Shape-Controlled Synthesis of Trimetallic Nanoclusters: Structure Elucidation and Properties Investigation.

The shape-controlled synthesis of metal nanoclusters (NCs) with precise atomic arrangement is crucial for tailoring the properties. In this work, we successfully control the shape of alloy NCs by altering the dopants in the alloying processes. The shape of the spherical [Pt1 Ag24 (SPhMe2 )18 ] NC is maintained when [AuI SR] is used as dopant. By contrast, the shape of Pt1 Ag24 is changed to be rodlike by alloying with [AuI (PPh3 )Br]. The structures of the trimetallic NCs were determined by X-ray crystallography and further confirmed by both DFT and far-IR measurements. The shape-preserved [Pt1 Au6.4 Ag17.6 (SPhMe2 )18 ] NC is in a tristratified arrangement-[Pt(center)@Au/Ag(shell)@Ag(exterior)]-and is indeed the first X-ray crystal structure of thiolated trimetallic NCs. On the other hand, the resulting rodlike NC ([Pt2 Au10 Ag13 (PPh3 )10 Br7 ]) exhibits a high quantum yield (QY=14.7 %), which is in striking contrast to the weakly luminescent Pt1 Ag24 (QY=0.1 %, about 150-fold enhancement). In addition, the thermal stabilities of both trimetallic products are remarkably improved. This study presents a controllable strategy for synthesis of alloy NCs with different shapes (by alloying heteroatom complexes coordinated by different ligands), and may stimulate future work for a deeper understanding of the morphology (shape)-property correlation in NCs.

Proteomic analysis of perfluorooctane sulfonate-induced apoptosis in human hepatic cells using the iTRAQ technique.

Perfluorooctane sulfonate (PFOS) is one of the most commonly used perfluorinated compounds, whose environmental exposure has been associated with a number of adverse health outcomes. However, the molecular mechanisms involved in PFOS toxicity are still not well elucidated. In the present study, we applied iTRAQ labeling quantitative proteomic technology to investigate the differential protein expression profiles of non-tumor human hepatic cells (L-02) exposed to PFOS. A total of 18 proteins were differentially expressed in a dose-dependent manner in PFOS-treated cells versus the control. Among these, 11 proteins were up-regulated and 7 were down-regulated. Gene ontology analysis indicated that PFOS would exert toxic effects on L-02 cells by affecting multiple biological processes, including protein biosynthesis and degradation, mRNA processing and splicing, transcription, signal transduction and transport. Furthermore, the proteomic results especially proposed that the inhibition of HNRNPC, HUWE1 and UBQLN1, as well as the induction of PAF1 is involved in the activation of the p53 and c-myc signaling pathways, which then trigger the apoptotic process in L-02 cells exposed to PFOS. Overall, these data will aid our understanding of the mechanisms responsible for PFOS-mediated hepatotoxicity, and develop useful biomarkers for monitoring and evaluating PFOS contamination in the environment.

Proteomic analysis of Ketogulonicigenium vulgare under glutathione reveals high demand for thiamin transport and antioxidant protection.

Ketogulonicigenium vulgare, though grows poorly when mono-cultured, has been widely used in the industrial production of the precursor of vitamin C with the coculture of Bacillus megaterium. Various efforts have been made to clarify the synergic pattern of this artificial microbial community and to improve the growth and production ability of K. vulgare, but there is still no sound explanation. In previous research, we found that the addition of reduced glutathione into K. vulgare monoculture could significantly improve its growth and productivity. By performing SEM and TEM, we observed that after adding GSH into K. vulgare monoculture, cells became about 4-6 folds elongated, and formed intracytoplasmic membranes (ICM). To explore the molecular mechanism and provide insights into the investigation of the synergic pattern of the co-culture system, we conducted a comparative iTRAQ-2-D-LC-MS/MS-based proteomic analysis of K. vulgare grown under reduced glutathione. Principal component analysis of proteomic data showed that after the addition of glutathione, proteins for thiamin/thiamin pyrophosphate (TPP) transport, glutathione transport and the maintenance of membrane integrity, together with several membrane-bound dehydrogenases had significant up-regulation. Besides, several proteins participating in the pentose phosphate pathway and tricarboxylic acid cycle were also up-regulated. Additionally, proteins combating intracellular reactive oxygen species were also up-regulated, which similarly occurred in K. vulgare when the co-cultured B. megaterium cells lysed from our former research results. This study reveals the demand for transmembrane transport of substrates, especially thiamin, and the demand for antioxidant protection of K. vulgare.

