Clopidogrel Plus Aspirin vs Aspirin Alone in Patients With Acute Mild to Moderate Stroke: The ATAMIS Randomized Clinical Trial.

Importance: Dual antiplatelet therapy has been demonstrated to be superior to single antiplatelet in reducing recurrent stroke among patients with transient ischemic attack or minor stroke, but robust evidence for its effect in patients with mild to moderate ischemic stroke is lacking. Objective: To evaluate whether dual antiplatelet therapy is superior to single antiplatelet among patients with mild to moderate ischemic stroke. Design, Setting, and Participants: This was a multicenter, open-label, blinded end point, randomized clinical trial conducted at 66 hospitals in China from December 20, 2016, through August 9, 2022. The date of final follow-up was October 30, 2022. The analysis was reported on March 12, 2023. Of 3065 patients with ischemic stroke, 3000 patients with acute mild to moderate stroke within 48 hours of symptom onset were enrolled, after excluding 65 patients who did not meet eligibility criteria or had no randomization outcome. Interventions: Within 48 hours after symptom onset, patients were randomly assigned to receive clopidogrel plus aspirin (n = 1541) or aspirin alone (n = 1459) in a 1:1 ratio. Main Outcomes and Measures: The primary end point was early neurologic deterioration at 7 days, defined as an increase of 2 or more points in National Institutes of Health Stroke Scale (NIHSS) score, but not as a result of cerebral hemorrhage, compared with baseline. The superiority of clopidogrel plus aspirin to aspirin alone was assessed based on a modified intention-to-treat population, which included all randomized participants with at least 1 efficacy evaluation regardless of treatment allocation. Bleeding events were safety end points. Results: Of the 3000 randomized patients, 1942 (64.6%) were men, the mean (SD) age was 65.9 (10.6) years, median (IQR) NIHSS score at admission was 5 (4-6), and 1830 (61.0%) had a stroke of undetermined cause. A total of 2915 patients were included in the modified intention-to-treat analysis. Early neurologic deterioration occurred in 72 of 1502 (4.8%) in the dual antiplatelet therapy group vs 95 of 1413 (6.7%) in the aspirin alone group (risk difference -1.9%; 95% CI, -3.6 to -0.2; P = .03). Similar bleeding events were found between 2 groups. Conclusions and Relevance: Among Chinese patients with acute mild to moderate ischemic stroke, clopidogrel plus aspirin was superior to aspirin alone with regard to reducing early neurologic deterioration at 7 days with similar safety profile. These findings indicate that dual antiplatelet therapy may be a superior choice to aspirin alone in treating patients with acute mild to moderate stroke. Trial Registration: ClinicalTrials.gov Identifier: NCT02869009.

Dendrobium huoshanense Polysaccharides Prevent Inflammatory Response of Ulcerative Colitis Rat through Inhibiting the NF-κB Signaling Pathway.

The polysaccharides of the Chinese herbal medicine Dendrobium huoshanense exhibit anti-inflammatory effects in multiple organs through regulating the immune responses. In the present study, we constructed ulcerative colitis (UC) model rats using dextran sulfate sodium to investigate the anti-inflammatory effects of D. huoshanense polysaccharides (DHP). After oral administration of DHP for two weeks, the indices of UC symptoms, including the ratio of colon weight to length, Disease Activity Index (DAI), and Colon Mucosal Damage Index (CMDI), all decreased significantly compared with the UC model group. The histological sections also revealed better cell orders in DHP treatments than in the UC model rats. Moreover, in treatment with high dose of DHP (200 mg/kg), the treatment efficacy arrived the similar levels to those in the treatment with 300 mg/kg sulfasalazine, which is a typical medicine to treat UC. These results indicated that DHP has a high efficacy to treat UC in model rats. Furthermore, serum levels of interleukin-1ß, tumor necrosis factor-α, interleukin-17, and transforming growth factor-ß were assessed using the enzyme linked immunosorbent assay (ELISA) method, and the levels of nuclear factor-κB in colon tissue sections were determined using the immunohistochemical method. The results showed that all these indices decreased significantly after administration of DHP in UC model rats, which might be the mechanisms underlying the DHP-suppressed UC inflammation. Overall, this study indicated that DHP might be directly used to treat UC and is a promising source to develop novel drugs against UC.

