Fullerene derivatives incorporating phosphoramidous ylide and phosphoramidate: synthesis and property.

The reaction of dimethyl acetylenedicarboxylate (DMAD) with C(60) in the presence of hexamethylphosphorous triamide (HMPT) or hexaethylphosphorus triamide (HEPT) results in fullerene derivatives incorporating HMPT or HEPT ylides. The ylide derivatives exhibit unusual electronic absorptions in the visible region (435-660 nm), likely due to the presence of the ylide moiety. Electrochemical studies revealed that the first reductive potential of these compounds was more negative relative to those of both C(60) (DeltaE = 130 mV) and a simple Bingel adduct (DeltaE = 90 mV). A phosphoramidate side product, which resulted from the addition of HMPT or HEPT to C(60) followed by hydrolysis, exhibited a featureless absorption spectrum in the visible region and a more negative first reductive potential (DeltaE = 70 mV) relative to that of C(60).

Nitrilotriacetic acid-coated magnetic nanoparticles as affinity probes for enrichment of histidine-tagged proteins and phosphorylated peptides.

We herein demonstrate superparamagnetic Fe3O4 nanoparticles coated with nitrilotriacetic acid derivative (NTA) that can bind with different immobilized metal ions are capable of probing diverse target species. Immobilized Ni(II) on the surfaces of the NTA-magnetic nanoparticles have the capability of selectively trapping histidine (His)-tagged proteins such as a mutated streptopain tagged with 6x His, i.e., C192S (MW approximately 42 kDa), from cell lysates. Enrichment was achieved by vigorously mixing the sample solution and the nanoparticles by pipetting in and out of a sample vial for only 30 s. After enrichment, the probe-target species could be readily isolated by magnetic separation. We also characterized the proteins enriched on the affinity probes using on-probe tryptic digestion under microwave irradiation for only 2 min, followed by matrix-assisted laser desorption/ionization mass spectrometry analysis. Using this enrichment and tryptic digestion, the target species can be rapidly enriched and characterized, reducing the time required for carrying out the complete analysis to less than 10 min. Furthermore, when either Zr(IV) or Ga (III) ions are immobilized on the surfaces of the NTA-magnetic nanoparticles, the nanoparticles have the capability of selectively enriching phosphorylated peptides from tryptic digests of alpha-, beta-caseins, and diluted milk. The detection limit for the tryptic digests of alpha- and beta-caseins is approximately 50 fmol.

Heavy chain of cytoplasmic dynein is a major component of the postsynaptic density fraction.

A protein with an apparent molecular size of 490 kDa was found in the postsynaptic density (PSD) fraction isolated from porcine cerebral cortices and rat forebrains, and this 490 kDa protein accounted for approximately 3% of the total protein of these samples. Matrix-assisted laser desorption ionization-time of flight mass spectrometric and Western blotting analyses consistently indicated that this 490 kDa protein consisted primarily of the heavy chain of cytoplasmic dynein (cDHC). Immunocytochemical analyses showed that cDHC was found in 92% and 89% of the phalloidin-positive protrusions that were themselves associated with discrete clusters of synaptophysin, a presynaptic terminal marker, and PSD-95, a postsynaptic marker, on neuronal processes, respectively. Quantitative Western blotting analyses of various subcellular fractions isolated from porcine cerebral cortices and rat forebrains further showed that not only the heavy but also the intermediate chains of dynein are enriched in the PSD fraction. Cytoplasmic dynein is a microtubule-associated motor protein complex that drives the movement of various cargos toward the minus ends of microtubules and plays many other diverse functions in the cell. Our results that cDHC is a major component of the PSD fraction, that both dynein heavy and intermediate chains are enriched in the PSD fraction and that cDHC is present in dendritic spines raise the possibilities that cytoplasmic dynein may play structural and functional roles in the postsynaptic terminal.

Affinity capture using vancomycin-bound magnetic nanoparticles for the MALDI-MS analysis of bacteria.

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) provides a straightforward means to differentiate microorganism species based on mass spectral fingerprinting. The pathogen cell concentration in an infected sample, however, is generally lower than that capable of being detected directly by MALDI-MS. Furthermore, the presence of proteins or metabolites in biological fluids always causes unavoidable interference for the identification of microorganism species. Vancomycin, which binds to D-Ala-D-Ala moieties on the cell walls of Gram-positive bacteria and, therefore, inhibits peptidoglycan synthesis, is one of the most potent antibiotics. Thus, we have employed vancomycin-modified magnetic nanoparticles as affinity probes to selectively trap Gram-positive pathogens from sample solutions; i.e., these bacteria can be isolated from sample solutions by applying a magnetic field. The isolated cells could then be characterized by MALDI-MS. This approach effectively reduces the interference of protein and metabolite signals in the mass spectra of Gram-positive bacteria because vancomycin has such high specificity for the D-Ala-D-Ala units of the cell walls. The lowest cell concentration we detected for both Staphylococcus saprophyticus and Staphylococcus aureus in a urine sample (3 mL) was approximately 7 x 10(4) cfu/mL.

Using biofunctionalized nanoparticles to probe pathogenic bacteria.