[Simultaneous analysis of 19 antibiotics in dairy products using ultra-performance liquid chromatography coupled with high resolution time-of-flight mass spectrometry].

An ultra-performance liquid chromatography coupled with high resolution time-of-flight mass spectrometry method (UPLC/HRTOF-MS) has been developed for the simultaneous analysis of 19 antibiotics in dairy products. The sample was treated with acetonitrile and acidic acetonitrile to remove protein and fat, and then the supernatant was concentrated with a concentrator system. The antibiotics in the prepared sample were separated on a BEH column, and then qualitatively and quantitatively analyzed by HRTOF-MS in positive ionization mode within 10 min. A screening database containing the qualitative information of the antibiotics was built with TargetAnalysis software. Matrix matching was used in the antibiotic analysis to compensate for the matrix effects that influence analytical response. The linear range of the antibiotics was 10-500 or 15-1 000 microg/L. The limits of detection (LOD) were from 3 to 5 microg/L. At the spiked levels of 20 and 100 microg/L, the average recoveries were from 68.4% to 96.7% with the relative standard deviations ranging from 2.1% to 12.5%. The screening results of a spiked milk sample showed that all the spiked antibiotics could be detected with their deviations of retention time < or = 0.1 min, the deviations of mass < 5 mDa, the degrees of isotope pattern match > or = 87.4%, and most spiked antibiotics were detected with high scores. Furthermore, the developed method was applied for the analysis of antibiotics in more than 40 milk and dairy products of seven manufacturers, and the target antibiotics were not detected in all the samples. The method is rapid, sensitive and easy to operate, and is suitable for the screening of antibiotic residues in milk and dairy products.

Highly specific enrichment of phosphopeptides by zirconium dioxide nanoparticles for phosphoproteome analysis.

Large-scale characterization of phosphoproteins requires highly specific methods for the purification of phosphopeptides because of the low abundance of phosphoproteins and substoichiometry of phosphorylation. A phosphopeptide enrichment method using ZrO2 nanoparticles is presented. The high specificity of this approach was demonstrated by the isolation of phosphopeptides from the digests of model phosphoproteins. The strong affinity of ZrO2 nanoparticles to phosphopeptides enables the specific enrichment of phosphopeptides from a complex peptide mixture in which the abundance of phosphopeptides is two orders of magnitude lower than that of nonphosphopeptides. Superior selectivity of ZrO2 nanoparticles for the enrichment of phosphorylated peptides than that of conventional immobilized metal affinity chromatography was observed. Femtomole phosphopeptides from digestion products could be enriched by ZrO2 nanoparticles and can be well detected by MALDI mass spectrometric analysis. ZrO2 nanoparticles were further applied to selectively isolate phosphopeptides from the tryptic digestion of mouse liver lysate for phosphoproteome analysis by nanoliter LC MS/MS (nano-LC-MS/MS) and MS/MS/MS. A total of 248 defining phosphorylation sites and 140 phosphorylated peptides were identified by manual validation using a series of rigid criteria.

Enrichment of peptides from plasma for peptidome analysis using multiwalled carbon nanotubes.

