42 production is distinct from its ability to block Ca2+-influx pathways, we provide evidence for a previously uncharacterized effect of this long known molecule also used in clinical practice.

Tyrosine is generally considered to be the physiological precursor of melanins and tyrosinase the enzyme responsible. However, recent studies have shown that also peroxidases are involved in the biosynthesis of melanins. These enzymes use hydrogen peroxide to oxidise various phenol substrates. In this paper, we used a substrate other than tyrosine, i.e. 5-hydroxytryptophan, to verify if its peroxidase/H2O2-mediated oxidation gave rise to the formation of melanin. We also subjected 5-hydroxytryptophan to the action of tyrosinase, for comparison purposes. We observed that both enzymes converted this substrate to melanin and that peroxidase, in the presence of hydrogen peroxide, was much more effective than tyrosinase in catalysing the oxidative polymerization of 5-hydroxytryptophan, with the formation of insoluble black melanin-like pigments. Samples deriving from the reaction-substrate enzyme were ultrafiltered at different times through an Amicon ultrafiltration cell equipped with an Amicon Diaflo XM-50 membrane, in order to remove the enzyme, and immediately lyophilised. The resulting samples were analysed by matrix assisted laser desorption/ionisation (MALDI) mass spectrometry, which clearly identified several oligomer species in the reaction mixture. This work was undertaken to investigate the possible precursors of neuromelanin and the enzyme responsible for melanogenesis in brain, since although the central nervous system does not contain tyrosinase, it is rich in peroxidase and hydrogen peroxide.

Recently an individual variability in the relationships between mean blood glucose levels and HbA1c has been observed among diabetic patients. The aim of this study was to provide an accurate description and evaluation of glycated and glyco-oxidated globins from diabetic subjects and their relationship with HbA1c and plasma glucose values.We studied 20 type 2 diabetic and 10 healthy subjects. Plasma samples were analysed by matrix-assisted laser desorption ionisation mass spectrometry.The presence of glycated and glyco-oxidated species of both alpha and beta globin was demonstrated. Values for these showed a good linear relationship with HbA1c values and the mean daily plasma glucose values for the 6 weeks preceding the investigation. Trends differed according to whether patients had chronic complications or not, differences being seen in the slopes of the plots relating HbA1c to the abundance of glycated and glyco-oxidated species.The data obtained are consistent with the concept that individuals have a different individual proclivity for oxidation and/or that different oxidation kinetics are related to behavioural and environmental factors. Our data are thus relevant to the analysis of phenotype differences in diabetic patients.

The precursors [M(ESDTM)Cl 2 ] (M=Pt(II), Pd(II); ESDTM=EtO 2 CCH 2 (CH 3 )NCS 2 Me, S -methyl(ethylsarcosinedithiocarbamate)) were synthesized as previously reported [J. Inorg. Biochem. 83 (2001) 31] and used to obtain [M(ESDT)Cl] n (ESDT=ethylsarcosinedithiocarbamate anion) species. The complexes formed through reaction between [M(ESDT)Cl] n and the two chiral amino-alcohols synephryne (Syn) and norphenylephrine (Nor) have been synthesized, with the ultimate goal of preparing mixed dithiocarbamate/amino metal complexes of the type [M(ESDT)(Am)Cl] (Am=Syn, Nor). These compounds have been isolated, purified and characterized by means of FT-IR, mono- and bidimensional NMR spectroscopy and mass spectrometry ESI/MS (electronspray mass spectra). The experimental data suggest that in all cases coordination of the dithiocarbamate ligand (ESDT) takes a place through the two sulfur atoms, the -NCSS moiety acting as a symmetrical bidentate chelating group, in a square-planar geometry around the M(II) ion, while the other two coordination positions are occupied by the chlorine atom and the amino-alcohol ligand, respectively. In particular, synephrine and norphenylephrine appear to be bound to the metal atom through the amino nitrogen atom by means of a dative bond. Finally, the biological activity of the new complexes has been studied by MTT (tetrazolio salt reduction) test and by detecting the inhibition of DNA synthesis and of clonal growth in various cancer cell lines. All Pd(II) derivatives showed a noticeable activity very close to that of cisplatin, used as reference drug. Moreover, they showed significantly reduced cross-resistance to cisplatin in a pair of cell lines (2008/C13*) with known acquired cisplatin resistance mechanisms.

