The Arabidopsis thaliana pho2 mutant, which is defective in a ubiquitin-conjugating E2 enzyme, displays inorganic phosphate (Pi) toxicity as a result of enhanced uptake and root-to-shoot ...translocation of Pi. To elucidate downstream components of the PHO2-dependent regulatory pathway, we identified two pho2 suppressors as carrying missense mutations in PHO1, which has been implicated in Pi loading to the xylem. In support of the genetic interaction between PHO1 and PHO2, we found that the protein level of PHO1 is increased in pho2, whereas such accumulation is ameliorated in both pho2 suppressors. Results from cycloheximide and endosomal Cys protease inhibitor E-64d treatments further suggest that PHO1 degradation is PHO2 dependent and involves multivesicular body-mediated vacuolar proteolysis. Using the transient expression system of tobacco Nicotiana tabacum) leaves, we demonstrated that PHO1 and PHO2 are partially colocalized and physically interact in the endomembranes, where the ubiquitin conjugase activity of PHO2 is required for PHO1 degradation. In addition, reduced PHO1 expression caused by PHO1 mutations impede Pi uptake, indicating a functional association between xylem loading and acquisition of Pi. Together, our findings uncover a pivotal molecular mechanism by which PHO2 modulates the degradation of PHO1 in the endomembranes to maintain Pi homeostasis in plants.
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 6× His, i.e., C192S (MW ∼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 α-, β-caseins, and diluted milk. The detection limit for the tryptic digests of α- and β-caseins is ∼50 fmol.
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 ∼3 × 105 cfu/mL, while the detectable cell concentration for S. saprophyticus in a urine sample was ∼3 × 107 cfu/mL.
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 ∼7 × 104 cfu/mL.
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 μJ) 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 7500 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 μJ) 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.
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).
The reaction of dimethyl acetylenedicarboxylate (DMAD) with C60 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 C60 (ΔE = 130 mV) and a simple Bingel adduct (ΔE = 90 mV). A phosphoramidate side product, which resulted from the addition of HMPT or HEPT to C60 followed by hydrolysis, exhibited a featureless absorption spectrum in the visible region and a more negative first reductive potential (ΔE = 70 mV) relative to that of C60.
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