Sample preparation of human serum for the analysis of tumor markers. Comparison of different approaches for albumin and gamma-globulin depletion

LC-MS is a powerful method for the sensitive detection of proteins and peptides in biological fluids. However, the presence of highly abundant proteins often masks those of lower abundance and thus generally prevents their detection and identification in proteomic studies. In human serum the most abundant proteins are albumin and gamma-globulins. We tested several approaches to specifically reduce the level of these proteins based on either specific antibodies, dye ligands (for albumin) and protein A or G (for gamma-globulins). The resulting, depleted serum was analyzed by sodium dodecylsulfate-polyacrylamide gel electrophoresis and LC-MS for the residual presence of these abundant proteins as well as for other serum proteins that should remain after depletion. To test the applicability of this method to real-life samples, depleted serum of a cervical cancer patient was analyzed for the presence of a specific tumor marker protein SCCA1 (squamous cell carcinoma antigen 1; P29508), which is present at ng/ml concentrations. The results demonstrate that SCCA1 can be detected by LC-MS in patient serum following depletion of albumin and gamma-globulins thus opening the possibility of screening patient sera for other, so far unknown, tumor markers.     

Femtosecond Spectroscopic Studies on the Red Light-Absorbing Form of Oat Phytochrome and 2,3-Dihydrobiliverdin

Abstract— The excited state behavior of the red light-absorbing form of phytochrome (Pr) was studied on the femtosecond time scale. After excitation of Pr with 75 fs laser pulses at 616 nm the kinetics of the transient absorption changes was recorded at selected wavelengths probing mainly the bleaching of the Pr ground-state absorption and the stimulated emission. The kinetic data obtained indicate the population of an excited state with a 3 ps lifetime immediately after excitation. This state precedes the formation of another excited state with a 32 ps lifetime. The decay of the latter state is followed by the appearance of a first product state that is assumed to represent lunii-R. In addition, 2,3-dihydrobiliverdin, which is considered to be an adequate model of the Pr chro-mophore, was included in the femtosecond studies. The absorption difference spectra recorded at various delay times show an immediate bleaching of the ground-state absorption. Simultaneously with bleaching a broad transient absorption appears between 410 and 525 nm. The data analysis yields similar kinetic components as they were observed in the decay of Pr. It is suggested from this finding that within the first tens of picoseconds after excitation the excited-state properties of Pr are mainly determined by the properties of the chromophore itself.     

Mild addition of carbon nucleophiles to pyridine and quinoline N-oxides under different activation conditions

In the course of our syntheses of functionalised pyridine and quinoline derivatives, we examined the reactivities of pyridine and quinoline N-oxides towards the nucleophilic addition of acidic carbon derivatives. Different activating reagents were used, such as PyBroP, triflic anhydride and a combination of pyrrolidine phosphoramide and triflic anhydride.     

Enantioselective Synthesis of a Tricyclic,sp3-Rich Diazatetradecanedione: an Amino Acid-Based Natural Product-Like Scaffold

6-, 7-, and 8-membered rings are assembled from a linear precursor by successive cyclisation reactions to construct a tricyclic diazatricyclo[6.5.1.0^{4, 9}]-tetradecanedione scaffold. Advanced building blocks based on d -aspartic acid and l -pyroglutamic acid were combined by a sp³−sp² Negishi coupling. A carbamate-guided syn-diastereoselective epoxidation followed by an intramolecular epoxide opening allowed the construction of the piperidine ring. An efficient one-pot hydroxyl-group protection twofold deprotection reaction prepared the ground for the cyclisation to the bicycle. A final deprotection of the orthogonal protecting groups and lactamisation led to the novel, sp³-rich tricycle. The final compound is a substrate mimic of peptidyl-prolyl cis-trans isomerases featuring a locked trans-amide bond. Cheminformatic analysis of 179 virtual derivatives indicates favourable physicochemical properties and drug-like characteristics. As proof of concept we, show a low micromolar activity in a fluorescence polarisation assay towards the FK506-binding protein 12.     

Mechanism of formation of cyclic species in cationic ring-opening polymerization of cyclodimethylsiloxanes

A general feature of the cationic polymerization of all cyclodimethylsiloxanes is the formation of various cyclic products (cyclics) together with that of a linear high polymer. However, the types of cyclics as well as their rate of formation may vary considerably according to the number x of D units ((CH₃)₂SiO units) in the monomer. The case of initiation by trifluoromethanesulfonic (triflic) acid in methylene chloride solution at 20°C has been particularly studied. With D₄, D₅, D₆ and D₇, for which the polymerization rate increases with the size of the ring, all types of cycles D_x are formed in relative amount decreasing with their size ([D₇] < [D₆] < [D₅] < [D₄]). The high polymer final concentration and molecular weight are independent from triflic acid concentration. This may result from a polymerization-depolymerization reaction, involving all the cyclics formed by back-biting reactions occurring with silyl triflates activated by the acid, and leading finally to an equilibrium. The situation with D₃ is completely different. The high polymer (HP) and the cyclics (D_3x multiples of D₃ like D₆, D₉, …) are formed simultaneously under kinetic control. The yields of the various cyclics (formed in amount often larger than that of the HP) are proportional to that of the linear HP. The latter is formed from the beginning of the reaction with a molecular weight proportional to HP yield and inversely proportional to the acid concentration. The opposite role of added water on the polymerization is discussed: an activating effect for D₃, and a desactivating one for D₄, D₅ and D₆. “Copolymerization” experiments between D₃ (or D₄) and tetramethyldisiloxane diol confirmed the effect of water and gave new informations about the occurrence - or absence - of condensation reactions in the mechanism of the growth of the polymer chains. A discussion leads to the conclusion that while the cationic polymerization of D₄ by triflic acid is propagated by activated triflic esters, that of D₃ may also involve the monomer activated by the higher hydrates of the acid and linear oligomeric silanol esters. The latter, formed continuously, may also give the D_3x cyclics by intramolecular heterocondensation.     

From holes to sponge at irradiated Ge surfaces with increasing ion energy—an effect of defect kinetics?

We show that hole patterns and sponge-like layers at irradiated Ge surfaces originate from the same driving force, namely the kinetics of ion beam induced defects in the amorphous Ge surface layer. Ge hole patterns reported earlier for irradiation with low energy (5 keV) Ga⁺ ions were reproduced for low energy Bi⁺ but also for Ge⁺ self-irradiation, which proves that the dominating driving force for morphology evolution cannot originate from the implanted impurities. At higher ion energies the well-known formation of sponge-like Ge surface layers after heavy ion irradiation was found for Bi⁺ irradiation and Ge⁺ self-irradiation, also. The transition from smooth surfaces via hole patterns to sponge-like morphologies with increasing ion energies was studied in detail. A model based on the kinetics of ion beam induced defects was developed and implemented in 3D kinetic Monte Carlo simulations, which reproduce the transition from hole patterns to sponge-like layers with increasing ion energy.     

An efficient solution method for Weber problems with barriers based on genetic algorithms

In this paper we consider the problem of locating one new facility with respect to a given set of existing facilities in the plane and in the presence of convex polyhedral barriers. It is assumed that a barrier is a region where neither facility location nor travelling are permitted. The resulting non-convex optimization problem can be reduced to a finite series of convex subproblems, which can be solved by the Weiszfeld algorithm in case of the Weber objective function and Euclidean distances. A solution method is presented that, by iteratively executing a genetic algorithm for the selection of subproblems, quickly finds a solution of the global problem. Visibility arguments are used to reduce the number of subproblems that need to be considered, and numerical examples are presented.