In order to study new and convenient sol–gel syntheses to homogeneous nanocomposites systems based on zinc sulphide clusters embedded in films of silica glass, a thiourea-functionalized silane ((EtO) 3 Si(CH 2 ) 3 NHC( S)NHPh, SilTu) was investigated as starting precursor. Electrospray ionization mass spectrometry (ESI-MS), FT-IR and NMR spectroscopies clearly evidence that SilTu coordinates Zn 2+ ions in solution through the thiourea function. Both zinc acetate (Zn(CH 3 COO) 2 ) and zinc chloride (ZnCl 2 ) were used as zinc source. The SilTu Zn 2+ complex is also stable under sol–gel conditions. Alcoholic solutions of SilTu, zinc acetate and tetraethoxysilane Si(OEt) 4 (TEOS) were used to prepare the thin films by dip-coating. Transparent, homogeneous and crack-free layers were obtained under annealing up to 600 °C. The samples were investigated by X-ray photoelectron spectroscopy (XPS) and secondary ion mass spectrometry (SIMS). Beside ZnS formation, the presence of oxygenated zinc species was observed inside the silica matrix.

Sulphur-functionalised trialkoxysilanes are suitable compounds for the sol–gel synthesis of silica glass embedding metal sulphide nanoclusters. In this study, three different thiourea-functionalised silanes, (EtO) 3 Si(CH 2 ) 3 NHC(S)NHPh (SilTu), 1,4-[(EtO) 3 Si(CH 2 ) 3 NHC(S)NH] 2 Ph (diSilTu) and (MeO) 3 Si(CH 2 ) 3 N[C(S)NH(C 6 H 5 )](CH 2 ) 2 NH[C(S)NH(C 6 H 5 )] (SildiTu), were used together with Zn 2+ ions to investigate their potential use in the preparation of ZnS–SiO 2 materials. Such precursors provide, in principle, a “molecule-to-nanocomposite” path by a direct anchoring of the metal cations to the forming silica network, via metal–sulphur (M–S) interactions. The sol–gel precursors were characterized by IR and NMR spectroscopies, and by electrospray mass spectrometry (ESI-MS). DiSilTu was also characterized by single crystal X-ray analysis. IR and 1 H , 13 C and 29 Si NMR measurements confirmed the formation of stable complexes with Zn 2+ ions, especially in the case of SilTu, also under sol–gel conditions.

The hydrolysis-polycondensation behaviour of alcoholic solutions containing Si(OEt)4 and Ti(OBun)4, in different molar ratios (Si/Ti = 10–0.2), was analysed by laser desorption/ionisation (LDI) and matrix-assisted laser desorption/ionisation (MALDI) mass spectrometry. The solutions were prepared using operating conditions usually employed in the sol-gel synthesis of SiO2-TiO2 materials. In accord with the well-known procedures for controlling the different chemical reactivities of the alkoxides, the pre-hydrolysis of the slower reacting silicon ethoxide and the chelation by acetylacetone of the faster reacting titanium butoxide were performed before mass spectrometric analysis. While LDI-MS did not provide evidence for the presence of mixed Si-Ti species in samples obtained from these reactions, MALDI-MS of samples diluted with chloroform and using 2,5-dihydroxybenzoic acid (DHB) as matrix led to detection of various oligomers with different contents of Si and Ti atoms. The results suggest that the formation of Si-Ti mixed oligomers seems to be the favoured process, especially for solutions in which one of the two components is diluted. Copyright © 2003 John Wiley & Sons, Ltd.

