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In this work we present for the first time the use of ion-exchange liquid chromatography to separate the native form and a partially structured intermediate of the folding of the amyloidogenic protein beta2-microglobulin. Using a strong anion-exchange column that accounts for the differences in charge exposure of the two conformers, a LC–UV method is initially optimised in terms of mobile phase pH, composition and temperature. The preferred mobile phase conditions that afford useful information were found to be 35 mM ammonium formate, pH 7.4 at 25 °C. The dynamic equilibrium of the two species is demonstrated upon increasing the concentration of acetonitrile in the protein sample. Then, the chromatographic method is transferred to MS detection and the respective charge state distributions of the separated conformers are identified. The LC–MS results demonstrate that one of the conformers is partially unfolded, compared with the native and more compact species. The correspondence with previous results obtained in free solution by capillary electrophoresis suggest that strong ion exchange LC–MS does not alter beta2-microglobulin conformation and maintains the dynamic equilibrium already observed between the native protein and its folding intermediate.

Soluble and toxic oligomers of amyloid beta (A beta) protein have been identified as the true neurotoxic species involved in Alzheimer's disease and considering them as targets to inhibit A beta aggregation might have a therapeutic value. We previously set up a CE method that enables the separation and quantification of transient oligomers of A beta protein-containing 42 amino acids (A beta(1-42)) along the pathway leading to fibrils and we now demonstrate how this method can be successfully applied to examine the in vitro inhibitory effects of small molecules on A beta oligomerization. To this end, we investigated mitoxantrone and pixantrone, two well-known anticancer drugs, as well as suramin and a suramin-like compound. By using CE, it is here shown how mitoxantrone and pixantrone either reduce or block A beta(1-42) oligomerization, while Thioflavin T spectrofluorimetric assay and transmission electron microscopy demonstrate how these two compounds also display antifibrillogenic activity. Interestingly, in vitro cell viability experiments indicated that pixantrone significantly reduces A beta(1-42) neurotoxicity.

Human beta2-microglobulin (beta2-m) is a small amyloidogenic protein responsible for dialysis-related amyloidosis, which represents a severe complication of long-term hemodialysis. A therapeutic approach for this amyloidosis could be based on the stabilization of beta2-m through the binding to a small molecule, to possibly inhibit protein misfolding and amyloid fibril formation. The search of a strong ligand of this protein is extremely challenging: by using CE in affinity and refolding experiments we study the effect that previously selected sulfonated molecules have on the equilibrium between the native form and an ensemble of conformers populating the slow phase of beta2-m folding. These data are correlated with the effect that the same molecules exert on in vitro fibrillogenesis experiments.

An essential oil, obtained by steam distillation of Clinopodium tomentosum (Kunth) Govaerts (Lamiaceae), collected in Ecuador, was analyzed by gas chromatography-olfactometry (GC-O) and GC-MS techniques. To our knowledge, the composition of this essential oil is described here for the first time, both from the chemical and olfactometric viewpoints. A preliminary analysis by GC-MS and using Kovats' retention indexes, lead to characterize and quantify the oil constituents, while GC-O was then applied for the identification of the main odorants. By the incremental dilution method (AEDA, CHARM Analysis), applied to the GC-O technique, the flavor dilution (FD) chromatogram was obtained. In order to calculate the TOC values of the main odorants, the relationship between the odorant concentration at the sniffing port and that one in the injected solution was established. This relationship was calculated by comparing the injected amount with the TOC value of a reference compound (limonene), obtained by dynamic dilution olfactometry. A good agreement was found between calculated and measured TOC values of few odorants.

The possibility to monitor, in solution, the steps of beta-amyloid (Abeta) nucleation and therefore to describe this dynamic process by using capillary electrophoresis and under optimized experimental conditions is described. Striking differences in the electrophoretic patterns of Abeta 1-42 and Abeta 1-40 over time are here shown, and different aggregation states are elucidated, which reflect the very diverse oligomerization behavior of two very similar peptides. The isolation of one aggregated species of high molecular weight by ultracentrifugation allowed us to assess its role as toxic oligomer. The perturbation of the existing equilibrium among the identified species by the addition of small molecules can in principle interfere with the aggregation process of the peptides and ultimately prevent the plaque formation in vitro.