Synthesis of tellurium nanosheet for use in matrix assisted laser desorption/ionization time-of-flight mass spectrometry of small molecules.

Two-dimensional tellurium nanosheets were prepared by a hydrothermal method and characterized by scanning electron microscopy, powder X-ray diffractometry, and UV-vis spectroscopy. The nanosheets were explored as a novel matrix for desorption/ionization of small molecules including nucleobases, fatty acids and amino acids by matrix assisted laser desorption/ionization time-of-flight mass spectrometry. The results show that the tellurium nanosheets have good UV light absorption, cause low matrix ion interference in the low-molecule-mass region, and have high desorption/ionization efficiency in the negative ion mode. Hence, they are a viable matrix for negative ion desorption/ionization in MALDI-TOF MS of small molecules. In order to investigate the desorption/ionization mechanisms, benzylpyridinium salt and bisphenol A were adopted as probes. The results show that both of the electronic transitions mechanism and laser-induced thermal mechanism play important roles in desorption/ionization process. Graphical abstract Two-dimensional tellurium (Te) nanosheet was synthesized by a hydrothermal method and explored as a novel matrix for desorption/ionization of small molecules by matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS).

Hydrothermally tailor-made chitosan fiber for micro-solid phase extraction of petroleum acids in crude oils.

Tailor-made chitosan fiber was prepared via hydrothermal treatment to serve as a micro-solid phase extraction (micro-SPE) sorbent for the analysis of petroleum acids (PAs) in crude oils. Chitosan fiber, which is commercial and cheap, has a diameter of about 10 µm and a length of a few centimeters. The fibrous property of the sorbent enables the micro-SPE to deal with viscous crude oil samples because of the low back-pressure during extraction, while the abundant hydroxyl groups and amino groups on the surface of chitosan fiber can provide high density of specific sites for adsorption of PAs. Moreover, it was found that hydrothermal treatment at certain conditions could tune the surface properties of chitosan fiber, leading to significant improvement of the capacity of the fiber in adsorption of PAs. Using hydrothermally treated chitosan fiber as sorbent, the micro-SPE was applied to the determination of PAs in crude oils, with the advantages of easy-operation, rapidness and high sensitivity (the limits of detection range from 0.7 ng/g to 5.4 ng/g). Furthermore, coupled with comprehensive two dimensional gas chromatography-mass spectrometry (GC × GCMS), the treated chitosan fiber packed micro-SPE method showed a great potential for comprehensive profiling of PAs in crude oils.

Magnetic extractant with an Fe3O4@SiO2 core and aqueous ammonia coating for microextraction of petroleum acids.

Magnetic aqueous ammonia (MAA) was prepared as a magnetic extractant for dispersive microextraction of petroleum acids (PAs). The amount of extractant in MAA was custom-made by a simple approach. In the MAA composed of an aqueous ammonia coating and Fe3O4@SiO2 core, the coating is a base extractant that can selectively extract acids, while the magnetic core serves as a support to achieve dispersion as well as rapid magnetic retrieval of the extractant during the extraction processes. This is the first use of reusable, stable and modifiable Fe3O4@SiO2 as a support instead of bare Fe3O4 in a magnetic particle assisted dispersive liquid-liquid microextraction technique. The parameters that affect extraction efficiency were investigated. The sampling step as well as the desorption step can be completed in 2 min. The linear ranges are 5-5000 ng g-1, while the limits of quantification range from 2.5 to 6.2 ng g-1. The recoveries in spiked crude oil samples are in the range of 79.1% to 112.1% with relative standard deviations less than 11.3% (intra-day) and 13.4% (inter-day). Finally, the proposed method was applied to the analysis of PAs in diluted crude oils with different maturities. In comparison to the existing methods for extraction of PAs, the proposed method provides superior performances including high throughput (12-well plate), high degree of sample clean-up, and low consumption of separation material, solvent and time.

Rapid and sensitive serum glucose determination using chemical labeling coupled with black phosphorus-assisted laser desorption/ionization time-of-flight mass spectrometry.