Determination of glyoxal and methylglyoxal levels in plasma is of great interest, since it allows us to evaluate oxidation processes occurring in glycated proteins. A method based on a simple derivatization procedure followed by gas chromatography/mass spectrometry (GC/MS) analysis has been developed. Ten diabetic patients were evaluated before and after improvement of glycemic control. Fasting plasma glucose, hemoglobin A1c (HbA1c), advanced glycation end products (AGE), pentosidine, glyoxal and methylglyoxal levels were measured. The percentage decreases of the levels of fasting plasma glucose, HbA1c and AGE were larger than those of pentosidine, glyoxal and methylglyoxal. These results may be explained by considering the different position of these compounds in the Maillard reaction pathways: these two sets of metabolic parameters give different pictures of patients' metabolic control. The measurement of glyoxal and methylglyoxal may be particularly important in the evaluation of the possible effect of oxidative stress. Other metabolic pathways can contribute to glyoxal production, and the observed minor decrease in these compounds can be, in principle, ascribed to such effect. However, a similar behavior of pentosidine indicates that these alternative pathways can be only partially responsible for glyoxal and methylglyoxal production.

New ferrocenyl derivatives (a β-ketoester and a β-diester) were synthesised and linked to fullerene C 60 , with the aim to elucidate factors involved in intramolecular electronic communication. These are the first examples of fullerene functionalised with ferrocenes via the cyclopropanation reaction. The resulting dyads were characterised.

The ESI mass spectrometric behaviour of a series of Pt(II) amidine complexes of the type cis- and trans-[PtCl2{NHC(R)NR′R″}2] (R=Me, Ph; R′=H, R″=Me, Et, But, Pri; R′=R″=Me, Et) and trans-[PtCl2(RCN){NHC(R)NR′R″] (R=Me, Ph; R′=R″=Me, Et) is reported. The bis-amidine complexes have been prepared by nucleophilic attack of primary and secondary amines on acetonitrile and benzonitrile ligands coordinated to Pt(II) substrates, while the mono-amidine complexes are obtained by reaction of secondary amines with trans-[PtCl2(RCN){NHC(R)NR′R″}] (R=Me, Ph; R′=R″=Me, Et). The protonated molecular ions or cationized ions are usually observed. The fragmentation processes are extremely reduced and in any case related to the original structure. As a general feature, cis and trans compounds give rise to different ESI spectra. Additions of Na+ and/or K+ are observed. From these data, ESI mass spectrometry, due to the soft ionisation, furtherly proposes as an efficient method for the characterisation of metal complexes in solution.

The Maillard reaction occurring between sugars and amino groups is important in living systems. When amino groups belonging to protein chains are involved, the Maillard reaction has been invoked as responsible for protein cross-linking and the production of 'toxic' compounds. The reaction leads to the production of a heterogeneous group of substances, usually called advanced glycation end products (AGEs). Classical analytical approaches, such as spectroscopic (ultraviolet, fluorescence) and mass spectrometric (matrix-assisted laser desorption/ionization, liquid chromatography/electrospray ionization mass spectrometry) methods, have shown that the digestion mixture is highly complex. However, there are clear differences between the digestion mixtures of glycated and unglycated human serum albumin (HSA). In the former case, possible glycated peptides belonging to the AGE peptide class may be identified. Tandem mass spectrometric experiments on selected species seemed to be promising as regards structural information, but it was thought of interest to undertake the present investigation, based on liquid chromatography/electrospray ionization Fourier transform mass spectrometry, in order to obtain definitive results on their elemental composition. Using this approach, about 20 glycated peptides were detected and their possible structures were postulated by examining the known sequence of HSA.