Human plasma contains a complex matrix of proteolytically derived peptides (plasma peptidome) that may provide a correlate of biological events occurring in the entire organism. Analyzing these peptides from a small amount of serum/ plasma is difficult due to the complexity of the sample and the low levels of these peptides. Here, we describe a novel peptidome analysis approach using multiwalled carbon nanotubes (MWCNTs) as an alternative adsorbent to capture endogenous peptides from human plasma. Harvested peptides were analyzed by using liquid chromatography-mass spectrometry as a means of detecting and assessing the adsorbed molecules. The improved sensitivity and resolution obtained by using liquid chromatography-mass spectrometry allowed detection of 2521 peptide features (m/z 300-1800 range) in about 50 microL of plasma. 374 unique peptides were identified with high confidence by two-dimensional liquid chromatography system coupled to a nano-spray ionization linear ion trap-mass spectrometer. High recovery of BSA digest peptides enriched with MWCNTs, in both standard buffer and high abundance protein solution, was observed. Comparative studies showed that MWCNTs were superior to C18 and C8 for the capture of the smaller peptides. This approach could hold promise of routine plasma peptidome analysis.

Preparation of a biochip on porous silicon and application for label-free detection of small molecule-protein interactions.

A new approach for the preparation of a biochip on porous silicon and the application of the biochip for detection of small molecule-protein interactions with desorption/ionization on porous silicon (DIOS) was demonstrated. The galvanostatically etched porous silicon substrates were chemically modified firstly to yield carboxylic acid terminated surfaces, and then the protein was covalently attached to the surface through amide bonding. By applying a solution of candidate chemicals to the surface and a subsequent wash step, the masses of captured compounds could be analyzed by DIOS. DIOS has advantages of being a direct detection tool compared to the classic fluorescence or chemiluminescence methods, because the process of labeling molecules employed in the fluorescence or chemiluminescence methods can sometimes alert the properties of the labeled molecule. The recognition between proteins and their binding partners is efficient and selective. A good tolerance to disturbance and high enrichment factor of the biochip to the analytes was observed. As an on-chip-based approach, the demonstrated method has a potential to perform in a high-throughput format.

Recent developments in methods and technology for analysis of biological samples by MALDI-TOF-MS.

Matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF-MS) is widely used in a variety of fields because it has the characteristics of speed, ease of use, high sensitivity, and wide detectable mass range for obtaining molecular weights and for structural characterization of macromolecules. In this article we summarize recent developments in matrix additives, new matrices, and sample-pretreatment methods using off-probe or on-probe techniques or nanomaterials for MALDI-TOF-MS analysis of biological samples.

Fe3+ immobilized metal affinity chromatography with silica monolithic capillary column for phosphoproteome analysis.

Immobilized metal affinity chromatography (IMAC) is a commonly used technique for phosphoproteome analysis due to its high affinity for adsorption of phosphopeptides. Miniaturization of IMAC column is essential for the analysis of a small amount of sample. Nanoscale IMAC column was prepared by chemical modification of silica monolith with iminodiacetic acid (IDA) followed by the immobilization of Fe3+ ion inside the capillary. It was demonstrated that Fe3+-IDA silica monolithic IMAC capillary column could specifically capture the phosphopeptides from tryptic digest of alpha-casein with analysis by MALDI-TOF MS. The silica monolithic IMAC capillary column was manually coupled with nanoflow RPLC/nanospray ESI mass spectrometer (muRPLC-nanoESI MS) for phosphoproteome analysis. The system was validated by analysis of standard phosphoproteins and then it was applied to the analysis of protein phosphorylation in mouse liver lysate. Besides MS/MS spectra, MS/MS/MS spectra were also collected for neutral loss peak. After database search and manual validation with conservative criteria, 29 singly phosphorylated peptides were identified by analyzing a tryptic digest of only 12 mug mouse liver lysate. The results demonstrated that the silica monolithic IMAC capillary column coupled with muRPLC-nanoESI MS was very suitable for the phosphoproteome analysis of minute sample.

3,4-diaminobenzophenone matrix for analysis of oligonucleotides by MALDI-TOF mass spectrometry.