The group of diseases caused by abnormalities of the process of protein folding and unfolding is rapidly growing and includes diseases caused by loss of function as well as diseases caused by gain of function of misfolded proteins. Amyloidoses are caused by gain of function of certain proteins that lose their native structure and self-assemble into toxic insoluble, extracellular fibrils. This process requires the contribution of multiple factors of which only a few are established, namely the conformational modification of the amyloidogenic protein, protein's post-translational modifications and the co-deposition of glycosaminoglicans and of serum amyloid P component. In parallel with the exponential growth of biochemical data regarding the key events of the fibrillogenic process, several reports have shown that small molecules, through the interaction with either the amyloidogenic proteins or with the common constituents, can modify the kinetics of formation of amyloid fibrils or can facilitate amyloid reabsorption. These small molecules can be classified on the basis of their protein target and mechanism of action, according to the following properties. 1) molecules that stabilize the amyloidogenic protein precursor 2) molecules that prevent fibrillogenesis by acting on partially folded intermediates of the folding process as well as on low molecular weight oligomers populating the initial phase of fibril formation 3) molecules that interact with mature amyloid fibrils and weaken their structural stability 4) molecules that displace fundamental co-factors of the amyloid deposits like glycosaminoglycans and serum amyloid P component and favor the dissolution of the fibrillar aggregate.

Dialysis-related amyloidosis is a disease in which partial unfolding of beta(2)-microglobulin plays a key pathogenetic role in the formation of the amyloid fibrils. We have recently demonstrated that a partially unfolded conformer of beta(2)-microglobulin is involved in fibrillogenesis and that this species is significantly populated under physiological conditions. In this work capillary electrophoresis has been used to measure the equilibrium between the native protein and this conformer in samples known to have a higher or lower amyloidogenic potential, namely full-length beta(2)-microglobulin, two truncated species and a mutant, created by replacing histidine in position 31 with thyrosine. In addition, for all protein species folding stability experiments have been carried out by monitoring the secondary structure by circular dichroism at increasing concentrations of guanidinium chloride. The values of free energy of unfolding in the absence of denaturant, obtained by elaboration of these experiments, were found to be inversely correlated to the area percent of the partially unfolded conformer, as measured by capillary electrophoresis. Affinity capillary electrophoresis experiments have been also carried out under nondenaturing conditions to assess the affinity of copper and suramin to either the native form or the conformational intermediate of full-length beta(2)-microglobulin.

In this work we used affinity capillary electrophoresis (ACE) to investigate the extent of interaction between a pool of drugs and wild-type transthyretin. After qualitative preliminary screening, attention was focused on the most promising molecules, flufenamic acid and flurbiprofen, which underwent a further stage of investigation, the determination of the binding constants, and, when possible, the assessment of the number of binding sites by ACE, frontal analysis (FA) capillary electrophoresis (CE) and parallel ultrafiltration (UF) experiments. Furthermore, our data demonstrate that FA CE is a suitable technique for identifying fibril ligands. This represents a novel CE application of pharmaceutical interest.

An ion-pair HPLC method is presented for the determination in plasma of 2,4- and 2,6-toluendiamine (TDA), known carcinogens. The chromatographic conditions consisted in isocratic elution on a reversed phase C18 column with 5 mM octanesulfonic acid in methanol-water, 45∶55, as mobile phase. UV detection was performed at 235 nm. Samples were analyzed after a simple single step liquid-liquid extraction and the method was validated by measurement of precision (interassay and intraassay), sensitivity, specificity, linearity, and recovery. The detection limit for both TDAs in plasma was 20 ng mL−1. The correlation coefficients based on the intrassay calibration curve were 0.998 and 0.997 for 2,6- and 2,4-TDA, respectively. The intraassay accuracy, expressed in terms of recovery, was found to be up to 89.91% and 97.05% for 2,6- and 2,4-TDA, respectively.

A new chiral stationary phase for high-performance liquid chromatography of quail egg white riboflavin binding protein is presented. Several chiral acidic, basic and uncharged drugs were analysed and the influence of the mobile phase's parameters on the retention times and enantioselectivity was evaluated. On the basis of the results obtained, the same protein was studied as a background electrolyte additive in free solution capillary electrophoresis, in order to evaluate if capillary electrophoresis (CE) could be used as a rapid scouting technique for screening the enantioselectivity of novel proteins without immobilisation on a solid support. To investigate if it is possible to directly compare the results obtained by each technique, the CE experiments were planned on the basis of both the findings and ideas originated in liquid chromatography.