Tyrosine is generally considered to be the physiological precursor of melanins and tyrosinase the enzyme responsible. However, recent studies have shown that also peroxidases are involved in the biosynthesis of melanins. These enzymes use hydrogen peroxide to oxidise various phenol substrates. In this paper, we used a substrate other than tyrosine, i.e. 5-hydroxytryptophan, to verify if its peroxidase/H2O2-mediated oxidation gave rise to the formation of melanin. We also subjected 5-hydroxytryptophan to the action of tyrosinase, for comparison purposes. We observed that both enzymes converted this substrate to melanin and that peroxidase, in the presence of hydrogen peroxide, was much more effective than tyrosinase in catalysing the oxidative polymerization of 5-hydroxytryptophan, with the formation of insoluble black melanin-like pigments. Samples deriving from the reaction-substrate enzyme were ultrafiltered at different times through an Amicon ultrafiltration cell equipped with an Amicon Diaflo XM-50 membrane, in order to remove the enzyme, and immediately lyophilised. The resulting samples were analysed by matrix assisted laser desorption/ionisation (MALDI) mass spectrometry, which clearly identified several oligomer species in the reaction mixture.This work was undertaken to investigate the possible precursors of neuromelanin and the enzyme responsible for melanogenesis in brain. since although the central nervous system does not contain tyrosinase, it is rich in peroxidase and hydrogen peroxide.

The product ion spectra obtained by electrospray ionization (ESI) ion trap instruments exhibit a higher number of fragment ions than those achieved by other ion-trap-based systems, indicating the presence of more effective energy deposition mechanisms. This behavior can be attributed to several different reasons, among which different initial internal energy of the precursor ions, pre-activation due to collisions taking place outside the trap, different target gas mixtures inside the trap, and different ion trap geometry were considered. Data obtained from experiments using a triple quadrupole instrument, CI-ion trap, and ESI-ion trap have been compared. The results so achieved seem to indicate that the presence inside the trap of neutral molecules of the solvent employed for the ESI process have a relevant role, promoting high energy deposition in the colliding ions.

The IgG glycation level of 30 healthy subjects and 60 type 2 diabetic patients with different degrees of metabolic control was evaluated by matrix-assisted laser desorption ionization mass spectrometry, a technique allowing the determination of mass increase of the IgG molecule. When applied to the digested mixture obtained by the action of papain on the plasma protein fraction, the same method established the mass increase of Fab and Fc fragments of IgG; for the former, a higher mass increase was found, possibly explained by its high reactivity to glucose. Experimental results were confirmed by molecular modeling calculations. Results suggest that the immunodeficiency observed in diabetic patients may be due to the inhibition of molecular recognition between antibody and antigen as a result of a change in functionality of the modified Fab fragment of IgG.

A new instrumental arrangement dedicated to the study of ion-molecule reactions and consists of by an electron impact/chemical ionization source mounted 420 mm away from an ion trap is described. It has been designed and developed to exclude diffusion into the ion trap of the neutral reactants from the EI/CI source and so to avoid undesired side-reactions. Its instrumental parameterization is described and some preliminary results are illustrated that show promise for the successful application of the system to the study of ion-molecule reactions.

Pancreatic adenocarcinoma causes diabetes mellitus by releasing factors interfering with glucose metabolism.We verified in isolated rat hepatocytes the molecular weight (MW) of the fraction from pancreatic cancer cell conditioned media (CM) that altered glucose metabolism and ascertained, using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) analysis, whether there is any common peptide in CM and in the sera of patients with pancreatic cancer.Sera was obtained from patients with pancreatic cancer ( n = 14) and chronic pancreatitis ( n = 9) and healthy control subjects ( n = 10). Conditioned medium (CM) was obtained from the following cell lines: MIA PaCa 2, PSK-1, PANC-1, and CAPAN-1. Two fractions (MW of less than 30,000 Da and less than 10,000 Da) were obtained from patients' sera, from CM, and from non-CM (NCM) after two-step ultrafiltration. Rat hepatocytes were incubated with CM and NCM. The peptide profile of patients' sera, CM, and NCM were analyzed using MALDI-MS.In rat hepatocytes, glucose metabolism was impaired by CM from all the pancreatic cancer cell lines and by CM with an MW of less than 10,000 Da. Two peptides (m/z 2030 and 2726) were found in CM and patients' sera. Only the peptide at m/z 2030 was found to be associated with the presence of diabetes.A peptide at m/z 2030 may be a putative pancreatic cancer-associated diabetogenic factor.

