Self-assembled copper nanoclusters used to mimic peroxidase for glucose detection.

A sensing system for glucose was established based on a self-assembled copper nanoclusters (Cu NCs)-based nano-enzyme and glucose oxidase (GOD). The assembled copper nanosheets (Cu NSs) were prepared in a one-step method using 2,3,4,5,6-pentafluorothiophenol (PFTP) as a reducing agent and protecting ligand. Cu NSs could be used to mimic the enzyme horseradish peroxidase. Cu NSs were endowed with excellent enzymatic catalytic activity in the oxidation of o-phenyldiamine (OPD) in the presence of H2O2. The latter could be generated in the aerobic oxidation of glucose catalyzed by GOD. Therefore, a detection method for glucose was constructed based on a Cu NSs-OPD-GOD catalytic system. This proposed sensing platform showed a standard linear range from 10 µM to 5 mM towards glucose, and the limit of detection was 5.5 µM. Finally, practical application of a sensor based on the Cu NSs nano-enzyme was verified in three sugared beverages as real samples. Our data reveal that the prepared Cu NSs could mimic peroxidase and be applied to a mixed catalytic system with GOD for glucose detection.

Quantification and isotope abundance determination of 13C labeled intracellular sugar metabolites with hydrophilic interaction liquid chromatography.

Metabolic flux analysis (MFA) using stable isotope labeled tracers is a powerful tool to estimate fluxes through metabolic pathways. It finds applications in studying metabolic changes in diseases, regulation of cellular energetics, and novel strategies for metabolic engineering. Accurate and precise quantification of the concentration of metabolites and their labeling states is critical for correct MFA results. Utilizing an ultra-high performance liquid chromatography-high resolution mass spectrometry (UHPLC-HRMS) system, an analytical method for simultaneously quantifying the concentration of sugar metabolites and their mass isotopologue distribution (MID) was developed. The method performs with good linearity and coefficient of determination (R2) > 0.99, while the detection limit ranged from 0.1 to 50 mg L-1. Seven sugar metabolites were detected in a labeled Brevibacterium flavum sample using the method. The detected quantities ranged from 6.15 to 3704.21 mg L-1, and 13C abundance was between 12.77% and 66.67% in the fermentation fluid and 16.28% and 91.93% in the bacterial body. Overall, the method is efficient, accurate, and suitable for analysis of labeled sugar metabolites in 13C MFA studies.

Unveiling the Biochar-Respiratory Growth of Methanosarcina acetivorans Involving Extracellular Polymeric Substances.

Biochar can be applied to diverse natural and engineered anaerobic systems. Biochar plays biogeochemical roles during its production, storage, and environmental dynamics, one of which is related to the global methane flux governed by methanotrophs and methanogens. Our understanding of relevant mechanisms is currently limited to the roles of biochar in methanotrophic growth, but less is known about the roles of biochar in methanogenic growth. Here, we demonstrated that biochar enhanced the methanogenic growth of a model methanogen, Methanosarcina acetivorans, and the role of biochar as an electron acceptor during methanogenic growth was confirmed, which is referred to as biochar-respiratory growth. The biochar-respiratory growth of M. acetivorans promoted the secretion of extracellular polymeric substances (EPS) with augmented electron transfer capabilities, and the removal of EPS significantly attenuated extracellular electron transfer. Identification and quantification of prosthetic cofactors for EPS suggest an important role of flavin and F420 in extracellular electron transfer. Transcriptomic analysis provided additional insights into the biochar-respiratory growth of M. acetivorans, showing that there was a positive response in transcriptional regulation to the favorable growth environment provided by biochar, which stimulated global methanogenesis. Our results shed more light on the in situ roles of biochar in the ecophysiology of methanogens in diverse anaerobic environments.

Effects of dietary supplementation of Anoectochilus roxburghii extract (ARE) on growth performance, abdominal fat deposition, meat quality, and gut microbiota in broilers.