In this paper, we report a method for fabricating biofunctionalized nanoparticles by attaching human immunoglobulin (IgG) onto their surfaces through either electrostatic interactions or covalent binding. We found that these IgG-presenting nanoparticles can bind selectively to the cell walls of pathogens that contain IgG-binding sites based on the investigation of transmission electron microscopy images. Our results demonstrate that such Au-IgG nanoparticles may serve as useful nanoscale probes for exploring the interactions between IgG and pathogens. Furthermore, the IgG-presenting magnetic nanoparticles have been employed as effective affinity probes for selectively concentrating traces of target bacteria from sample solutions. The trapped bacteria were then characterized by using matrix-assisted laser desorption/ionization mass spectrometry. The lowest cell concentration we detected for both Staphylococcus saprophyticus and Staphylococcus aureus in aqueous sample solutions (0.5 mL) was approximately 3 x 10(5) cfu/mL, while the detectable cell concentration for S. saprophyticus in a urine sample was approximately 3 x 10(7) cfu/mL.

Gold nanoparticles as selective and concentrating probes for samples in MALDI MS analysis.

MALDI mass spectrometry is used widely in various fields because it has the characteristics of speed, ease of use, high sensitivity, and wide detectable mass range, but suppression effects between analyte molecules and interference from the sample matrix frequently arise during MALDI analysis. The suppression effects can be avoided if target species are isolated from complicated matrix solutions in advance. Herein, we proposed a novel method for achieving such a goal. We describe a strategy that uses gold nanoparticles to capture charged species from a sample solution. Generally, ionic agents, such as anionic or cationic stabilizers, encapsulate gold nanoparticles to prevent their aggregation in solution. These charged stabilizers at the surface of the gold particles are capable of attracting oppositely charged species from a sample solution through electrostatic interactions. We have employed this concept to develop nanoparticle-based probes that selectively trap and concentrate target species in sample solutions. Additionally, to readily isolate them from solution after attracting their target species, we used gold nanoparticles that are adhered to the surface of magnetic particles through S-Au bonding. A magnet can then be employed to isolate the Au@magnetic particles from the solution. The species trapped by the isolated particles were then characterized by MALDI MS after a simple washing. We demonstrate that Au@magnetic particles having negatively charged surfaces are suitable probes for selectively trapping positively charged proteins from aqueous solutions. In addition, we have employed Au@magnetic particle-based probes successfully to concentrate low amounts of peptide residues from the tryptic digest products of cytochrome c (10(-7) M).

Glass-chip-based sample preparation and on-chip trypic digestion for matrix-assisted laser desorption/ionization mass spectrometric analysis using a sol-gel/2,5-dihydroxybenzoic acid hybrid matrix.

A glass-chip-based sample preparation method for matrix-assisted laser desorption/ionization mass spectrometric (MALDI-MS) analysis of tryptic digests of proteins and intact cells is described. A MALDI matrix, 2,5-dihydroxybenzoic acid (2,5-DHB), was hybridized with sol-gels to generate a sol-gel-derived material. Taking advantage of the characteristics of sol-gels, the sol-gel-derived material readily adhered to the surface of a glass chip through covalent bonding. Only one step of sample preparation, deposition of the sample solution on the glass chip, was required before MALDI-MS analysis. Because 2,5-DHB was homogeneously dispersed on the sol-gel network structure, good spot-to-spot reproducibility was obtained in MALDI analysis using this approach and the analyte signals were uniform throughout the chip. The modified glass chips were robust and effective for at least 1 week. This glass-chip-based matrix preparation method provides a straightforward approach to developing techniques for analyzing the on-chip enzymatic digestion of proteins and intact cells of microorganisms. Cytochrome C and Escherichia coli were used as analytes to demonstrate the feasibility of this approach. The products of the on-chip enzymatic digests were identified through protein database searches.

Laser desorption/ionization time-of-flight mass spectrometry on sol-gel-derived 2,5-dihydroxybenzoic acid film.

This work presents a novel method for direct desorption/ ionization of analytes from sol-gel-derived film. 2,5-Dihydroxy benzoic acid (DHB), a common MALDI matrix, was incorporated into a sol-gel polymeric structure. The sol-gel-derived DHB thin film can assist the mass analysis of analytes by laser desorption/ionization, with a matrix interference-free background in the mass spectra. The sol-gel-derived film can function as an energy absorber during laser irradiation because it contains DHB molecules. Furthermore, laser irradiation with normal laser power (70-110 microJ) is not likely to generate any background ions from this sol-gel-DHB derived film. The samples were prepared straightforwardly. After a thin film was formed on a Parafilm membrane from the sol-gel-derived DHB solution coating, the sample solution was directly added to the top of the film, for laser desorption/ ionization mass analysis. The analyte signals were homogeneously obtained on the sol-gel-derived DHB film. Experimental results show that the optimum concentrations of DHB incorporated in the sol-gel solution were between 7,500 ppm and 10,000 ppm, providing a matrix interference-free background. Analytes, including small proteins, peptides, amino acids, and small organics, were used to demonstrate the effectiveness of the proposed method. However, a higher laser power (> 110 microJ) than normal was required to desorb small proteins from the sol-gel-derived DHB film. Therefore, a few matrix ions desorbed from the thin film were generated during laser irradiation. The detection limit for both small molecules and proteins, using this sol-gel-assisted laser desorption/ ionization (SGALDI) mass spectrometry (MS), was as low as 81 fmol. However, a mass spectrometer with cutoff-mass selection could detect 8.1 fmol of cytochrome c. The largest analyte observed by the SGALDI-MS in this study was myoglobin.