The oligomerization of Sn(OBut)4 in the sol-gel process has been studied by several different analytical approaches. Electrospray ionization (ESI) mass spectrometry was highly effective in describing the first species produced by the hydrolysis-polycondensation reactions. Mossbauer spectroscopy has elucidated the fast behavior of the hydrolytic oligomerization process, while calorimetric data have demonstrated the high enthalpy of the reaction in the presence of different solvents. Copyright © 2001 John Wiley & Sons, Ltd.

Fast atom bombardment (FAB), matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) and plasma desorption (PD) mass spectra of newly synthesized polyethylene glycols (PEGs), (Mw 600–4000 Da) chemically modified with biologically active (2-benzothiazolon-3-yl)acetyl end-groups are described (products 1–6). The spectra were also used for the determination of the molecular mass characteristics (number average (Mn) and weight average (Mw) molecular masses) of the initial and modified PEGs. As expected, Mn and Mw of the modified samples are higher than those of the nonmodified samples. However, it is shown that molecular mass dispersity (determined by the comparison of the polydispersity indices (PDI = Mw/Mn) of both types of PEGs) essentially do not change during this modification. The FAB mass spectra, together with molecular species, show the presence of abundant [M + Na]+ ions of product 1 and [M + Na + H]+ species of 2 and 3, and [M + Na + 2H]+ of product 4. Two main series of fragment ions, derived from the cleavage of the ether bonds, are observed. The number fractions of the molecular adduct ions and fragment adduct ions, determined from the FAB and PD mass spectra of the modified PEGs, are compared. The MALDI-TOF mass spectra of compounds 1–6 show the presence of two series of polymers. The most abundant peaks are due to [M + Na]+ and [M + K]+ ions originating from the polymers, in which the two terminal hydroxyl groups of PEGs are esterified with (2-benzothiazolon-3-yl)acetic acid. The less abundant peaks are due to the monosubstituted polymers. Copyright © 2001 John Wiley & Sons, Ltd.

A simple method, based on the use of a vacuum cell, is described for the coupling of an Nd: YAG Q-switched laser with 600 μm fibre optics. By such a device the usually observed damage of the fibre optical surface is completely avoided.

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.

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.

Breast-feeding is the nutritional model in humans, and is continued after birth for variable periods. Milk represents an essential source of growth from both nutritional and functional points of view. When nursing is not possible, indicated or sufficient, artificial feeding becomes essential. Substitutes for mother's milk are usually obtained by modifying the composition of cow's milk either qualitatively or quantitatively. The changes usually involve enzymatic and/or thermal treatment, and for this reason a description of the protein profiles of milk formulae is of great interest. After examination of the results obtained by matrix-assisted laser desorption/ionization (MALDI) in the characterization of protein profiles of cow's milk after different thermal treatments, the application of this analytical technique in the above context appeared interesting. MALDI data for eleven milk formulae, directly acquired from the market, are described and discussed. Results indicate that MALDI mass spectrometry is a particularly powerful method which may be employed either during the production stages of milk formulae or to study the relationships between milk protein content and clinical formula evaluation. Copyright © 1999 John Wiley & Sons, Ltd.

In previous work we demonstrated that gp20, a sialoglycoprotein of human sperm is homologous to the leukocyte antigen CD52 and that anti-gp20 recognizes an antigen of the same molecular weight as that recognized by CAMPATH-1 (anti CD52) in leukocytes and sperm, but with some differences. In this study we used anti-gp20 to perform immunoblot analysis of many different sperm, seminal plasma and leukocyte samples. The sperm and seminal plasma antigens were similar and appeared to consist of two components, whereas the leukocyte antigen is unique. Evidence of the presence of two components of the sperm antigen, running respectively at about 19 and 21 kDa, was obtained by analyzing the purified antigen stained with Coomassie brilliant blue and by immunoblot analysis of the antigen after two-dimensional electrophoresis. Both components had an isoelectric point (pI) between 3 and 6. MALDI analysis of the purified antigen confirmed the presence of two components and indicated masses (Mr) of 8243 and 10908. The possible relationship between these findings and the presence of two forms of the CD52 gene differing at two aminoacids C-terminal to the GPI-anchor site has been discussed.