The broiler industry frequently encounters 2 common problems: excessive deposition of abdominal fat and poor quality of meat. However, there are limited nutritional manipulation strategies to address these issues. While Anoectochilus roxburghii (Wall.) Lindl., a traditional Chinese herb, has been shown to have multiple beneficial effects in humans, its potential roles in broiler chickens remain unexplored. In this study, the effects of dietary supplementation with Anoectochilus roxburghii extract (ARE) on growth performance, abdominal fat deposition, meat quality, blood indices, and gut microbiota were investigated in yellow-feather broiler chickens. A total of 90 twenty-one-day-old yellow-feather broilers were randomly divided into 3 treatments, and each treatment included 5 replicates with 6 birds per replicate. Birds were fed a basal diet supplemented with 0, 0.15, or 0.30% ARE for 6 wk. The results showed that the inclusion of ARE in the diet did not have any significant effect on meat yield (P > 0.05). However, it did lead to a reduction in abdominal fat deposition and an improvement in meat quality (P < 0.05). Mechanistically, the addition of ARE inhibited lipid biosynthesis and enhanced lipid breakdown in both the liver and adipose tissue of the broilers. Furthermore, ARE supplementation increased the antioxidase activities in the muscle and serum of the broilers (P < 0.05). In addition, the supplementation of ARE optimized the diversity and composition of the cecal microbiota, particularly by lowering the ratio of Firmicutes to Bacteroidetes (P < 0.05). Moreover, the abundance of some bacteria that were positively correlated with abdominal fat deposition was reduced by ARE, and vice versa (P < 0.05). Collectively, the results suggest that ARE is a promising candidate as a feed additive for reducing abdominal fat deposition and improving meat quality in the broiler industry.

BLTSA: pseudotime prediction for single cells by branched local tangent space alignment.

MOTIVATION: The development of single-cell RNA sequencing (scRNA-seq) technology makes it possible to study the cellular dynamic processes such as cell cycle and cell differentiation. Due to the difficulties in generating genuine time-series scRNA-seq data, it is of great importance to computationally infer the pseudotime of the cells along differentiation trajectory based on their gene expression patterns. The existing pseudotime prediction methods often suffer from the high level noise of single-cell data, thus it is still necessary to study the single-cell trajectory inference methods. RESULTS: In this study, we propose a branched local tangent space alignment (BLTSA) method to infer single-cell pseudotime for multi-furcation trajectories. By assuming that single cells are sampled from a low-dimensional self-intersecting manifold, BLTSA first identifies the tip and branching cells in the trajectory based on cells' local Euclidean neighborhoods. Local coordinates within the tangent spaces are then determined by each cell's local neighborhood after clustering all the cells to different branches iteratively. The global coordinates for all the single cells are finally obtained by aligning the local coordinates based on the tangent spaces. We evaluate the performance of BLTSA on four simulation datasets and five real datasets. The experimental results show that BLTSA has obvious advantages over other comparison methods. AVAILABILITY AND IMPLEMENTATION: R codes are available at https://github.com/LiminLi-xjtu/BLTSA. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Prevalence of latent tuberculosis infection and incidence of active tuberculosis in school close contacts in Shanghai, China: Baseline and follow-up results of a prospective cohort study.

