Rapid Quantification of Polyhydroxybutyrate Polymer from Single Bacterial Cells with Mass Spectrometry.

Polyhydroxyalkanoate (PHA) is one of the biocompatible and biodegradable plastics that can be produced and accumulated as granules inside microorganisms. In this study, a new approach to rapidly quantify a short-chain-length PHA, polyhydroxybutyrate (PHB), produced from genetically engineered Escherichia coli containing phaCAB is presented. The mass of each bacterial cell was measured using a laser-induced radio frequency (rf) plasma charge detection quadrupole ion trap mass spectrometer (LIRFP CD QIT-MS), and then, the PHB contents were determined by calculating the change in cellular mass. The quantitative results showed that the PHB contents measured by LIRFP CD QIT-MS were consistent with those by reference analysis, gas chromatography (GC). The PHB content of each bacterial sample can be obtained within 20 min from sampling using LIRFP CD QIT-MS while GC analysis takes 2 days. In addition, LIRFP CD QIT-MS does not use any hazardous chemicals in cellular mass quantification as compared to GC. This indicates that LIRFP CD QIT-MS has potential in routine monitoring of PHB production.

Synthesis of Cu-doped carbon dot/chitosan film composite as a catalyst for the colorimetric detection of hydrogen peroxide and glucose.

The use of colloidal nanoparticles suffers from the drawbacks of potential color interference and substrate-induced aggregation. To overcome the limitations, a catalyst was developed by crosslinking Cu-doped carbon dots (Cu-CDs) with chitosan. Cu-CDs with high peroxidase activity were prepared by using a rapid microwave-assisted method. The Cu-CDs containing 6.88% of Cu had an average particle size of 2.25 nm and exhibited 9% of fluorescence quantum yield. The nanozyme/film composite was prepared by crosslinking between the amino groups of Cu-CDs and those of chitosan via a glutaraldehyde linker. A H2O2-mediated tetramethylbenzidine (TMB) oxidation reaction was use to evaluate the peroxidase activity of the film. Based on the TMB color changes, colorimetric assays were developed for the detection of H2O2 and glucose at an absorption wavelength 652 nm. Under the optimal conditions, the linear ranges for H2O2 and glucose were 0.625-40 µM and 1.9-125 µM, respectively, and the detection limits were 0.12 µM and 0.69 µM, respectively. The colorimetric assay was also applied to analyze diluted human serum samples spiked with glucose. Furthermore, this biodegradable, non-toxic, and easy-to-handle nanozyme composite could be stored for over 4 weeks without a significant decrease in activity.

Selective Capture and Identification of Methicillin-Resistant Staphylococcus aureus by Combining Aptamer-Modified Magnetic Nanoparticles and Mass Spectrometry.

A nucleic acid aptamer that specifically recognizes methicillin-resistant Staphylococcus aureus (MRSA) has been immobilized on magnetic nanoparticles to capture the target bacteria prior to mass spectrometry analysis. After the MRSA species were captured, they were further eluted from the nanoparticles and identified using matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS). The combination of aptamer-based capture/enrichment and MS analysis of microorganisms took advantage of the selectivity of both techniques and should enhance the accuracy of MRSA identification. The capture and elution efficiencies for MRSA were optimized by examining factors such as incubation time, temperature, and elution solvents. The aptamer-modified magnetic nanoparticles showed a capture rate of more than 90% under the optimized condition, whereas the capture rates were less than 11% for non-target bacteria. The as-prepared nanoparticles exhibited only a 5% decrease in the capture rate and a 9% decrease in the elution rate after 10 successive cycles of utilization. Most importantly, the aptamer-modified nanoparticles revealed an excellent selectivity towards MRSA in bacterial mixtures. The capture of MRSA at a concentration of 102 CFU/mL remained at a good percentage of 82% even when the other two species were at 104 times higher concentration (106 CFU/mL). Further, the eluted MRSA bacteria were successfully identified using MALDI mass spectrometry.

Boron, and nitrogen co-doped carbon dots as a multiplexing probe for sensing of p-nitrophenol, Fe (III), and temperature.