A reversed-phase HPLC method has been developed for the urinary determination of mutagenic 2,4- and 2,6-toluendiamines. These amines are degradation products of polyurethane, a material used to cover textured silicone breast implants. FMOC-Cl was used as fluorescent derivatising agent in order to obtain a limit of detection of 15 ng/ml in urine. Pre-treatment of urine samples was by liquid/liquid extraction and urine specimens of patients after surgury were analysed.

A chiral stationary phase for high-performance liquid chromatography based on hen egg yolk riboflavin binding protein is introduced. The purified protein was immobilized on activated 5NH2 Nucleosil silica. Chiral acidic, basic and uncharged drugs were chromatographed and the influence of the mobile phase parameters on the retention times and enantioselectivity was studied. Thirteen out of the twenty compounds tested were partially or baseline resolved. These encouraging preliminary results suggest that this chiral stationary phase may be applicable to a wide range of drug enantiomers in the reversed-phase mode.

A high-performance liquid chromatographic method using a μBondapak C 18 column and a UV detector at 230 nm was developed for the simultaneous determination of 5-aminosalicylic acid (5-ASA) and related compounds (4-aminophenol, 3-aminosalicylic acid and 3-aminobenzoic acid). A method for the determination of 5-ASA in pharmaceutical preparations (tablets, granules and rectal suspension) is also described. Calibration graphs for both 5-ASA and related compounds are reported. The standard deviation is ± 0.623 ( n = 10).

The development of an integrated chromatographic system for complete phosphoprotein analysis is described. The digestion of phosphoproteins with trypsin- or pronase-based monolithic bioreactors is carried out on-line with selective enrichment on a TiO 2 trap and separation of the produced phosphopeptides by reversed-phase liquid chromatography-multiple mass spectrometry (RPLC/MS n ). A detailed study on the selective extraction of peptides with different degrees of phosphorylation on TiO 2 cartridges is discussed. This analytical strategy has been optimized using β-casein as a standard phosphoprotein, and then applied to the identification of phosphorylation sites in insulin-like grow factor-binding protein 1 (IGFBP-1) isolated from amniotic fluid.

The applicability of a trypsin-based monolithic bioreactor coupled on-line with LC/MS/MS for rapid proteolytic digestion and protein identification is here described. Dilute samples are passed through the bioreactor for generation of proteolytic fragments in less than 10 min. After digestion and peptide separation, electrospray ionization tandem mass spectrometry is used to generate a peptide map and to identify proteolytic peptides by correlating their fragmentation spectra with amino acid sequences from a protein database. By digesting picomoles of proteins sufficient data from ESI and MS/MS were obtained to unambiguously identify proteins alone and in serum samples. This approach was also extended to locate mutation sites in beta-lactoglobulin A and B variants. Keywords: immobilized trypsin • in-column digestion • LC−MS/MS • variant identification

In this work, a new type of penicillin G acylase (PGA)-based monolithic silica support was developed and evaluated for the chiral separation in HPLC. The preparation procedure consisted of two steps: preparation of an epoxy derivatized monolithic silica column and chemical modification of the epoxide groups with the enzyme chiral selector. The epoxy Silica-Rod column for the immobilization of PGA was prepared with the in situ modification process by using epoxy-silanes and the identification of the species bound to the surface was achieved by solid-state nuclear magnetic resonance. The enzyme was covalently immobilized to the surface of the derivatized monolithic column. The enantioselectivity and the performance of the developed column are discussed and compared to the corresponding experimental data obtained with a PGA-based microparticulate (5 μm) silica column.

The preparation and characterization of a new trypsin-based bioreactor is here described for on-line protein digestion and peptide analysis. Trypsin was immobilized on an epoxy-modified silica monolithic support with a single reaction step and the amount of immobilized enzyme was found to be 66.07 mg (+/-11.75 S.D.)/column (n = 6). The bioreactor was coupled through a switching valve to an analytical column for the on-line digestion, peptide separation and identification of test proteins by ESI-MS-MS. The influence of various parameters (flow rate, temperature, buffer pH and molarity, etc.) on enzymatic activity was investigated by an experimental design and the mostly significant factor was found to be the flow rate. The efficacy of the reported on-line bioreactor for tryptic mapping is reported for somatostatin and myoglobin, selected as model compounds. Tryptic peptide maps obtained by on-line digestion of myoglobin were compared to those obtained by traditional off-line digestion. Sequence coverage obtained with the on-line protocol (21 peptides, 75.16% coverage of myoglobin sequence) was found to be comparable to the one obtained with the off-line protocol (18 peptides, 76.47% coverage). Sensitivity for myoglobin digestion and identification was 0.1 mg/ml. The reproducibily of the peptide maps in terms of retention time was from 1.53 to 4.31%, R.S.D.