Monitoring the concentration of blood glucose in patients is a key component of good medical diagnoses. Therefore, developing an accurate, rapid and sensitive strategy for monitoring blood glucose is of vital importance. We proposed a strategy for serum glucose determination combining 2-(4-boronobenzyl) isoquinolin-2-ium bromide chemical labeling with black phosphorus assisted laser desorption ionization-time of flight mass spectrometry (CL-BP/ALDI-TOF MS). The entire analytical process consisted of 1min of protein precipitation and 3min of chemical labeling in a microwave oven prior to the BP/ALDI-TOF MS analysis. The analysis can be completed in 5min with high throughput and extremely low sample consumption. Good linearity for glucose was obtained with a correlation coefficient (R) of 0.9986. The limit of detection (LOD) and limit of quantification (LOQ) were 11.5 fmol and 37.5 fmol, respectively. Satisfied reproducibility and reliability were gained by evaluation of the intra- and inter-day precisions with relative standard deviations (RSDs) less than 7.2% and relative recoveries ranging from 87.1% to 108.1%, respectively. The proposed strategy was also applied for the analysis of endogenous glucose in various serum samples and the results were consistent with those obtained using the hexokinase method in a clinical laboratory. Considering the results, the proposed CL-BP/ALDI-TOF MS strategy has proven to be reliable, fast, and sensitive for quantitative analysis of serum glucose.

High Strength and Hydrophilic Chitosan Microspheres for the Selective Enrichment of N-Glycopeptides.

Protein glycosylation is an important post-translational modification that plays a crucial role in many biological processes. Because of the low abundance of glycoproteins and high complexity of clinical samples, the development of methods to selectively capture glycoproteins/glycopeptides is crucial to glycoproteomics study. In this work, a kind of highly cross-linked chitosan microspheres (CSMs) was prepared using epichlorhydrine as a cross-linker from chitosan solution in an alkaline/urea aqueous system. The results showed that CSMs had high amino groups content, large surface area, mesoporous structure, good acidic resistance, and high strength by various tests. On the basis of hydrophilic interaction between the polar groups (amino groups and hydroxyl groups) on CSMs and glycan moieties on glycopeptides, the prepared CSMs were applied to specific capture of N-glycopeptides from standard protein digests and complex biological samples (body fluids and tissues). The CSMs exhibited high selectivity (HRP/BSA = 1:100), good sensitivity (4.5 × 10-10 M of HRP), good recovery yield (74.9-106.4%), and high binding capacity (100 mg g-1) in glycopeptides enrichment. Because of the excellent performance in glycopeptides enrichment, CSMs were applied to selectively enrich N-glycopeptides from tryptic digests of human serum and rat brain followed by nanoLC-MS/MS analysis. We identified 194 and 947 unique N-glycosylation sites from 2 µL of human serum and 0.1 mg of rat brain, respectively. Additionally, the extraction time of our method was much shorter than the previously reported methods. Therefore, the fabricated CSMs with desirable properties will find broad application in large-scale and in-depth N-glycoproteome analysis.

Polyoxometalate incorporated polymer monolith microextraction for highly selective extraction of antidepressants in undiluted urine.

In this work, a polyoxometalate (POM) incorporated polymer monolith microextraction (PMME) was successfully proposed and employed in the selective extraction of basic antidepressants in undiluted urine sample. This hybrid monolith exhibited strong cation-exchange interaction (SCX) with positively charged antidepressants when pH was 3.0, because of the multiple ionizable moieties on polyanionic POM. As such, antidepressants in complex sample matrices were efficiently extracted by the monolith, and the matrix effect was significantly reduced. In addition, due to the high amount of anionic POM, the monolith exhibited remarkable extraction capacities for target antidepressants ranging from 4.7 to 5.8mg/g. Further, the POM incorporated PMME was coupled with high-performance liquid chromatography-ultraviolet (HPLC-UV). Thus, antidepressants in undiluted urine sample was efficiently extracted under optimized extraction conditions online. The limits of detection (LODs) for the target antidepressants ranged from 0.7 to 1.4ng/mL, and the linear range was 5-1000ng/mL with determination coefficients (R2) higher than 0.9960. The recoveries ranged from 86.8% to 104.0% with relative standard deviations (RSDs) of 0.4-10.1%. The proposed procedure was successfully applied to determine antidepressant in human urine. Taken together, the developed method presented a new strategy for the analysis of basic drugs in undiluted urine sample, which could be used for monitoring medicines in pharmacokinetic analysis.

Pyridoxal 5'-phosphate mediated preparation of immobilized metal affinity material for highly selective and sensitive enrichment of phosphopeptides.