Background: The management of latent tuberculosis infection (LTBI) is a key action for the realization of the "End tuberculosis (TB) Strategy" worldwide, and it is important to identify priority populations. In this prospective cohort study, we evaluated the prevalence of LTBI and incidence of active TB among close contacts and explored the suitable TB control strategy in schools. Methods: We designed a cohort with 2 years of follow-up, recruiting freshman/sophomore TB patients' close contacts from three administrative districts in Shanghai. These were chosen based on different levels of TB incidence reported in 2019. Questionnaires were included and all participants received both tuberculin skin test (TST) and QuantiFERON-TB Gold (QFT) at baseline, then tracked the outcomes of them during the follow-up period. Results: The prevalence of LTBI was 4.8% by QFT. Univariate analysis showed that the risk of LTBI was higher in those contacting bacteriologically confirmed patients or did not have BCG scars, including smokers. The risk increased with poor lighting and ventilation conditions at contact sites. Multivariate analysis showed that those contacting with bacteriologically confirmed patients (OR=4.180; 95%CI, 1.164-15.011) or who did not have BCG scars (OR=5.054; 95%CI, 2.278-11.214) had a higher risk of being LTBI, as did the current smokers (OR=3.916; 95%CI, 1.508-10.168) and those who had stopped smoking (OR=7.491; 95%CI, 2.222-25.249). During the 2-year follow-up period, three clinically diagnosed cases of TB were recorded, the 2-year cumulative incidence was 0.4% (95%CI 0.1-1.2), the median duration for TB occurrence was 1 year, the incidence rate of active TB was 2.0 per 1000 person-years with a total of 1497.3 observation person-years. For those LTBI, no one initiated preventive treatment, in the QFT (+) cohort, 1 TB case was observed, 71 person-years with an incidence rate of 14.1 14.1 (95%CI 2.5-75.6) per 1000 person-years, in the TST (+++) cohort, 2 TB cases were observed 91.5 person-years with an incidence rate of 21.9 (95%CI 6.0-76.3) per 1000 person-years. Conclusions: The results suggest that school close contacts are one of the key populations for LTBI management. Measures should be taken to further reduce the prevalence of LTBI and the incidence of active TB among them.

Discovery of New Inhibitors of eEF2K from Traditional Chinese Medicine Based on In Silico Screening and In Vitro Experimental Validation.

Eukaryotic elongation factor 2 kinase (eEF2K) is a highly conserved α kinase and is increasingly considered as an attractive therapeutic target for cancer as well as other diseases. However, so far, no selective and potent inhibitors of eEF2K have been identified. In this study, pharmacophore screening, homology modeling, and molecular docking methods were adopted to screen novel inhibitor hits of eEF2K from the traditional Chinese medicine database (TCMD), and then cytotoxicity assay and western blotting were performed to verify the validity of the screen. Resultantly, after two steps of screening, a total of 1077 chemicals were obtained as inhibitor hits for eEF2K from all 23,034 compounds in TCMD. Then, to verify the validity, the top 10 purchasable chemicals were further analyzed. Afterward, Oleuropein and Rhoifolin, two reported antitumor chemicals, were found to have low cytotoxicity but potent inhibitory effects on eEF2K activity. Finally, molecular dynamics simulation, pharmacokinetic and toxicological analyses were conducted to evaluate the property and potential of Oleuropein and Rhoifolin to be drugs. Together, by integrating in silico screening and in vitro biochemical studies, Oleuropein and Rhoifolin were revealed as novel eEF2K inhibitors, which will shed new lights for eEF2K-targeting drug development and anticancer therapy.

Effects of Dietary Inclusion of ß-Hydroxy-ß-Methylbutyrate on Growth Performance, Fat Deposition, Bile Acid Metabolism, and Gut Microbiota Function in High-Fat and High-Cholesterol Diet-Challenged Layer Chickens.

Excessive lipid deposition in layer chickens due to inappropriate feeding adversely affects egg production; however, nutritional manipulation methods to deal with this issue are still limited. ß-hydroxy-ß-methylbutyrate (HMB), a metabolite of L-leucine, was recently reported as a lipid-lowering nutrient in mice and pigs, although its role in layers had not been investigated. Here, we employed high-fat and high-cholesterol diet (HFHCD)-challenged growing layers as an obese model to explore HMB function in the regulation of lipid metabolism and the potential mechanisms involved. We found that dietary supplementation with (0.05% or 0.10%) HMB significantly reduced HFHCD-induced bodyweight growth in layers, mainly due to reduction in abdominal fat deposition. Mechanistically, HMB supplementation enhanced hepatic bile acid synthesis from cholesterol through elevating expression of Cyp7a1, a gene coding a key enzyme in bile acid synthesis. Furthermore, 16S rRNA gene sequencing revealed that HMB supplementation remodeled the diversity and composition of the layers' cecal microbiota, and the abundance of Bacteroidetes at the phylum level were especially affected. Correlation analysis further indicated a strong negative association between Bacteroidetes abundance and lipid metabolism-related parameters. Taken together, these data suggest that dietary HMB supplementation could improve abdominal fat deposition in layers, probably through modulating hepatic bile acid synthesis and gut microbiota function.