Boron and nitrogen co-doped carbon dots (B, N-CDs) were fabricated through a simple, one-step hydrothermal reaction of citric acid, boric acid, and tris base. The obtained B, N-CDs exhibit excitation-dependent fluorescence, high quantum yield (QY), biocompatibility, photostability, and aqueous solubility. The QY was substantially increased to 57% by doping boron atoms. Furthermore, the fluorescence intensity of B, N-CDs was temperature-dependent and decreased linearly from 283 to 333 K. The prepared B, N-CDs were used as a fluorescence probe for the detection ofpara-nitrophenol (p-NP) and Fe (III) ions with low detection limits of 0.17µM and 0.30µM, respectively. Moreover, the presence of p-NP could be further confirmed by a colorimetric assay. The fluorescent probe has been applied to determine p-NP and Fe (III) in a spiked serum sample and spiked water samples (lake and tap water). Moreover, the as-prepared B, N-CDs were of low toxicity and capable of bioimaging.

Mesoporous Polydopamine Nanoparticles Attenuate Morphine Tolerance in Neuropathic Pain Rats by Inhibition of Oxidative Stress and Restoration of the Endogenous Antioxidant System.

Oxidative stress resulting from reactive oxygen species (ROS) is known to play a key role in numerous neurological disorders, including neuropathic pain. Morphine is one of the commonly used opioids for pain management. However, long-term administration of morphine results in morphine antinociceptive tolerance (MAT) through elevation of ROS and suppression of natural antioxidant defense mechanisms. Recently, mesoporous polydopamine (MPDA) nanoparticles (NPS) have been known to possess strong antioxidant properties. We speculated that morphine delivery through an antioxidant nanocarrier might be a reasonable strategy to alleviate MAT. MPDAs showed a high drug loading efficiency of ∼50%, which was much higher than conventional NPS. Spectral and in vitro studies suggest a superior ROS scavenging ability of NPS. Results from a rat neuropathic pain model demonstrate that MPDA-loaded morphine (MPDA@Mor) is efficient in minimizing MAT with prolonged analgesic effect and suppression of pro-inflammatory cytokines. Additionally, serum levels of liver enzymes and levels of endogenous antioxidants were measured in the liver. Treatment with free morphine resulted in elevated levels of liver enzymes and significantly lowered the activities of endogenous antioxidant enzymes in comparison with the control and MPDA@Mor-treated group. Histopathological examination of the liver revealed that MPDA@Mor can significantly reduce the hepatotoxic effects of morphine. Taken together, our current work will provide an important insight into the development of safe and effective nano-antioxidant platforms for neuropathic pain management.

Fluorescent Mesoporous Nanoparticles for ß-Lactamase Screening Assays.

We present a sensitive and rapid screening method for the determination of ß-lactamase activity of antibiotic-resistant bacteria, by designing a pH-sensitive fluorescent dye-doped mesoporous silica nanoparticle encapsulated with penicillin G as a substrate. When penicillin G was hydrolysed by ß-lactamase and converted into penicilloic acid, the acidic environment resulted in fluorescence quenching of the dye. The dye-doped mesoporous nanoparticles not only enhanced the ß-lactamase-catalyzed reaction rate but also stablized the substrate, penicillin G, which degrades into penicilloic acid in a water solution without ß-lactamase. Twentyfive clinical bacterial samples were tested and the antibiotic resistant and susceptible strains were identified. The proposed method may detect the presence of ß -lactamases of clinically relevant samples in less than 1 hour. Moreover, the detection limit of ß-lactamase activity was as low as 7.8×10-4 U/mL, which was determined within two hours.

Mass spectrometry combined with affinity probes for the identification of CP4 EPSPS in genetically modified soybeans.