Phosphorylation is a crucial post-translational modification, which plays pivotal roles in various biological processes. Analysis of phosphopeptides by mass spectrometry (MS) is intractable on account of their low stoichiometry and the ion suppression from non-phosphopeptides. Thus, enrichment of phosphopeptides before MS analysis is indispensable. In this work, we employed pyridoxal 5'-phosphate (PLP), as an immobilized metal affinity chromatography (IMAC) ligand for the enrichment of phosphopeptides. PLP was grafted onto several substrates such as silica (SiO2), oxidized carbon nanotube (OCNT) and silica coated magnetic nanoparticles (Fe3O4@SiO2). Then the metal ions Fe3+, Ga3+ and Ti4+ were incorporated for the selective enrichment of phosphopeptides. It is indicated that Fe3O4@SiO2-PLP-Ti4+ has a superior selectivity towards phosphopeptides under as much as 1000-fold interferences of non-phosphopeptides. Further, Fe3O4@SiO2-PLP-Ti4+ exhibited high efficiency in selective enrichments of phosphopeptides from complex biological samples, including human serum and tryptic digested non-fat milk. Finally, Fe3O4@SiO2-PLP-Ti4+ was successfully employed in the sample pretreatment for profiling phosphopeptides in a tryptic digest of rat brain proteins. Our experimental results evidenced a great potential of this new chelator-based material in phosphoproteomics study.

Specific PCR Identification between Peucedanum praeruptorum and Angelica decursiva and Identification between Them and Adulterant Using DNA Barcode.

BACKGROUND: The traditional Chinese medicine (TCM) Qianhu and Zihuaqianhu are the dried roots of Peucedanum praeruptorum and Angelica decursiva, respectively. Since the plant sources of Qianhu and Zihuaqianhu are more complex, the chemical compositions of P. praeruptorum and A. decursiva are significantly different, and many adulterants exist because of the differences in traditional understanding and medication habits. Therefore, the rapid and accurate identification methods are required. OBJECTIVE: The aim was to study the feasibility of using DNA barcoding to distinguish between Traditional Chinese medicine Qianhu (Peucedanum praeruptorum), Zihuaqianhu (Angelica decursiva), and common adulterants, based on internal transcribed spacer (ITS) sequences, as well as specific PCR identification between P. praeruptorum and A. decursiva. MATERIALS AND METHODS: The ITS sequences of P. praeruptorum, A. decursiva, and adulterant were studied, and a phylogenetic tree was constructed. Based on the ITS barcode, the specific PCR primer pairs QH-CP19s/QH-CP19a and ZHQH-CP3s/ZHQH-CP3a were designed for P. praeruptorum and A. decursiva, respectively. The amplification conditions were optimized, and specific PCR products were obtained. RESULTS: The results showed that the phylogenetic trees constructed using the BI and MP methods were consistent, and P. praeruptorum and A. decursiva sequence haplotypes formed their own monophyly. The experimental results showed that in PCR products, the target bands appeared in the genuine drug and not in the adulterant, which suggests the high specificity of the two primer pairs. CONCLUSION: The ITS sequence was ideal DNA barcode to identify P. praeruptorum, A. decursiva, and adulterant. The specific PCR is a quick and effective method to distinguish between P. praeruptorum and A. decursiva. SUMMARY: Peucedanum praeruptorum and Angelica decursiva sequence haplotypes formed their own monophyly.The ITS sequence was ideal DNA barcode to identify P. praeruptorum, A. decursiva, and adulterant.Specific PCR is a quick and effective method to distinguish between P. praeruptorum and A. decursiva. Abbreviations used: TCM: The traditional Chinese medicine, P.: Peucedanum, A.: Angelica, ITS: The internal transcribed spacer, PCR: Polymerase chain reaction, NCBI: National Center for Biotechnology Information, NI: Number of individuals, HN: Haplotype number; GAN: Gen Bank accession numbers, L.: Ligusticum, O.: Ostericum, A.: Angelica, P.: Pimpinella, BI: Bayesian inference, MP: Maximum parsimony, AIC: Akaike Information Criterion, MCMC: Markov Chains Monte Carlo, TBR: Tree bisection-reconnection, LPP: Length of PCR product, PRP: PCR reaction procedure, SNP: Single nucleotide polymorphisms, PP: Posterior probability, BS: Bootstrap.Qun Zhao.

Graft modification of cotton with phosphate group and its application to the enrichment of phosphopeptides.