The monitoring of glycemic control in diabetic subjects has been greatly improved by the development of analytical methods for the measurement of glycohemoglobin concentrations, which reflect average serum glucose concentrations 4–8 weeks preceding the analysis. In particular, because HbA1c is considered one of the most powerful markers in such evaluations (1), optimized conditions for its determination still remain one of the major goals of clinical chemistry, as recently emphasized by Miedema (2). The development of methods without the limitations inherent in chromatographic, electrophoretic, and immunological procedures (3) is certainly of great interest. These methods may be used to measure HbA1c directly and/or to determine the extent of glycation of the “universal calibrator”. Choosing between these two different approaches is necessarily related to the feasibility (and to the price/performance ratio) of each method. In this context, Roberts et al. (4) recently showed the potentialities of electrospray ionization mass spectrometry (ESI/MS) for quantifying glycohemoglobin. ESI mass spectra from the analysis of whole blood samples were used to evaluate the percentage of glycohemoglobin directly, and comparison of the ESI data with those from established affinity chromatographic procedures showed a particularly good linear relationship. These evaluations were based on the four ionic species detected in ESI conditions after baseline subtraction, i.e., glycated and nonglycated α- and β-globins. A different, highly specific approach for unequivocal HbA1c evaluation, which was proposed recently by Kobold et al. (5), is based on enzymatic cleavage of hemoglobin by an endoproteinase. This treatment yielded the β-N-terminal peptides of HbA1c and HbA, and their separation and quantification was performed by ESI/MS and capillary electrophoresis. This procedure may be used as a reference method for HbA1c measurement. In previous investigations (6)(7)(8)(9)(10), we demonstrated that matrix-assisted laser …

Matrix-assisted laser desorption ionization (MALDI) mass spectrometry (MS) has been applied to achieve a qualitative and quantitative evaluation of glycated globins on a wide number of healthy and diabetic subjects. The method allowed us to establish that both alpha- and beta-globins are glycated and that, in addition to simply glycated products, other species are detected. Investigations by different sample treatments and by analysis of the glycated beta-globin fraction obtained by preparative chromatography indicated that these species correspond to glyco-oxidized globins. Consequently MALDI-MS can be validly employed to evaluate not only the glycation level, but also the degree of oxidative stress. The percentages of glycated and glycooxidized species were calculated from the related MALDI spectra by the measurement of the related peak areas, without any other treatment of data. A linear relationship between HbA1c values and the total percentage of glycated and glyco-oxidized globins has been found, and its slope (< 1) has been rationalized by the uncorrected evaluation of glycated globins content in the standard samples employed for HbA1c measurements.

Ewe milk and ewe cheese samples were analysed by matrix-assisted laser desorption/ionization mass spectrometry and their protein profiles were compared with those obtained from bovine milk and bovine cheeses. Various mixtures of bovine and ewe cheeses in different weight ratios were analysed, leading to a reproducible calibration curve, which has been successfully employed in determining the percentage of bovine milk fraudulently added to ewe milk in the production of marketed ewe cheese.

The male reproductive system of the mollusc bivalve Unio elongatulus contains two distinct forms of α-L-fucosidase, one present in the gonad fluid and a second one associated with the sperm plasma membrane. Both activities were purified to homogeneity. The soluble seminal plasma enzyme had an oligomeric MW of 56 kDa as determined by MALDI-TOF mass spectrometry, whereas the enzyme purified from sperm plasma membranes had an MW of 68 kDa. Analyzed by lectin blotting with ConA and PNA, the 68 kDa enzyme did not bind either lectin, whereas the 56 kDa form bound ConA only. Both fucosidases followed a Michaelis-Menten kinetics with the K m of the sperm-bound enzyme being 7.1 × 10 −4 M and that of the seminal enzyme being 9.1 × 10 −4 M. Both had a pH optimum of 5.0.