Determination of isotope abundance for deuterium-labeled compounds by quantitative 1 H NMR + 2 H NMR.

Deuterated reagents have been used in many research fields. Isotope abundance, as the feature parameter of deuterated reagents, the precise quantification, is of great importance. Based on quantitative nuclear magnetic resonance technology, a novel method that combines 1 H NMR + 2 H NMR was systematically established to determine the isotopic abundance of deuterated reagents. The results showed that the isotopic abundance of partially labeled and fully labeled compounds calculated by this new method was even more accurate than that calculated by classical 1 H NMR and mass spectrometry (MS) methods. In brief, this new method is a robust strategy for the determination of isotope abundance in large-scale deuterated reagents.

Design and application of hydrophilic bimetallic metal-organic framework magnetic nanoparticles for rapid capture of exosomes.

Functional materials with good biocompatibility have been widely used in the study of genomics, proteomics and disease diagnosis, which has improved the progress of life science. In this paper, the material not only exhibited a strong affinity to the phosphate groups on the exosomal membrane due to the coexistence of Zr-O clusters and Ti4+, but also owned great hydrophilicity to reduce non-specific adsorption of contaminated proteins, achieving the separation and purification of exosomes from complex biosamples. The model exosomes extracted by ultracentrifugation (UC) were used to evaluate the feasibility of Fe3O4@UiO-66-NH2@PA-Ti4+ capturing exosomes. The process of Fe3O4@UiO-66-NH2@PA-Ti4+ capturing exosomes was simple to operate with a high recovery rate (97.3%) within a short time (5 min). Then Fe3O4@UiO-66-NH2@PA-Ti4+ was further applied to capture exosomes in media and urine followed by the downstream proteomics analysis. 348 and 284 exosomal proteins were identified for cell medium and urine, respectively. This work shows great potential of the material for subsequent function research of disease-related exosomes by separating exosomes rapidly and efficiently.

Mutagenicity, health risk, and disease burden of exposure to organic micropollutants in water from a drinking water treatment plant in the Yangtze River Delta, China.

A wide variety of organic micropollutants in drinking water pose a serious threat to human health. This study was aimed to reveal the characteristics of organic micropollution profiles in water from a drinking water treatment plant (DWTP) in the Yangtze River Delta, China and investigate the mutagenicity, health risk and disease burden through mixed exposure to micropollutants in water. The presence of organic micropollutants in seven categories in organic extracts (OEs) of water from the DWTP was determined, and Ames test was conducted to test the mutagenic effect of OEs. Meanwhile, health risk of exposure to organic micropollutants in finished water through three exposure routes (ingestion, dermal absorption and inhalation) was assessed with the method proposed by U.S. EPA, and disability-adjusted life years (DALYs) were combined to estimate the disease burden of cancer based on the carcinogenic risk (CR) assessment. The results showed that 28 organic micropollutants were detected in the raw and finished water at total concentrations of 967.28 ng/L and 1073.45 ng/L, respectively, of which phthalate esters (PAEs) were the dominant category (95.79% in the raw water and 96.61% in the finished water). Although the results of the Ames test for OEs were negative and the non-carcinogenic hazard index of the organic micropollutants in the finished water was less than 1 in all age groups, the total CR was 2.17 × 10-5, higher than the negligible risk level (1.00 × 10-6). The total DALYs caused by the organic micropollutants in the finished water was 2945.59 person-years, and the average individual DALYs was 2.21 × 10-6 per person-year (ppy), which was 2.21 times the reference risk level (1.00 × 10-6 ppy) defined by the WHO. Exposure to nitrosamines (NAms) was the major contributor to the total CR (92.06%) and average individual DALYs (94.58%). This study demonstrated that despite the negative result of the mutagenicity test with TA98 and TA100 strains, the health risk of exposure to organic micropollutants in drinking water should not be neglected.