Grafting copolymerization, especially "grafting from" approach, has attracted a great attention for the preparation of cellulose-based materials with various functionalities. In this study, a novel phosphate group-containing cotton fiber-based material, CF-NH2-AZO-p(VPA-x), was successfully synthesized by a "grafting from" radical polymerization approach using vinyl phosphonic acid (VPA) as monomer. The phosphate group content of CF-NH2-AZO-p(VPA-x) could be easily regulated by adjusting the concentration of VPA monomer. Subsequently, titanium ions immobilized CF-NH2-AZO-p(VPA-x) (CF-NH2-AZO-p(VPA-x)-Ti4+) was prepared and used as an immobilized metal affinity chromatography (IMAC) adsorbent for the selective enrichment of phosphopeptides from various biological samples. The relationship between enrichment efficiency and phosphate group content of CF-NH2-AZO-p(VPA-x)-Ti4+ was investigated. The optimized CF-NH2-AZO-p(VPA-2)-Ti4+ (x=2) exhibited high selectivity and extraction capacity in phosphopeptides enrichment from standard peptides mixture, non-fat milk digests and protein-rich human serum. In addition, CF-NH2-AZO-p(VPA-2)-Ti4+ was further applied for the specific capture of phosphopeptides from tryptic digests of rat brain lysate, and 3241 unique phosphopeptides were identified from 0.5mg of rat brain by combining CF-NH2-AZO-p(VPA-2)-Ti4+ pretreatment with liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. We expect the proposed method can promote the development of "grafting from" approach in the preparation of cellulose-based adsorbents and broaden the application of cellulose-based adsorbents for biological analysis.

Facial synthesis of nickel(II)-immobilized carboxyl cotton chelator for purification of histidine-tagged proteins.

Immobilized metal affinity chromatography (IMAC) technique is frequently used in the purification of histidine-tagged (His-tagged) recombinant proteins. In this study, nickel(II)-immobilized carboxyl cotton chelator (CCC-Ni2+) fibers was synthesized by a simple method based on the coordination effect between Ni2+ and carboxyl group. The nickel content of the CCC-Ni2+ fibers was determined to be 5 times larger than that of Ni2+-immobilized sulfhydryl cotton fiber (SCF-Ni2+) fibers developed in our previous work. The prepared CCC-Ni2+ fibers were then applied for the selective and rapid separation of His-tagged protein from escherichia coli (E. coli) cell lysates on the basis of the high affinity of Ni2+ to 6×His with a lab-in-syringe format. Benefiting from the good biological compatibility and high nickel content, the results showed that CCC-Ni2+ fibers were able to selectively capture His-tagged proteins from complex E. coli cell lysates and exhibited a relatively large adsorption capacity toward His-tagged protein. The recoveries of His-tagged GFP in E. coli cell lysates were in the range of 89.8%-106.7% with the relative standard deviations (RSDs) less than 9.4% (intra-day) and 10.3% (inter-day). Taken together, this efficient approach for the purification of recombinant proteins extends the application of CCC-based fibrous materials in biological analysis.

Immobilization of zirconium-glycerolate nanowires on magnetic nanoparticles for extraction of urinary ribonucleosides.

The authors have immobilized nanowires made from zirconium glycerolate (ZrGly) on magnetite (Fe3O4) nanoparticles by applying a solvothermal growth process using metal-glycerolate as a precursor. The structure and the dissolution-recrystallization mechanism of the resulting Fe3O4@ZrGly composite were investigated by attenuated total reflection-FTIR, energy-dispersive X-ray analysis, thermogravimetric analysis and solid-state cross polarization/magic angle spinning 13C NMR spectroscopy. The interaction between the zirconium glycerolate in Fe3O4@ZrGly and cis-diols leads to efficient adsorption of riboncleosides which then can be quantified by HPLC with UV detection. The sorbent was successfully applied to the selective enrichment of adenosine, cytidine, uridine and guanosine from spiked human urine samples. The detection limit of the method is in the range from 1.7 to 19 ng·mL-1 of nucleosides in spiked human urine, with relative standard deviations of lower than 12.4% and recoveries ranging from 90.6 to 113%. Graphical abstract Fe3O4@ZrGly with high selectivity towards ribonucleosides was designed and applied for quantitation of urinary ribonucleosides.