Sample preparation methods used for genetically modified organisms (GMOs) analysis are often time consuming, require extensive manual manipulation, and result in limited amounts of purified protein, which may complicate the detection of low-abundance GM protein. A robust sample pretreatment method prior to mass spectrometry (MS) detection of the transgenic protein (5-enolpyruvylshikimate-3-phosphate synthase [CP4 EPSPS]) present in Roundup Ready soya is investigated. Liquid chromatography-multiple reaction monitoring tandem MS (nano LC-MS/MS-MRM) was used for the detection and quantification of CP4 EPSPS. Gold nanoparticles (AuNPs) and concanavalin A (Con A)-immobilized Sepharose 4B were used as selective probes for the separation of the major storage proteins in soybeans. AuNPs that enable the capture of cysteine-containing proteins were used to reduce the complexity of the crude extract of GM soya. Con A-sepharose was used for the affinity capture of ß-conglycinin and other glycoproteins of soya prior to enzymatic digestion. The methods enabled the detection of unique peptides of CP4 EPSPS at a level as low as 0.5% of GM soya in MRM mode. Stable-isotope dimethyl labeling was further applied to the quantification of GM soya. Both probes exhibited high selectivity and efficiency for the affinity capture of storage proteins, leading to the quantitative detection at 0.5% GM soya, which is a level below the current European Union's threshold for food labeling. The square correlation coefficients were greater than 0.99. The approach for sample preparation is very simple without the need for time-consuming protein prefractionation or separation procedures and thus presents a significant improvement over existing methods for the analysis of the GM soya protein.

Identification of bacteria in juice/lettuce using magnetic nanoparticles and selected reaction monitoring mass spectrometry.

Ensuring food safety requires a rapid and reliable method for detecting food-borne pathogens. Mass spectrometry has been demonstrated as a powerful tool to classify pure bacterial species. However, matrix interference from food backgrounds may lead to false results because of the suppression of microbial signals. It is useful to develop a method for bacterial enrichment and marker identification in food samples. Magnetic zirconia nanoparticles were used to concentrate spiked microorganisms from apple juice/lettuce under specific conditions (pH 4.5). Bacterial identification was achieved using nanoLC-MS. Selected reaction monitoring of bacteria-related peptides was applied for the first time to identify bacteria including Staphylococcus aureus and Escherichia coli. This study presents an accurate means for bacterial identification in food matrixes using MS. The analysis time is less than 90 min and the minimum concentration of E. coli detected was 5 × 103 CFU/mL. The interaction between bacteria and the magnetic nanoparticles was electrostatic and nonspecific, in contrast to immunoassays which require specific antibodies. The targeted peptide analysis focuses on the bacterial markers, thus significantly simplifying the analysis and leading to an accurate identification of bacteria.

Quantitative analysis of genetically modified soya using multiple reaction monitoring mass spectrometry with endogenous peptides as internal standards.

A simple label-free method was developed for the quantification of the herbicide-resistant gene-related protein 5-enolpyruvylshikimate-3-phosphate synthase using multiple reaction monitoring liquid chromatography-mass spectrometry. Sample pretreatment procedures including ion exchange chromatography and CaCl2 precipitation were used to purify the 5-enolpyruvylshikimate-3-phosphate synthase protein. Quantification of various percentages of genetically modified soya (0.5-100%) was performed by selecting suitable endogenous soybean peptides as internal standards. Results indicated that Gly P (QGDVFVVPR) and Lec P (LQLNK) are useful internal standards for the quantification of low and high percentages of genetically modified soya, respectively. Linear regression analysis of both calibration curves yielded good linearity with R2 of 0.99. This approach is a convenient and accurate quantification method for genetically modified soya at a level as low as 0.5% (less than the current EU threshold for labeling genetically modified soya).

Studying the effect of microwave heating on the digestion process and identification of proteins.

The impact of microwave irradiation on the in-solution digestion processes and the detection limit of proteins are systematically studied. Kinetic processes of many peptides produced through the trypsin digestion of various proteins under microwave heating at 50°C were investigated with MALDI-MS. This study also examines the detection limits and digestion completeness of individual proteins under microwave heating at 50°C and at different time intervals (1, 5 and 30 min) using LC-MS. We conclude that if the peptides without missed cleavage dictate the detection limit, conventional digestion will lead to a better detection limit. The detection limit may not differ between the microwave and conventional heating if the peptides with missed cleavage sites and strong intensity are formed at the very early stage (i.e., less than 1 min) and are not further digested throughout the entire digestion process. The digestion of Escherichia coli lysate was compared under conventional and short time (microwave) conditions. The number of proteins identified under conventional heating exceeded that obtained from microwave heating over heating periods less than 5 min. The overall results show that the microwave-assisted digestion is not complete. Although the sequence coverage might be better, the detection limit might be worse than that under conventional heating.

