Targeted protein quantitation in human body fluids by mass spectrometry.

Human body fluids (biofluids) contain various proteins, some of which reflect individuals' physiological conditions or predict diseases. Therefore, the analysis of biofluids can provide substantial information on novel biomarkers for clinical diagnosis and prognosis. In the past decades, mass spectrometry (MS)-based technologies have been developed as proteomic strategies not only for the identification of protein biomarkers but also for biomarker verification/validation in body fluids for clinical applications. The main advantage of targeted MS-based methodologies is the accurate and specific simultaneous quantitation of multiple biomarkers with high sensitivity. Here, we review MS-based methodologies that are currently used for the targeted quantitation of protein components in human body fluids, especially in plasma, urine, cerebrospinal fluid, and saliva. In addition, the currently used MS-based methodologies are summarized with a specific focus on applicable clinical sample types, MS configurations, and acquisition modes.

Current progress on the production, modification, and applications of bacterial cellulose.

Adoption of biomass for the development of biobased products has become a routine agenda in evolutionary metabolic engineering. Cellulose produced by bacteria is a "rising star" for this sustainable development. Unlike plant cellulose, bacterial cellulose (BC) shows several unique properties like a high degree of crystallinity, high purity, high water retention, high mechanical strength, and enhanced biocompatibility. Favored with those extraordinary properties, BC could serve as ideal biomass for the development of various industrial products. However, a low yield and the requirement for large growth media have been a persistent challenge in mass production of BC. A significant number of techniques has been developed in achieving efficient BC production. This includes the modification of bioreactors, fermentation parameters, and growth media. In this article, we summarize progress in metabolic engineering in order to solve BC growth limitation. This article emphasizes current engineered BC production by using various bioreactors, as well as highlighting the structure of BC fermented by different types of engineered-bioreactors. The comprehensive overview of the future applications of BC, aims to provide readers with insight into new economic opportunities of BC and their modifiable properties for various industrial applications. Modifications in chemical composition, structure, and genetic regulation, which preceded the advancement of BC applications, were also emphasized.

Enzymatic degradation of ginkgolic acid by laccase immobilized on novel electrospun nanofiber mat.

BACKGROUND: Ginkgo biloba leaf extract contains many active ingredients that are beneficial for health. However, ginkgolic acid, one of the major components found in G. biloba extract, may cause serious allergic and toxic side effects. The purpose of this study is to immobilize the laccase system on the electrospun nylon fiber mat (NFM) to hydrolyze the ginkgolic acid in G. biloba leaf extract efficiently. RESULTS: Novel electrospinning technology successfully produced high-quality nanoscopic fiber mats made of a mixture of multi-walled carbon nanotube and nylon 6,6. Laccase that was immobilized onto the NFM exhibited much higher efficiency in the catalyzation of 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) than nylon 6,6 pellets. After being immobilized onto the NFM, the pH and temperature stability of laccase were significantly improved. The NFM-immobilized laccase could maintain more than 50% of its original activity even after 40 days of storage or 10 operational cycles. The kinetic parameters, including rate constant (K), the time (τ50) in which 50% of ginkgolic acid hydrolysis was reached, the time (τcomplete) required to achieve complete ginkgolic acid hydrolysis, Km and Vmax were determined, and were 0.07 ± 0.01 min-1 , 8.97 ± 0.55 min, 45.45 ± 2.79 min, 0.51 ± 0.09 mM and 0.49 ± 0.03 mM min-1 mg-1 , respectively. CONCLUSION: The result successfully demonstrated the strong potential of using novel electrospun nanofiber mats as enzyme immobilization platforms, which could significantly enhance enzyme activity and stability. © 2020 Society of Chemical Industry.

The use of atmospheric-pressure chemical ionization for pesticide analysis using liquid chromatography mass spectrometry.

Based on the regulations of the Ministry of Health and Welfare (MOHW) of Taiwan in 2017, an analysis of 373 pesticides in food was conducted using the MOHW official method. The analyses involved the use of either liquid chromatography mass spectrometry (LC-MS) with electrospray ionization (ESI) or gas chromatography mass spectrometry (GC-MS) with electron ionization (EI). In this study, the applicability of detecting pesticides using atmospheric pressure chemical ionization (APCI) was investigated and evaluated. The pesticides were separated using an aqueous solution of ammonium formate with methanol as the mobile phase, and ionization efficiency was compared between APCI, ESI, and EI coupled with triple quadrupole mass spectrometer using multiple reaction monitoring (MRM) acquisition. Among the 196 pesticides that were originally analyzed by ESI, 164 could be successfully detected by APCI with 6 showing a higher sensitivity when APCI was used. Among the 177 pesticides that were analyzed by EI, 43 could be successfully detected by APCI. The results also showed that APCI gave superior ionization efficiency for pesticides containing triazine, imidazole, triazole, and pyrazole groups.