This unit contains procedures for analysis of oligonucleotides by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry with 3,4-diaminobenzophenone (DABP) as a matrix. This new matrix has demonstrated advantages in the analysis of oligonucleotides. With DABP as a matrix, intact oligonucleotide ions can be readily produced with lower laser powers, resulting in better detection limits, less fragmentation, and fewer alkali-metal ion adducts compared with results obtained using conventional matrices. Minimal fragmentation and fewer alkali-metal ion adducts were seen even at low oligonucleotide concentrations. It was also found that samples prepared with DABP are highly homogenous, therefore reducing the need to find "sweet spots" in MALDI. Finally, excellent shot-to-shot reproducibility, resolution, and signal-to-noise ratio are observed using the DABP matrix.

Enrichment of phosphopeptides by Fe3+-immobilized mesoporous nanoparticles of MCM-41 for MALDI and nano-LC-MS/MS analysis.

Fe3+-immobilized mesoporous molecular sieves MCM-41 with particle size of ca. 600 nm and pore size of ca. 3 nm is synthesized and applied to selectively trap and separate phosphopeptides from tryptic digest of proteins. For the capture of phosphopeptides, typically 10 microL of tryptic digest solution was first diluted to 1 mL by solution of ACN/0.1% TFA (50:50, v/v) and incubated with 10 microL of 0.1% acetic acid dispersed Fe3+-immobilized MCM-41 for 1 h under vibration. Fe3+-immobilized MCM-41 with trapped phosphopeptides was separated by centrifugation. The deposition was first washed with a volume of 300 microL of solution containing 100 mM NaCl in ACN/0.1% TFA (50:50, v/v) and followed by a volume of 300 microL of solution of 0.1% acetic acid to remove nonspecifically bound peptides. The nanoparticles with trapped phosphopeptides are mixed with 2,5-dihydroxybenzoic acid (2,5-DHB) and deposited onto the target for analysis by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). It was found that phosphopeptides from tryptic digest of alpha-casein and beta-casein are effectively and specifically trapped on Fe3+-immobilized MCM-41 with few peptides nonspecifically adsorbed. After the extraction by Fe3+-immobilized MCM-41, the suppression to the detection of phosphopeptides caused by abundant nonphosphopeptides from tryptic digest is effectively eliminated, and the detection of phosphopeptides by MALDI is greatly enhanced with the value of signal-to-noise (S/N) increased by more than an order of magnitude. It is demonstrated that the mechanism of the adsorption of phosphopeptides on Fe3+-immobilized MCM-41 is based on the interaction between the Fe3+ and the phosphate group. Finally, Fe3+-immobilized MCM-41 is applied to extract phosphopeptides from tryptic digest of the lysate of mouse liver for phosphoproteome analysis by nano-LC-MS/MS.

Zirconium phosphonate-modified porous silicon for highly specific capture of phosphopeptides and MALDI-TOF MS analysis.

Phosphorylation is one of the most important post-translational modifications of proteins, which modulates a wide range of biological functions and activity of proteins. The analysis of phosphopeptides is still one of the most challenging tasks in proteomics research by mass spectrometry. In this study, a novel phosphopeptide enrichment approach based on the strong interaction of zirconium phosphonate (ZrP) modified surface with phosphopeptides has been developed. ZrP modified porous silicon (ZrP-pSi) wafer was prepared to specifically capture the phosphopeptides from complex peptide mixtures, and then the captured phosphopeptides were analyzed by MALDI-TOF MS by directly placing the wafer on a MALDI target. The phosphopeptide enrichment and MALDI analysis were both performed on the ZrP-pSi wafer which significantly reduced the sample loss and simplified the analytical procedures. The prepared ZrP-pSi wafer has been successfully applied for the enrichment of phosphopeptides from the tryptic digest of standard phosphoproteins beta-casein and alpha-casein. The excellent selectivity of this approach was demonstrated by analyzing phosphopeptides in the digest mixture of beta-casein and bovine serum albumin with molar ratio of 1:100. High detection sensitivity has been achieved for the analysis of the phosphopeptides from tryptic digestion of 2 fmol beta-casein on the ZrP-pSi surface.