Study of microwave effects on the lipase-catalyzed hydrolysis.

The effect of microwave heating on lipase-catalyzed reaction remains controversial. It is not clear whether the reaction rate enhancements are purely due to thermal/heating effects or to non-thermal effects. Therefore, quantitative mass spectrometry was used to conduct accurate kinetic analysis of lipase-catalyzed hydrolysis of triolein by microwave and conventional heating. Commercial lipases from Candida rugosa (CRL), Porcine Pancreas (PPL), and Burkholderia cepacia (BCL) were used. Hydrolysis reactions were performed at various temperatures and pH levels, along with various amounts of buffer and enzymes. Hydrolysis product yields at each time point using an internal-standard method showed no significant difference between microwave and conventional heating conditions when the reaction was carried out at the same temperature. CRL showed optimum catalytic activity at 37 °C, while PPL and BCL had better activities at 50 °C. The phosphate buffer was found to give a better hydrolysis yield than the Tris-HCl buffer. Overall results prove that a non-thermal effect does not exist in microwave-assisted lipase hydrolysis of triolein. Therefore, conventional heating at high temperatures (e.g., 50 °C) can be also used to accelerate hydrolysis reactions.

Phenologic variation of major triterpenoids in regular and white Antrodia cinnamomea.

BACKGROUND: Antrodia cinnamomea and its host Cinnamomum kanehirae are both endemic species unique to Taiwan. Many studies have confirmed that A. cinnamomea is rich in polysaccharides and triterpenoids that may carry medicinal effects in anti-cancer, anti-inflammation, anti-hypertension, and anti-oxidation. Therefore it is of interest to study the chemical variation of regular orange-red strains and white strains, which included naturally occurring and blue-light induced white A. cinnamomea. RESULTS: The chemical profiles of A. cinnamomea extracts at different growth stages were compared using thin layer chromatography (TLC) and high performance liquid chromatography (HPLC). The TLC and HPLC profiles indicated that specific triterpenoids varied between white and regular strains. Moreover, the compounds of blue-light induced white strain were similar to those of naturally occurring white strain but retained specific chemical characteristics in more polar region of the HPLC chromatogram of regular strain. CONCLUSIONS: Blue-light radiation could change color of the regular A. cinnamomea from orange-red to white by changing its secondary metabolism and growth condition. Naturally occurring white strain did not show a significantly different composition of triterpenoid profiles up to eight weeks old when compared with the triterpenoid profiles of the regular strain at the same age. The ergostane-type triterpenoids were found existing in both young mycelia and old mycelia with fruiting body in artificial agar-plate medium culture, suggesting a more diversified biosynthetic pathway in artificial agar-plate culture rather than wild or submerged culture.

Functionalized magnetic iron oxide (Fe3O4) nanoparticles for capturing gram-positive and gram-negative bacteria.

The development of nanotechnology in biology and medicine has raised the need for conjugation of nanoparticles (NPs) to biomolecules. In this study, magnetic and functionalized magnetic iron oxide nanoparticles were synthesized and used as affinity probes to capture Gram-positive/negative bacteria. The morphology and properties of the magnetic NPs were examined by transmission electron microscopy, Fourier transform infrared spectroscopy, and zeta potential measurements. Furthermore, this study investigated the interaction between functionalized magnetic nanoparticles and Gram positive/negative bacteria. The positively and negatively charged magnetic nanoparticles include functionalities of Fe3O4, SiO2, TiO2, ZrO2, poly ethyleneimine (PEI) and poly acrylic acid. Their capture efficiencies for bacteria were investigated based on factors such as zeta potential, concentration and pH value. PEI particles carry a positive charge over a range of pH values from 3 to 10, and the particles were found to be an excellent candidate for capturing bacteria over such pH range. Since the binding force is mainly electrostatic, the architecture and orientation of the functional groups on the NP surface are not critical. Finally the captured bacteria were analyzed using matrix-assisted laser desorption/ionization mass spectrometry. The minimum detection limit was 10(4) CFU/mL and the analysis time was reduced to be less than 1 hour. In addition, the detection limit could be reduced to an extremely low concentration of 50 CFU/mL when captured bacteria were cultivated.

