Protein alkylation in the presence/absence of thiourea in proteome analysis: a matrix assisted laser desorption/ionization-time of flight-mass spectrometry investigation.

Although it is highly recommended that reduction and alkylation of free -SH groups in proteins should be performed prior to any electrophoretic step (including the first isoelectric focusing/immobilized pH gradient (IEF/IPG) dimension), it is here reported that one component of the sample solubilization cocktail adopted recently (namely thiourea) strongly quenches such alkylation process (as typically carried out with iodoacetamide, IAA). The present matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) analysis demonstrates that thiourea is an effective scavenger of IAA, since its sulfur atom reacts as efficiently as the ionized, free -SH group of Cys in proteins at alkaline pH values (pH 8.5-9.0). As a result of this reaction, free IAA is quickly depleted by thiourea, via the formation of an intermediate adduct, which is rapidly deamidated to form the cyclic compound thiazolinidone monoimine. This reaction strongly competes with the direct addition reaction of IAA onto the -SH group in proteins, resulting in poorly alkylated proteins. It is, therefore, recommended that, whenever possible and compatible with the type of sample, thiourea should be omitted from the solubilizing cocktail in proteome analysis. However, after proper sample reduction and alkylation, thiourea can be incorporated into the IEF/IPG gel, where it will have the beneficial effect of augmenting protein solubility at their pI values and scavenging the excess of free IAA.

Coupling of a supercritical fluid chromatography system to a hybrid (Q-TOF 2) mass spectrometer: on-line accurate mass measurements.

This note describes a simple and economical method to couple a supercritical fluid chromatography (SFC) system (Berger Instruments, US) with a high-resolution hybrid mass spectrometer (Q-TOF 2; Micromass, UK). This experimental arrangement has three distinct advantages: (1) coupling between the two systems can be effected without the need for an interface or hardware modifications of either system, (ii) this experimental arrangement provides on-line accurate mass SFC/MS measurements which are indispensable for the characterisation of new chemical entities and unknown metabolites, and (iii) the characteristically fast spectral acquisition rate of the time-of-flight (TOF) analyser renders the present arrangement an important contribution to future semipreparative fraction collection setups which use mass spectrometry as a detector.

Substituted analogues of GV150526 as potent glycine binding site antagonists in animal models of cerebral ischemia.

A series of analogues of the indole-2-carboxylate GV150526, currently in clinical trials as a potential neuroprotective agent for the control of the cerebral damage after stroke onset, was designed based on previous studies dealing with the electronic features of the north-east region of the glycine binding site associated with the NMDA receptor. In particular, the substitution of the para position of the terminal phenyl ring of GV150526 with suitable hydrophilic groups resulted in the identification of a new class of glycine antagonists. These compounds exhibited nanomolar in vitro affinity to the glycine binding site, high receptor selectivity, and outstanding in vivo potency. In particular, 3-[(E)-2-[(4-ureidomethylphenyl)aminocarbonyl]ethenyl]-4, 6-dichloroindole-2-carboxylic acid was found to be highly effective in the middle cerebral artery occlusion (MCAo) model in the rat, an animal model of focal ischemia, when given both prior to and after the occlusion of the middle cerebral artery. Notably, a significant neuroprotective effect was seen in this model postischaemia, when the administration of this compound was delayed up to 6 h from the occlusion of the middle cerebral artery, further confirming the wide therapeutic window seen for GV150526A.

Liquid chromatography/tandem mass spectrometry to monitor acrylamide adducts with bovine beta-lactoglobulin B.

Complexation of acrylamide with bovine beta-lactoglobulin B and some of its tryptic fragments have been examined by liquid chromatography coupled to tandem mass spectrometry. Such complexation was investigated both in the presence and in the absence of dithiothreitol as a reducing agent. Under the latter conditions, the intact protein exhibited a single cysteine-acrylamide complex which both the present work and previous studies attribute to Cys160. The involvement of this particular residue is tentatively attributed to an intramolecular disulphide exchange which results in its disengagement from the S-S bridge to offer a free SH group for reaction with the acrylamide monomer. In the absence of dithiothreitol, both free and complexed cysteine-containing tryptic fragments were present, while in its presence, one of the tryptic fragments, which contains three cysteine residues was fully absent, instead a part of this fragment containing two cysteines complexed with two acrylamide monomers was observed. The absence of any analytical information in the literature regarding the latter complexes underlines the potential of liquid chromatography coupled to mass spectrometry in the characterization of this commonly occurring modification.

A new matrix in esthetic posterior restorations.

