Identification by mass spectrometry of two-dimensional gel electrophoresis-separated proteins extracted from lager brewing yeast.

As two-dimensional (2-D) electrophoresis allows the separation of several hundred proteins in a single gel, this technique has become an important tool for proteome studies and for investigating the cellular physiology. In order to take advantage of information provided by the comparison of proteome pictures, the mass spectrometry technique is the way chosen for a rapid and an accurate identification of proteins of interest. Unfortunately, in the case of industrial yeasts, due to the high level of complexity of their genome, the whole DNA sequence is not yet available and all encoded protein sequences are still unknown. Nevertheless, this study presents here 30 lager brewing yeast proteins newly identified with matrix assisted laser desorption/ionization-time of flight (MALDI-TOF), tandem mass spectrometry (MS/MS) and database searching against the protein sequences of Saccharomyces cerevisiae. The identified proteins of the industrial strain correspond to proteins which do not comigrate with known proteins of S. cerevisiae separated on 2-D gels. This study presents an application of the MS technique for the identification of industrial yeast proteins which are only homologous to the corresponding S. cerevisiae proteins.

Synthesis, mechanism of action, and antiviral activity of a new series of covalent mechanism-based inhibitors of S-adenosyl-L-homocysteine hydrolase.

A direct method for the preparation of 5'-S-alkynyl-5'-thioadenosine and 5'-S-allenyl-5'-thioadenosine has been developed. Treatment of a protected 5'-acetylthio-5'-deoxyadenosine with sodium methoxide and propargyl bromide followed by deprotection gave the 5'-S-propargyl-5'-thioadenosine 4. Under controlled base-catalysis with sodium tert-butoxide in tert-butyl alcohol 4 was quantitatively converted into 5'-S-allenyl-5'-thioadenosine 5 or 5'-S-propynyl-5'-thioadenosine 6. Incubation of recombinant human placental AdoHcy hydrolase with 4, 5, or 6 resulted in time- and concentration-dependent inactivation of the enzyme (K(i): 45 +/- 0.5, 16 +/- 1, and 15 +/- 1 microM, respectively). Compound 4 caused complete conversion of the enzyme from its E-NAD(+) to E-NADH form during the inactivation process. This indicates that 4 is a substrate for the 3'-oxidative activity of AdoHcy hydrolase (type I inhibitor). In contrast, the NAD(+)/NADH content of the enzyme was not affected during the inactivation process with 5 and 6, and their mechanism of inactivation was further investigated. Addition of enzyme-sequestered water on the S-allenylthio group of 5 or S-propynylthio group of 6 within the active site should lead to the formation of the corresponding thioester 7. This acylating-intermediate agent could then undergo nucleophilic attack by a protein residue, leading to a type II mechanism-based inactivation. ElectroSpray mass spectra analysis of the inactivated protein by 5 supports this mechanistic proposal. Further studies (MALDI-TOF and ESI/MS(n) experiments) of the trypsin and endo-Lys-C proteolytic cleavage of the fragments of inactivated AdoHcy hydrolase by 5 were carried out for localization of the labeling. The antiviral activity of 4, 5, and 6 against a large variety of viruses was determined. Significant activity (EC(50): 1.9 microM) was noted with 5 against vaccinia virus.

Determination by electrospray mass spectrometry of the outersphere association constants of DNA/platinum complexes using 20-mer oligonucleotides and ([Pt(NH(3))(4)](2+), 2Cl(-)) or ([Pt(py)(4)](2+), 2Cl(-)).

The cytotoxic effects of cisplatin, cis-diamminedichloroplatinum(II), are generally ascribed to the formation of DNA adducts. Several in vitro as well as in vivo studies showed that cisplatin binds preferentially to guanines belonging to (G)(n) sequences (n > or = 2). After mono- or diaquation of cisplatin, giving the cationic complexes cis-[PtCl(NH(3))(2)(H(2)O)](+) and cis-[Pt(NH(3))(2)(H(2)O)(2)](2+), DNA platination occurs in two steps: the cationic complex gives an outersphere association with DNA and the actual coordination then occurs by substitution of one aqua ligand by guanine-N7. For a better understanding of the (G)(n) selectivity of cisplatin giving the biologically active adducts, also necessary for the conception of new platinum drugs, the respective contribution of the outersphere association and actual guanine platination must be investigated. To check the role of outersphere association in the overall platination process, we used electrospray mass spectrometry (ESMS) to detect and quantify outersphere association between 20-mer oligonucleotides and platinum complexes. The 20-mer oligonucleotides were single- or double-stranded, with the same number of guanines either isolated or adjacent to each other. To deal only with outersphere association and check the influence of platinum ligands, the [Pt(NH(3))(4)](2+) and [Pt(py)(4)](2+) complexes were used. We characterized by ESMS all the different outersphere association species formed during titration of each oligonucleotide with the various platinum complexes and evaluated their affinity constants. The ESMS results demonstrate that the outersphere association depends on electrostatic interactions and on the ability of the platinum ligands to participate to hydrogen bonding, particularly within the duplex form.

Hb Ernz [beta123(H1)Thr-->Asn] and Hb Renert [beta133(H11)Val-->Ala]: two new neutral variants revealed by reversed phase high performance liquid chromatography analysis.

Analysis of globin chains by reversed phase high performance liquid chromatography, used as an additional tool for characterizing hemoglobin variants, has led to the discovery of a new class of variants that display only differences in hydrophobicity. Two such variants are here described. Hb Ernz was found in a man of Italian origin who was polycythemic, and in two of his three daughters who were hematologically normal. Hb Renert, a slightly unstable variant, was found in a man from Cape Verde who also carried Hb S and presented with chronic hemolysis. The structural abnormalities were characterized by protein structure methods involving reversed phase high performance liquid chromatographic separations of globins and peptides, followed by mass spectrometry studies (electrospray, ion trap, tandem mass spectrometry).