Synthesis of Polyethylenimine Functionalized Mesoporous Silica for In-Pipet-Tip Phosphopeptide Enrichment.

Synthesis of functionalized mesoporous silica material with large particle size remains a chanllenge. In this work, polyethylenimine (PEI) functionalized mesoporous silica (PFMS) with particle size as large as 100 µm was successfully synthesized by a facile method. In the synthesis process, PEI served as four roles simultaneously, including functionalized reagent, alkaline catalyst, template for particle formation, and pore-structure-directing agent. The surface areas of the products were higher than 260 m2/g. Benefiting from the large particle size and high surface area, PFMS was packed in a pipet tip to fabricate a convenient and miniaturized solid phase extraction apparatus for sample preparation. Additionally, based on the extremely abundant basic sites in the organic units of PFMS, the in-pipet-tip system was used as an anion-exchanger for phosphopeptide enrichment. The specificity of the developed method was investigated by capture of phosphopeptides from tryptic digests of standard protein mixtures, tryptic digests of nonfat milk, and human serum. Furthermore, the method was utilized to analyze phosphopeptides in tryptic digests of rat brain lysate, and 2251 unique phosphopeptides were successfully detected.

Black phosphorus-assisted laser desorption ionization mass spectrometry for the determination of low-molecular-weight compounds in biofluids.

Quantitative analysis of small molecules by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been a challenging task due to matrix-derived interferences in low m/z region and poor reproducibility of MS signal response. In this study, we developed an approach by applying black phosphorus (BP) as a matrix-assisted laser desorption ionization (MALDI) matrix for the quantitative analysis of small molecules for the first time. Black phosphorus-assisted laser desorption/ionization mass spectrometry (BP/ALDI-MS) showed clear background and exhibited superior detection sensitivity toward quaternary ammonium compounds compared to carbon-based materials. By combining stable isotope labeling (SIL) strategy with BP/ALDI-MS (SIL-BP/ALDI-MS), a variety of analytes labeled with quaternary ammonium group were sensitively detected. Moreover, the isotope-labeled forms of analytes also served as internal standards, which broadened the analyte coverage of BP/ALDI-MS and improved the reproducibility of MS signals. Based on these advantages, a reliable method for quantitative analysis of aldehydes from complex biological samples (saliva, urine, and serum) was successfully established. Good linearities were obtained for five aldehydes in the range of 0.1-20.0 µM with correlation coefficients (R (2)) larger than 0.9928. The LODs were found to be 20 to 100 nM. Reproducibility of the method was obtained with intra-day and inter-day relative standard deviations (RSDs) less than 10.4 %, and the recoveries in saliva samples ranged from 91.4 to 117.1 %. Taken together, the proposed SIL-BP/ALDI-MS strategy has proved to be a reliable tool for quantitative analysis of aldehydes from complex samples. Graphical Abstract An approach for the determination of small molecules was developed by using black phosphorus (BP) as a matrix-assisted laser desorption ionization (MALDI) matrix.

Magnetic graphitic carbon nitride anion exchanger for specific enrichment of phosphopeptides.

Anion-exchange chromatography (AEX) is one of the chromatography-based methods effectively being used for phosphopeptide enrichment. However, the development of AEX materials with high specificity toward phosphopeptides is still less explored as compared to immobilized metal affinity chromatography (IMAC) or metal oxide affinity chromatography (MOAC). In this work, magnetic graphitic carbon nitride (MCN) was successfully prepared and introduced as a promising AEX candidate for phosphopeptide enrichment. Due to the extremely abundant content of nitrogen with basic functionality on the surface, this material kept excellent retention for phosphopeptides at pH as low as 1.8. Benefiting from the large binding capacity at such low pH, MCN showed remarkable specificity to capture phosphopeptides from tryptic digests of standard protein mixtures as well as nonfat milk and human serum. In addition, MCN was also applied to selective enrichment of phosphopeptides from the tryptic digests of rat brain lysate and 2576 unique phosphopeptides were successfully identified.

Hydrophilic Carboxyl Cotton Chelator for Titanium(IV) Immobilization and Its Application as Novel Fibrous Sorbent for Rapid Enrichment of Phosphopeptides.