A matrix of 3,4-diaminobenzophenone for the analysis of oligonucleotides by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

A new matrix of 3,4-diaminobenzophenone (DABP) was demonstrated to be advantageous in the analysis of oligonucleotides by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. With DABP as a matrix, intact oligonucleotide ions can be readily produced with lower laser powers, resulting in better detection limits, less fragmentation and fewer alkali metal ion adducts compared with the results obtained with conventional matrices. Importantly, minimal fragmentation and fewer alkali metal ion adducts were seen even at low concentrations of oligonucleotides. It was also found that samples prepared with DABP are highly homogenous and therefore reducing the need for finding 'sweet' spots in MALDI. In addition, excellent shot-to-shot reproducibility, resolution and signal-to-noise ratio were seen with DABP as the matrix.

Monitoring enzyme reaction and screening enzyme inhibitor based on MALDI-TOF-MS platform with a matrix of oxidized carbon nanotubes.

A matrix assisted laser desorption/ionization time-of-flight mass spectrometry platform for quantitatively monitoring enzyme activity and screening enzyme inhibitors has been demonstrated. The described method employs a new matrix of oxidized carbon nanotubes. Compared with the traditional fluorescence approach, this label-free method has the advantage of directly identifying the substrates and products in enzymatic reactions. Moreover, the method could be conveniently carried out with any commercial mass spectrometer without modification. We quantitatively monitored the acetylcholinesterase activity and screened acetylcholinesterase inhibitors with a detection rate of about 3.3 s per sample.

Iminodiacetic acid derivatized porous silicon as a matrix support for sample pretreatment and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis.

Iminodiacetic acid (IDA)-1,2-epoxy-9-decene has been synthesized and covalently linked to the surface of porous silicon wafer through a photochemical reaction. The negatively charged carboxylic acid groups on the porous silicon wafer are capable of binding oppositely charged species from sample solutions through electrostatic interactions. This allows the removal of contaminants prior to matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) by simply washing the porous silicon surface. The carboxylic acid end groups on porous silicon can be used to selectively bind and concentrate target species in sample solutions. Furthermore, Fe(3+)-IDA-derivatized porous silicon was prepared to specifically and effectively concentrate phosphopeptides from the tryptic digests of phosphoproteins, followed by MALDI-MS analysis.

Determination of DL-tetrahydropalmatine in Corydalis yanhusuo by L-tetrahydropalmatine imprinted monolithic column coupling with reversed-phase high performance liquid chromatography.

A method for direct determination of DL-tetrahydropalmatine (DL-THP) in Corydalis yanhusuo, a traditional Chinese herb, by L-THP imprinted monolithic precolumn on-line/off-line coupling with reversed-phase high performance liquid chromatography (RP-HPLC) was developed. The L-THP imprinted monolithic column has been prepared by in situ polymerization using methacrylic acid (MAA) and ethylene dimethacrylate (EDMA) as functional monomer and cross-linker, respectively. With the optimization of chromatographic conditions, such as mobile phase composition, flow rate, column temperature and sample loading, for the separation of enantiomer, DL-THP was base-line separated on the MIP. The imprinted monolithic column was used as a precolumn for fractionation of the C. yanhusuo extract. Both the non-retained and retained fractions were separated by RP-HPLC. Meanwhile, the D-THP and L-THP can be detected in the non-retained and retained fractions, respectively. Additionally, direct determination of L-THP using molecularly imprinted monolith on-line coupling with a reversed-phase column was acquired.

Matrix with high salt tolerance for the analysis of peptide and protein samples by desorption/ionization time-of-flight mass spectrometry.