A metal oxide affinity probe derived from MIL-125 for selective enrichment of endogenous phosphopeptides.

Highly effective enrichment of endogenous phosphopeptides from complex biological samples is an essential and crucial theme in the analysis of phosphopeptidomics. Herein, an ordered mesoporous TiO2/C composite (denoted as Ti-MCM) was prepared by the pyrolysis of MIL-125 under a N2 atmosphere. The obtained Ti-MCM possesses a high specific surface area (165 m2 g-1), a uniform pore size (3.75 nm), and a large amount of Ti (46%). By utilizing the selective chelation between Ti-MCM and phosphopeptides, 25 phosphopeptides were detected in α-casein digest after enrichment. The material shows good selectivity even in the presence of 2000-fold excess of interference peptides. It was also used to enrich endogenous phosphopeptides from the complex samples of human serum and saliva and showed a good performance.

Effects of leaf removal on the accumulation of anthocyanins and the expression of anthocyanin biosynthetic genes in Cabernet Sauvignon (Vitis vinifera L.) grapes.

BACKGROUND: Anthocyanins, a major flavonoid class, determine the color and quality of wine. Recent research revealed that basal leaf removal can increase the content of these compounds. This study determined the effects of basal leaf removal on the anthocyanin profiles of Cabernet Sauvignon grapes. RESULTS: The effects of basal leaf removal on anthocyanin composition in Cabernet Sauvignon grapes were investigated over two growing seasons. Leaf removal at 5% veraison (VB6) and at 100% veraison (VC6) was compared with a control. Reducing sugar and total anthocyanin contents in the leaf removal group were significantly higher than in the control group at harvest for both vintages. Leaf removal increased the content of individual anthocyanins and significantly improved the malvidin-3-O-glucoside (Mv-3-glc), peonidin-3-O-glucoside (Pn-3-glc), and malvidin-3-O-(6-acetyl)-glucoside (Mv-3-acglc) content of the VB6 group. At harvest, VB6 treatment increased the transcript abundance of structural and regulator genes in the anthocyanin pathway, especially VvF3'5'H, VvLDOX, and VvDFR. CONCLUSIONS: Our results suggest that leaf removal at 5% veraison may be useful for improving the anthocyanin content in grapes. © 2020 Society of Chemical Industry.

Preparation and application of peptide molecularly imprinted material based on mesoporous metal-organic framework.

In this study, a new molecularly imprinted material, MIP@UiO-66-NH2, was synthesized with glutathione (GSH) as template and mesoporous metal organic framework (UiO-66-NH2) as matrix. The molecularly imprinted polymer was modified on the surface and into the pores of the UiO-66-NH2 by surface molecular imprinting method with thin polymer layer. Based on high specific surface area (1091.93 m2 g-1) and appropriate pore size (35 nm) of the ordered mesoporous UiO-66-NH2, the adsorption capacity for GSH reached 94.43 mg g-1, and the adsorption equilibrium could be achieved within 30 min. The adsorption isotherm data of MIP@UiO-66-NH2 could be described well by Freundlich model and the kinetic data complied well with pseudo-second-order model. In addition, the MIP@UiO-66-NH2 showed low adsorption capacity to GSH structural analogs (QL-cys = 6.51 mg g-1), suggesting great selectivity for GSH recognition. Finally, the MIP@UiO-66-NH2 was successfully applied for selective separation of GSH from BSA, skim milk and egg white tryptic digest.