Analysis of heterocyclic aromatic amines using selective extraction by magnetic molecularly imprinted polymers coupled with liquid chromatography - Mass spectrometry.

Heterocyclic aromatic amines (HCAs) are highly carcinogenic and mutagenic chemicals. This study reports on the development of magnetic molecularly imprinted polymers (MMIPs) for the purification and quantification of HCAs. A novel magnetic molecularly imprinted polymer was successfully prepared using a surface molecular imprinting method using functionalized Fe particles as the magnetic cores. 2-Amino-3-methylimidazo[4,5-f]quinoline (IQ) was used as a molecular template; methacrylic acid (MAA), ethylene glycol dimethyl acrylate (EGDMA), 2, 2'-Azobis (2-methylpropionitrile) were used as the functional monomer, crosslinker, and initiator, respectively. The use of the template/functional monomer/crosslinking agent at a ratio of 1:4:20 resulted in a product with better adsorption properties (3.24 mg/g). The HCAs were successfully detected and quantified in processed meat samples by MISPE and LC-MS/MS. Under the final optimized detection conditions, the proposed method offered good linearity (R > 0.995) for the five HCAs with an acceptable level of precision, and an LOQ of 0.05 ng/g was successfully achieved.

Extracts of Antrodia cinnamomea mycelium as a highly potent tyrosinase inhibitor.

BACKGROUND: Ganoderma has been known as a cure for diseases since ancient times, and been used as a medicinal mushroom for more than 2000 years. By many accounts, Ganoderma lucidum extracts from fruit bodies exhibited the comparable tyrosinase inhibition activity. AIMS: To validate A. cinnamomea mycelia anti-melanogenesis activity. Ethanolic extracts of A. cinnamomea mycelia were evaluated using in vitro cell-free tyrosinase assay, cell-based and zebrafish phenotype-based method. Meanwhile, safety assessment was also conducted to ensure the feasibility as the novel ingredients in cosmetic and pharmaceutic industries. METHODS: The major regulatory enzymes being in charge of cutaneous pigmentation, was investigated in both cell-free and cellular enzyme systems, and in phenotype-based zebrafish model. A high-throughput TLC in vitro screening system was introduced to perform the initial evaluation of those with anti-melanin formation activity. RESULTS: Among the fractions, 50% ethanol extracted fraction (AC_Et50_Hex) exhibited highest anti-melanin formation activity. AC_Et50_Hex (at 100 ppm) reduced 30% intracellular melanin of B16-F10 cells through suppression of tyrosinase activity and its protein expression. For animal study, not only does AC_Et50_Hex exhibited similar depigmenting efficacy to kojic acid (56.1% vs 52.3%) with lower dosage (50 ppm vs 1400 ppm), but showed less toxicity to zebrafish. CONCLUSION: A. cinnamomea mycelium extracts can be an ideal candidate/substitute for skin-whitening since kojic acid has been reported with carcinogenic effect. AC_Et50_Hex was recognized as a potential tyrosinase inhibitor throughout in vitro and in vivo analysis studies. The mass production of A. cinnamomea mycelium from agitated fermentation realizes the natural mushroom extracts for commercial application.

Enhanced bioethanol production using atmospheric cold plasma-assisted detoxification of sugarcane bagasse hydrolysate.

The current study used acid hydrolysis of lignocellulosic materials to obtain fermentable sugar for bioethanol production. However, toxic compounds that inhibit fermentation are also produced during the process, which reduces the bioethanol productivity. In this study, atmospheric cold plasma (ACP) was adopted to degrade the toxic compounds within sulfuric acid-hydrolyzed sugarcane bagasse. After ACP treatment, significant decreases in toxic compounds (31% of the formic acid, 45% of the acetic acid, 80% of the hydroxymethylfurfural, and 100% of the furfural) were observed. The toxicity of the hydrolysate was low enough for bioethanol production using Kluyveromyces marxianus. After adopting optimal ACP conditions (200 W power for 25 min), the bioethanol productivity improved from 0.25 to 0.65 g/L/h, which means that ACP could effectively degrade toxic compounds within the hydrolysate, thereby enhancing bioethanol production. Various nitrogen substitute was coordinated with detoxified hydrolysate, and chicken meal group presented the highest bioethanol productivity (0.45 g/L/h).

Hydrolyzation of mogrosides: Immobilized ß-glucosidase for mogrosides deglycosylation from Lo Han Kuo.