High concentrations of urea and guanidine hydrochloride are commonly used for the denaturation of protein, which was digested by enzymatic proteolysis for the identification by MS analysis. The presence of these contaminants seriously suppresses the ion signal of analytes in MALDI-TOF MS analysis. Herein, a novel MALDI matrix, 3, 4-diaminobenzophenone (DABP), has been found with high tolerance for these contaminants in MALDI MS analysis. The ion signal of analyte insulin can be detected in the presence of 2 M guanidine hydrochloride and 1.5 M urea using DABP as matrix. The tryptic digest of BSA (400 fmol) in 1 M guanidine hydrochloride or 1 M urea was successfully analyzed without any pretreatment prior to MS analysis. Furthermore, it has been found that this matrix can also effectively suppress the cation ion adduction of the peptides in the presence of high concentrations of metal ions in sample solution.

Quantitative determination of oxidized carbon nanotube probes in yeast by capillary electrophoresis with laser-induced fluorescence detection.

Short oxidized multi-walled carbon nanotubes were functionalized with fluorescein isothiocyanate to form carbon nanotube probes (CNTP). The distribution of CNTP in yeast was quantitatively determined by capillary electrophoresis coupled with laser-induced fluorescence detection. The detection sensitivity for CNTP was greatly improved comparing with UV absorbance and Raman detection. The time- and temperature-dependent influx patterns of CNTP into yeast were obtained. The apparent permeability coefficient for influx of CNTP into yeast was calculated, which suggested that CNTP might permeate into yeast through endocytosis. This study implies that CNTP could be a fine drug transporter and might be wildly used in multidrug resistance research and microorganism detection.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry with a matrix of carbon nanotubes for the analysis of low-mass compounds in environmental samples.

The use of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) for environmental analysis has been mainlyfocused on qualitative analysis of high-mass molecules, such as toxins, humic acid, and microorganisms. Herein,we describe a novel MALDI-TOF-MS method with a matrix of oxidized carbon nanotubes for analysis of low-mass compounds in environmental samples. A number of chemicals in the environment were qualitatively analyzed by the present method, and it was found that most of them, especiallythe highly polar chemicals, were measurable with high sensitivity. With the intrinsic ability to measure high-mass chemicals, this method can compensate for the current shortage of methods for environmental analysis for the measurement of highly polar or high-mass chemicals. For sample analysis, arsenic speciation in Chinese traditional medicines was qualified and diphenylolpropane in water samples was quantified. With the relatively high tolerance of the method to interfering molecules, a simple pretreatment or even no pretreatment could be employed before MS detection. Furthermore, this method can be employed in a high-throughput format.

Integration of ion-exchange chromatography fractionation with reversed-phase liquid chromatography-atmospheric pressure chemical ionization mass spectrometer and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry for isolation and identification of compounds in Psoralea corylifolia.

An approach for the separation and identification of components in a traditional Chinese medicine Psoralea corylifolia was developed. Ion-exchange chromatography (IEC) was applied for the fractionation of P. corylifolia extract, and then followed by concentration of all the fractions with rotary vacuum evaporator. Each of the enriched fractions was then further separated on an ODS column with detection of UV absorbance and atmospheric pressure chemical ionization mass spectrometer (APCI/MS), respectively, and also analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS) with matrix of oxidized carbon nanotubes. Totally more than 188 components in P. corylifolia extract were detected with this integrated approach, and 12 of them were preliminary identified according to their UV spectra and mass spectra performed by APCI/MS and MALDI-TOF/MS. The obtained analytical results not only demonstrated the powerful resolution of integration IEC fractionation with reversed-phase liquid chromatography (RPLC)-APCI/MS and MALDI-TOF/MS for analysis of compounds in a complex sample, but also exhibited the superiority of APCI/MS and MALDI-TOF/MS for identification of low-mass compounds, such as for study of traditional Chinese medicines (TCMs) and metabolome.