Using composite resins in posterior esthetic restorations has become more acceptable in recent years as a result of improvements in materials. For this reason, innovative products such as Luciwedge and the SuperMat System have gained the attention of many practitioners. SuperMat, an improved translucent matrix, offers a new approach to matrices for use with multimaterial fillings. It makes the procedure easier and offers many other advantages, particularly in composite-compomer restorations.

On-line liquid chromatography/electrospray tandem mass spectrometry to investigate acrylamide adducts with cysteine residues: implications for polyacrylamide gel electrophoresis separations of proteins.

Adduction between acrylamide and cysteine residues is a post-translational modification associated with proteins separated by gel electrophoresis. In the present article, three model peptides containing 2-4 cysteine residues were reduced with dithiothreitol, incubated with acrylamide monomers and examined by on-line liquid chromatography coupled to electrospray tandem mass spectrometry. Each of the solutions examined in this work revealed the presence of four distinct components: the free peptide, two different peptide-acrylamide 1:1 adducts involving two cysteine residues at different positions within the same sequence, and the peptide-acrylamide 1:2 adducts. The use of liquid chromatography allowed the separation of components which differed only by the site of complexation of acrylamide, while the application of tandem mass spectrometry furnished reliable sequencing information permitting the identification of most cysteine residues involved in such complexation.

Crudes of synthesis of neuropeptide Y and beta-amyloid peptide examined by liquid chromatography/electrospray tandem mass spectrometry.

The two peptides, neuropeptide Y (M(r) = 4254) and beta (1-39) amyloid (M(r) = 4230) are attracting pharmaceutical and biomedical interest for different yet equally important reasons. The first is recognized for its various physiological functions in the peripheral and central nervous system, while the second has been identified as one of the components of the cerebral amyloid deposits characteristic of Alzheimer's disease. High level purity of both peptides is considered of prime importance for correct interpretation of data obtained by biological and pharmaceutical assays and binding tests. Solid-phase synthesis of both peptides resulted in crudes of reaction containing both the target peptides as well as a number of undesired side products. The use of liquid chromatography/electrospray-tandem mass spectrometry (LC/ESI-MS/MS) furnished reliable information on the target peptides and provided sequencing information on a number of side products. Furthermore, LC/MS/MS data of doubly and triply protonated sequences yielded a number of C-terminal fragment ions exhibiting the loss of NH3 considered to be an important process for the understanding of fragmentation mechanism(s) of multiply protonated peptides.

Six years of in vitro/in vivo experience with Composipost.

Some years ago, we created the following classifications for endodontic posts: (1) first generation posts (self-threaded posts, screw posts, serrated-carved posts); (2) second generation posts (passive posts); (3) third generation posts (nonmetallic passive posts). In this last group, we the placed carbon-fiber posts, which have a modulus of elasticity very similar to the modulus of elasticity of dentine and can realize a tooth-post-core monobloc instead of an assemblage of heterogeneous materials. This is quite a new philosophy in rebuilding endodontically treated teeth and is based on the use of integratable materials that homogeneously distribute masticatory loads and reduce stress.

Investigation of beta-amyloid peptide by on-line high-performance liquid chromatography coupled to electrospray ionization mass spectrometry.

beta(1-39) amyloid peptide is one of the components of the cerebral amyloid deposits that are characteristic of Alzheimer's disease. Solid-phase synthesis of this peptide resulted in a fairly complex crude product containing both the target peptide and a number of side products. High-performance liquid chromatography coupled to electrospray ionization mass spectrometry allowed rapid and reliable identification of both the desired peptide and most of the side products which were found to have relative molecular masses above and below that of the target peptide.

[New research on endodontic carbon-fiber posts]. / Nuove ricerche sui perni endocanalari in fibra di carbonio.

The authors present their clinical experience with fibre-carbon posts, which has been carried on for more than 4 years, supporting it with studies made in European and U.S.A. Universities, specially regarding the mechanical resistance and retention of those posts. Tests show luting procedures as the critical and improvable point. No real retention increase results using Sealbond and Flexi-Flow composite cements; failure always occurs at cement-post junction. Using a special fibre-carbon post, named Composipost Retentive, which presents circumferential grooves, very superior retention is obtained. Using 5 types of composite cement (Sticky Post, Flexi-Flow, Sealbond, Super Bond and All Bond II), we reach a very high retention. Super Bond and All Bond II specially show a tensile strength over 120 kg measured with Instrom machine. Those two composite cements massively penetrate in dentinal tubules, giving best results in cement-root channel interface.