Quantification of genetically modified soya using strong anion exchange chromatography and time-of-flight mass spectrometry.

Stable-isotope dimethyl labeling was applied to the quantification of genetically modified (GM) soya. The herbicide-resistant gene-related protein 5-enolpyruvylshikimate-3-phosphate synthase (CP4 EPSPS) was labeled using a dimethyl labeling reagent, formaldehyde-H2 or -D2. The identification and quantification of CP4 EPSPS was performed using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). The CP4 EPSPS protein was separated from high abundance proteins using strong anion exchange chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Then, the tryptic peptides from the samples and reference were labeled with formaldehyde-H2 and formaldehyde-D2, respectively. The two labeled pools were mixed and analyzed using MALDI-MS. The data showed a good correlation between the peak ratio of the H- and D-labeled peptides and the GM soya percentages at 0.5, 1, 3, and 5 %, with R (2) of 0.99. The labeling reagents are readily available. The labeling experiments and the detection procedures are simple. The approach is useful for the quantification of GM soya at a level as low as 0.5 %.

Antrodia cinnamomea profoundly exalted the reversion of activated hepatic stellate cells by the alteration of cellular proteins.

The direct modulation of Antrodia cinnamomea (AC) on the prominent role of liver fibrosis-hepatic stellate cells (HSCs) in situ remains unclear. Firstly, the administration of A. cinnamomea mycelial extract (ACME) could improve liver morphology and histological changes including collagen formation and GPT activity in the liver of thioacetamide (TAA)-injured rats. The morphology and fatty acid restore of TAA-induced HSCs (THSCs) returned to the non-chemical induced HSCs (NHSCs) type as measured by immunofluorescence and Oil Red O staining. PPARγ was upregulated associated with the lowering of α-SMA protein in NHSC-ACME. ACME inhibited the MMP-2 activity in NHSCs by gelatin Zymography. After LC-MS/MS, the cytoskeleton (tubulin, lamin A) and heat shock protein 8 in NHSC-ACME, and guanylate kinase, brain-specific kinase, SG-II and p55 proteins were downregulated in THSC-ACME. Whereas MHC class II, SMC6 protein, and phospholipase D were upregulated in NHSC-ACME. Furthermore, PKG-1 was downregulated in NHSC-ACME and upregulated in THSC-ACME. SG-II and p55 proteins were downregulated in NHSC-ACME and THSC-ACME by Western blotting. Taken together, the beneficial effect of A. cinnamomea on the induction of HSC cellular proteins is potentially applied as an alternative and complementary medicine for the prevention and amelioration of a liver injury.

Inhibition of aldolase A blocks biogenesis of ATP and attenuates Japanese encephalitis virus production.

Viral replication depends on host proteins to supply energy and replication accessories for the sufficient production of viral progeny. In this study, we identified fructose-bisphosphate aldolase A as a binding partner of Japanese encephalitis virus (JEV) untranslated regions (UTRs) on the antigenome via RNA affinity capture and mass spectrometry. Direct interaction of aldolase A with JEV RNAs was confirmed by gel mobility shift assay and colocalization with active replication of double-stranded RNA in JEV-infected cells. Infection of JEV caused an increase in aldolase A expression of up to 33%. Knocking down aldolase A reduced viral translation, genome replication, and viral production significantly. Furthermore, JEV infection consumed 50% of cellular ATP, and the ATP level decreased by 70% in the aldolase A-knockdown cells. Overexpression of aldolase A in aldolase A-knockdown cells increased ATP levels significantly. Taken together, these results indicate that JEV replication requires aldolase A and consumes ATP. This is the first report of direct involvement of a host metabolic enzyme, aldolase A protein, in JEV replication.

Evaluating the potential nonthermal microwave effects of microwave-assisted proteolytic reactions.