Core-shell magnetic microporous covalent organic framework with functionalized Ti(iv) for selective enrichment of phosphopeptides.

Selective, sensitive and efficient phosphopeptide enrichment is significant in understanding phosphorylation-regulated processes and finding potential biomarkers. Magnetic materials have significant advantages for the separation and enrichment of modified peptides and proteins. In this work, we fabricated a core-shell magnetic Ti4+-functionalized covalent organic framework composite (denoted as Fe3O4@SiO2@TpPa-Ti4+) to selectively capture phosphopeptides in biosamples. The specific porous structure makes high surface area, which provides space and more affinity sites to binding titanium ions. The loading capacity of titanium ions was as high as 14% (wt%). Calculated by the MS results of α-casein tryptic digests, the binding capability could reach 200 mg g-1. Fe3O4@SiO2@TpPa-Ti4+ nanoparticles also demonstrate high sensitivity (above 0.2 fmol µL-1) and selectivity (α-casein : BSA = 1 : 5000). 1083 phosphopeptides were identified within two LC-MS/MS replicates with 91.8% specificity (phosphopeptides/all identified peptides). These MS results demonstrate that with the aid of the magnet, this method based on the magnetic titanium-functionalized covalent organic framework composite is applicable to achieve rapid, selective and efficient phosphopeptide analysis.

Core-shell magnetic bimetallic MOF material for synergistic enrichment of phosphopeptides.

In proteomics, phosphorylation is an important process for protein post-translational modification (PTM), which greatly improves the diversity of proteomes. The PTM regulates almost all physiological and pathological processes such as signal transduction, cell division, proliferation, differentiation and metabolism. The abnormal expression of protein phosphorylation is also associated with cellular metabolic disorders and a range of diseases. However, in mass spectrometry-based phosphorylated peptideomics studies, phosphorylated peptide signals were inhibited by a high abundance of non-phosphorylated peptides; thus, highly selective enrichment was required. In this study, a newly designed material named Fe3O4@MIL(Fe/Ti) was synthesized using a layer-by-layer self-assembly technique that coats the surface of magnetic oxide nanospheres with bimetallic MOF of iron and titanium. The synergistic synthetic coating of the bimetallic MOF gives the material a large surface area and excellent hydrophilicity, which endow the nanoparticles with excellent phosphopeptide enrichment ability, high selectivity (ß-casein/BSA molar ratio 1:500), a low detection limit (3 fmol), high recovery rate (85%), strong binding capacity, size exclusion ability, and ideal batch-to-batch repeatability. For comparison, we used Fe3O4@MIL(Fe/Ti) and two single-metal MOF materials Fe3O4@MIL-100(Fe) and Fe3O4@MIL-125(Ti), to enrich α-casein in the middle. Thus, the iron-titanium bimetallic MOF can not only enrich all the phosphorylated peptides enriched by Fe3O4@MIL-100(Fe) and Fe3O4@MIL-125(Ti), but can also specifically enrich four phosphorylated peptides. Encouraged by the excellent results of characterization and standard protein enrichment, we used this material to analyze human serum and found that bimetallic materials can effectively enrich all four phosphorylated peptides and exclude high molecular proteins. These experimental results indicate that the novel bimetallic MOF is a good candidate to analyze protein phosphorylation in complex samples.

[Preparation of magnetic nanoparticles based on MIL-100(Fe) and its application in the enrichment of endogenous peptides].

Core-shell magnetic nanoparticles, Fe3O4@MIL-100(Fe), were synthesized by layer-by-layer self-assembly. The final material, Fe3O4@MC, was obtained after high temperature calcination. Fe3O4@MC magnetic nanoparticles exhibited large pore size (17.78 nm) and high carbon content (6.79%). Thirty-three peptides of bovine serum albumin (BSA) digest were enriched by Fe3O4@MC magnetic nanomaterial. Fe3O4@MC magnetic nanomaterial also showed high selectivity for peptides when the mass ratio of BSA digest to BSA was 1:400. In addition, the nanomaterial material exhibited excellent enrichment performance for the endogenous peptides of human serum.