An immobilized enzyme system for bioconversion of Lo Han Kuo (LHK) mogrosides was established. ß-Glucosidase which was covalently immobilized onto the glass spheres exhibited a significant bioconversion efficiency from pNPG to pnitrophenol over other carriers. Optimum operational pH and temperature were determined to be pH 4 and 30°C. Results of storage stability test demonstrated that the glass sphere enzyme immobilization system was capable of sustaining more than 80% residual activity until 50 days, and operation reusability was confirmed for at least 10 cycles. The Michaelis constant (K m) of the system was determined to be 0.33 mM. The kinetic parameters, rate constant (K) at which Mogrosides conversion was determined, the τ 50 in which 50% of mogroside V deglycosylation/mogroside IIIE production was reached, and the τ complete of complete mogroside V deglycosylation/mogroside IIIE production, were 0.044/0.017 min-1, 15.6/41.1 min, and 60/120 min, respectively. Formation of the intermediates contributed to the kinetic differences between mogroside V deglycosylation and mogroside IIIE formation.

Characteristics of fucose-containing polysaccharides from submerged fermentation of Agaricus blazei Murill.

Fucose is one of important residues of recognition pattern for many immune cells. In this study, we characterized bioactive fucose-containing acidic polysaccharides from submerged fermentation of Agaricus blazei Murill. We obtained the polysaccharides through a cell-based activity-guided strategy, and used carbohydrate recognition monoclonal antibodies based Enzyme-Linked Immuno Sorbent Assay (ELISA) along with methylation and NMR analyses to investigate the structural characteristics of the polysaccharides. The polysaccharides had Mw of 3.5 × 105 Da. The major sugars were l-fucose, l-arabinose, d-galactose, d-xylose, and d-galacturonic acid in the molar ratio of 6.4, 15.5, 28.5, 14.7, and 25.0% with a small amount of d-glucose, d-mannose, l-rhamnose, and d-glucuronic acid. Results indicated that the bioactive polysaccharides consisted of a (1,4)-Galp and (1,4)-GalAp back bone; (1,2)-Xyl and (1,2)-Rha might also comprise backbone or constitute side chain; linkage (1,5)-Ara and terminal fucosyl residues were also involved in the polysaccharides. Regarding bioactivity, removal of the terminal l-fucosyl residues reduced the TNF-α cytokine stimulating activity of the polysaccharides in a RAW 264.7 macrophage cell-line test, whereas NF-κB and TLR4 affected the polysaccharide-induced TNF-α production.

Parallel minisequencing followed by multiplex matrix-assisted laser desorption/ionization mass spectrometry assay for beta-thalassemia mutations.

Beta-thalassemia is a common monogenic disease caused by mutations in the human beta-globin gene (HBB), many of which are differentially represented in human subpopulations stratified by ethnicity. This study describes an efficient and highly accurate method to screen for the eight most-common disease-causing mutations, covering more than 98% of HBB alleles in the Taiwanese population, using parallel minisequencing and multiplex assay by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The MALDI-TOF MS was optimized for sensitivity and resolution by "mass tuning" the PinPoint assay for eight HBB SNPs. Because of the close proximity and clustering of mutations in HBB, primer extension reactions were conducted in parallel. Efficient sequential desalting using POROS and cationic exchange chromatography allowed for an unambiguous multiplex genotyping by MALDI-TOF MS. The embellishing SNP assay allowed for highly accurate identification of the eight most-common beta-thalassemia mutations in homozygous normal control, carrier, and eight heterozygous carrier mixtures, as well as the diagnosis of a high-risk family. The results demonstrated a flexible strategy for rapid identification of clustering SNPs in HBB with a high degree of accuracy and specificity. It can be adapted easily for high-throughput diagnosis of various hereditary diseases or to establish family heritage databases for clinical applications.

Specific recognition of DNA bulge structure by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

This study reports a novel approach utilizing an octahedral CoII(HAPP)(TFA)2 reagent in the presence of H2O2 with analysis by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) to serve as an efficient probe for bulged DNA structures. Elucidation of DNA bulge-specific recognition pathways and cleavage mechanisms is demonstrated by characterization of bulge-specific cleavage products and other backbone lesion fragments. The cleavage specificity of CoII(HAPP)(TFA)2/H2O2 arises from sugar oxidative strand scission, for which the position of the abstracted hydrogen is unambiguously determined as the 4'-H of the deoxyribose moiety. Furthermore, differentiation between bulge-specific recognition and diffusion-controlled non-selective cleavage can be clarified through time-dependent MALDI-TOFMS studies. The present results demonstrate that MALDI-TOFMS can be a sensitive and efficient technique for complex mechanistic studies of this kind, providing information for future rational drug design targeting bulged DNA structures.