Molecularly imprinted composite materials were evaluated as chiral stationary phases in capillary electrochromatography (CEC). These consisted of spherical silica particles of different sizes and of different porosities, containing a surface-immobilized layer of molecularly imprinted polymer (MIP) targeted to bind L-phenylalanine anilide. Fused silica capillaries were packed over a length of 8.5 cm, using a pneumate amplification pump, and the stationary phase thus obtained was tested with respect to its electrochromatographic performance. The electroendosmotic flow (EOF) mobility was evaluated with respect to the content of grafted polymer, as well as the ionic strength and the acetonitrile content of the electrolyte. Moreover, the influence of the layer thickness and of the stationary phase porosity on the performance and on the sample load capacity was investigated. The packings exhibited different relative efficiencies for the two enantiomers. The results were discussed in terms of differencies in accessibility to the binding sites of the packings and of the mechanism of EOF generation. In the wide context of the different approaches so far proposed for MIP stationary phases in CEC, these materials can be a good alternative, worthy of further development and application, not restricted to chiral separations.

The chiral recognition properties of a new chiral stationary phase based on immobilized penicillin G acylase were investigated using 35 acidic racemates. Twenty-seven compounds were resolved with high separation factors. The influences of mobile phase pH, type of organic modifier and ionic strength on enantioselective retention were studied. The most important tool for affecting the enantioselectivity was the mobile phase pH and interestingly the retention order of the enantiomers of some analytes could be controlled by this parameter. The analysis time for resolving enantiomers could be adjusted with a minor decrease in enantioselectivity using a high ionic strength mobile phase buffer while both retention and enantioselectivity decreased by adding organic modifier to the mobile phase. Displacement studies have demonstrated that the enzymatically active site and the chiral adsorption site overlap.

A preliminary evaluation of the enantioselective properties of quail egg yolk riboflavin binding protein (qRfBP) was carried out in capillary electrophoresis by using the complete filling technique. The most promising results obtained by this screening of nineteen chiral drugs were singled out with the aim of optimizing enantiomer separations by applying the partial filling technique, which allows operating at much higher protein concentrations without detection problems. The building of the separation zone in the partial filling technique has been modified in order to enable on-line monitoring, before each run, of the actual protein plug application velocity and, consequently, the building of a plug of the desired length. The electrophoretic conditions chosen gave opposite migration directions for the chiral selector and the analytes, with qRfBP migrating away from the detector. A polyvinyl alcohol-coated capillary was first totally filled with protein and the optimal plug length was obtained by further applying negative pressure together with positive voltage for the time needed. Separations of basic drugs were optimized by using protein concentrations ranging from 200 microM up to 900 microM and different plug lengths, while the running buffer pH (6.0), temperature (25 degrees C) and operating voltage (+20 kV) were kept constant. The enantioresolution of all solutes was affected by both the chiral selector concentration and protein plug length. Baseline separations were obtained for oxprenolol, prilocaine and bupivacaine.

The application of frontal affinity chromatography-mass spectrometry (FAC-MS), along with molecular modeling studies, to the screening of potential drug candidates toward the recently deorphanized G-protein-coupled receptor (GPCR) GPR17 is shown. GPR17 is dually activated by uracil nucleotides and cysteinyl-leukotrienes, and is expressed in organs typically undergoing ischemic damage (i.e., brain, heart and kidney), thus representing a new pharmacological target for acute and chronic neurodegeneration. GPR17 was entrapped on an immobilized artificial membrane (IAM), and this stationary phase was used to screen a library of nucleotide derivatives by FAC-MS to select high affinity ligands. The chromatographic results have been validated with a reference functional assay ([(35)S]GTPgammaS binding assay). The receptor nucleotide-binding site was studied by setting up a column where a mutated GPR17 receptor (Arg255Ile) has been immobilized. The chromatographic behavior of the tested nucleotide derivatives together with in silico studies have been used to gain insights into the structure requirement of GPR17 ligands.