Transition metal embedded C3N monolayers as promising catalysts for the hydrogen evolution reaction.

Transition metal (TM) doped or TM, N co-doped carbon materials have attracted increasing attention as efficient catalysts for the hydrogen evolution reaction (HER), to replace Pt or reduce the usage of Pt. By using first-principles calculations, the TM-embedded C3N monolayer (TM@C3N) has been theoretically investigated for HER, for which eighteen TMs are selected from the 3d, 4d, and 5d rows. The M-CC catalysts, with the TM atom embedded into the C-C double atomic vacancy, are the most stable among the various TM@C3N materials. All the M-CC catalysts show metallic conductivity and high thermal stability. The hydrogen binding free energy for the M-CC catalysts can be optimized to be close to 0 eV by choosing a suitable TM, and the kinetic barrier under the Tafel mechanism for further gaseous hydrogen evolution can be reduced to as low as 0.58 eV. These results suggest that the HER catalytic activities of the M-CC catalysts are likely comparable or even higher than those of the well-explored MoS2 nanostructures or Pt catalysts. Moreover, the HER activities of the M-CC catalysts can be illustrated by the electronic state distribution near the Fermi level of the catalytically active sites. This study provides a new possibility for cost-efficient HER catalysts of high activity and for the application of C3N nanostructures.

Preparation of mesoporous carbon material derived from Metal-Organic Frameworks and its application in selective capture of endogenous peptides from human serum.

Highly selective and efficient extraction of endogenous peptides from complex biological systems plays an important role in proteomics. In this study, an ordered mesoporous magnetic material (denoted as CZIF) with micron size (1 µm) was facilely synthesized by direct pyrolyzing ZIF-67 under N2 atmosphere for efficient and selective enrichment of endogenous peptides. The CZIF keeps the morphology of the original ZIF-67 crystal with enlarged pore size of 3.67 nm and a specific surface area of 156.58 m2/g, and shows weak magnetism (18 emu/g). The loading buffer and eluting buffer were optimized to improve the sensitivity and selectivity of the CZIF. Benefiting from the highly graphitization of the CZIF, the limit detection of BSA digest as low as 0.2 fmol/µL was achieved, showing good enrichment sensitivity for peptides. The uniform mesoporous help to achieve high selectivity by excluding proteins (BSA tryptic digest/BSA = 1:1000). The CZIF can be repeatedly used (n ≥ 5) without further treatment. Moreover, 402 endogenous peptides were enriched from human serum by CZIF, which indicated that CZIF was a promising candidate for the extraction of endogenous peptides from complex biologicals.

Carrier ampholyte-free free-flow isoelectric focusing for separation of protein.

Free-flow isoelectric focusing (FFIEF) has the merits of mild separation conditions, high recovery and resolution, but suffers from the issues of ampholytes interference and high cost due to expensive carrier ampholytes. In this paper, a home-made carrier ampholyte-free FFIEF system was constructed via orientated migration of H+ and OH- provided by electrode solutions. When applying an electric field, a linear pH gradient from pH 4 to 9 (R2 = 0.994) was automatically formed by the electromigration of protons and hydroxyl ions in the separation chamber. The carrier ampholyte-free FFIEF system not only avoids interference of ampholyte to detection but also guarantees high separation resolution by establishing stable pH gradient. The separation selectivity was conveniently adjusted by controlling operating voltage and optimizing the composition, concentration and flow rate of the carrier buffer. The constructed system was applied to separation of proteins in egg white, followed by MADLI-TOF-MS identification. Three major proteins, ovomucoid, ovalbumin and ovotransferrin, were successfully separated according to their pI values with 15 mmol/L Tris-acetic acid (pH = 6.5) as carrier buffer at a flow rate of 12.9 mL/min.