Microwave-assisted proteolytic digestion methods have evolved into a highly effective approach and serve as an alternative to conventional overnight digestion. This approach typically exploits the unique microwave properties to facilitate the digestion of proteins into their peptides within minutes. Conventional digestion is carried out at 37°C while microwave-assisted digestion requires much higher and sometimes inconsistent temperatures. Thus, this study aims to investigate whether the faster reaction rate is due to the microwave quantum effect or the thermal effect. Quantitative mass spectrometry was used to conduct kinetic analysis of tryptic digestion for several proteins by microwave and conventional heating. The percentages of digestion products relative to internal standards showed no significant difference between microwave and conventional heating conditions at the same digestion temperature. The optimum temperature for tryptic digestion was determined to be 50°C. Furthermore, this study compares the digestion completeness indicators of several proteins under microwave and conventional heating. Again, the values obtained from microwave and conventional heating were similar given identical temperatures. The overall results prove that a nonthermal effect does not exist in microwave-assisted tryptic digestion. Therefore, conventional heating at high temperatures (50°C) can be also used to accelerate digestion reactions.

Advances in mass spectrometry for the identification of pathogens.

Mass spectrometry (MS) has become an important technique to identify microbial biomarkers. The rapid and accurate MS identification of microorganisms without any extensive pretreatment of samples is now possible. This review summarizes MS methods that are currently utilized in microbial analyses. Affinity methods are effective to clean, enrich, and investigate microorganisms from complex matrices. Functionalized magnetic nanoparticles might concentrate traces of target microorganisms from sample solutions. Therefore, nanoparticle-based techniques have a favorable detection limit. MS coupled with various chromatographic techniques, such as liquid chromatography and capillary electrophoresis, reduces the complexity of microbial biomarkers and yields reliable results. The direct analysis of whole pathogenic microbial cells with matrix-assisted laser desorption/ionization MS without sample separation reveals specific biomarkers for taxonomy, and has the advantages of simplicity, rapidity, and high-throughput measurements. The MS detection of polymerase chain reaction (PCR)-amplified microbial nucleic acids provides an alternative to biomarker analysis. This review will conclude with some current applications of MS in the identification of pathogens.

Use of polyethylenimine-modified magnetic nanoparticles for highly specific enrichment of phosphopeptides for mass spectrometric analysis.

Phosphopeptides have been isolated and concentrated by use of polyethyleneimine (PEI)-modified magnetic nanoparticles as an extremely specific affinity probe. The particles specifically captured phosphopeptides from a tryptic digest of a protein mixture that contained 0.07% (mole/mole) phosphoproteins, which is the highest specificity obtained to date. The time required for enrichment of the phosphopeptides was 1 min only. PEI-modified magnetic nanoparticles carry positive charges over a wide range of pH-between 3 and 11. This feature means the particles are effectively dispersed in solution during phosphopeptide capture. Mass spectrometric analysis revealed the very high efficiency of enrichment of phosphopeptides that contain both single and multiply-phosphorylated sites. The detection limit in the analysis of phosphopeptides obtained from both bovine α-casein and ß-casein by matrix-assisted laser desorption/ionization mass spectrometry was 5 fmol. This approach was also used to enrich the phosphopeptides in a protein digest obtained from non-fat milk.

ß-Actin is a downstream effector of the PI3K/AKT signaling pathway in myeloma cells.

Interleukin 6 is the in vivo growth factor of myeloma cells. In response to IL-6 stimulation, the PI3K/AKT signaling pathway is activated in these cells. With comparative proteomic approaches, this study reveals many putative downstream effectors of the PI3K/AKT pathway. Mass spectrometry analysis of excised protein spots from 2-dimensional gel allowed the identification of proteins such as ß-Actin, cyclophilin A, E3 SUMO-protein ligase PIAS-NY protein, HSP 27, PML, and transforming growth factor ß-2. Among these putative effectors, ß-Actin was chosen for further characterization. Phosphorylation of ß-Actin by AKT upon IL-6 stimulation was confirmed by western blotting using a phospho-AKT substrate antibody. Interestingly, IL-6 significantly increased cell migration (P < 0.05) and the content of filamentous actin (P < 0.05). Therefore, IL-6 stimulation could have effects on the migration of myeloma cells, and the phosphorylation of ß-Actin is probably involved in the process.