Sample preparation methods with high selectivity, efficiency, and matrix resistance are essential for phosphoproteomic analysis. In this study, carboxyl cotton chelator-titanium(IV) (CCC-Ti4+) fibers, a novel CCC-based fibrous sorbent with excellent biocompatibility, were successfully synthesized on the basis of the coordination effect between double carboxyl groups on CCC and Ti4+. The synthesis of CCC-Ti4+ fibers was easy, and the incorporated titanium content was high. On the basis of immobilized metal ion affinity chromatography (IMAC), CCC-Ti4+ fibers were used for specific capture of phosphopeptides using a lab-in-syringe solid-phase extraction (SPE) from multiple biological samples, including standard protein digests, nonfat milk digests, human serum, and animal tissue. The proposed sorbent exhibited high selectivity (ß-casein/BSA=1:1000) and good sensitivity (10 fmol) in phosphopeptides analysis. Meanwhile, the lab-in-syringe SPE greatly simplified the entire process of enrichment. Thanks to the good biocompatibility of CCC-based material, CCC-Ti4+ fibers showed excellent performance in phosphopeptide enrichment from protein-rich human serum. Finally, CCC-Ti4+ fibers were applied for selective capture of phosphopeptides from tryptic digests of rat brain lysate followed by LC-MS/MS analysis. Using the proposed method, we identified 3950 unique phosphosites from 1 mg of rat brain in a single experiment, which is much better than previously reported IMAC-based strategies. Taken together, this efficient method will find broad application in large-scale phosphoproteomics analysis because of the rapid (3 min) convenient procedure and excellent performance.

Nickel(II)-immobilized sulfhydryl cotton fiber for selective binding and rapid separation of histidine-tagged proteins.

In the current study, a novel nickel(II)-immobilized sulfhydryl cotton fiber (SCF-Ni(2+)) was prepared in a simple way based on the coordination effect between Ni(2+) and thiol group on the surface of SCF. The composition and element mapping of SCF-Ni(2+) fibers were demonstrated by energy-dispersive X-ray (EDX) spectroscopy. Based on the high affinity of Ni(2+) to 6×His on histidine-tagged (His-tagged) proteins, SCF-Ni(2+) fibers were then further used as an immobilized metal ion affinity chromatography (IMAC) adsorbent for selective binding and rapid separation of His-tagged proteins using an in- pipette-tip SPE format. Our results showed that SCF-Ni(2+) adsorbent can selectively capture His-tagged proteins from protein mixture and Escherichia coli cell lysates. Taken together, the developed method provides a rapid, convenient and efficient approach for the purification of His-tagged proteins.

Facile synthesis of polyaniline-coated SiO2 nanofiber and its application in enrichment of fluoroquinolones from honey samples.

In this study, polyaniline coated SiO2 nanofibers (PANI/SiO2) was prepared by combining electrospinning technique with in-situ polymerization. The proposed strategy for the preparation of PANI/SiO2 can eliminate the aggregation of PANI and the yield of PANI/SiO2 was high. Scanning electron microscopy (SEM) images showed that PANI nanoparticles were uniformly coated on the surface of SiO2 nanofibers. The as-prepared PANI/SiO2 nanofibers were then applied as the sorbent for in-syringe dispersive solid-phase extraction (dSPE) for the extraction of fluoroquinolones (FQs) from honey samples. The influence of SiO2 amount on the formation of PANI/SiO2 and several parameters that affect the extraction efficiency were investigated. Under optimized conditions, a rapid, simple and effective method for the determination of FQs in honey sample was developed by coupling with liquid chromatography-fluorescence detector (LC-FLD) analysis. Due to the fast extraction equilibrium, the whole sample pretreatment process could be accomplished within 4 min. The limits of detection (LODs) for the target FQs were found to be 0.1-1.3 ng/g. The recoveries in honey sample were in the range of 81.4-118.1% with the RSDs of 0.8-14.4% (intra-day) and 1.4-14.9% (inter-day). This study offers a new strategy for the preparation of functional SiO2 nanofibers using post-electrospinning modification by in-situ polymerization, which could be generally applied in the preparation of various separation materials with electrospun nanofibers.

Electrospun highly ordered mesoporous silica-carbon composite nanofibers for rapid extraction and prefractionation of endogenous peptides.

A simple method was developed for the preparation of ordered mesoporous silica-carbon composite nanofibers (OMSCFs). The OMSCFs exhibited high carbon content, continuously long fibrous properties, uniform accessible mesopores, and a large surface area. The OMSCFs were also found to have ion-exchange capacity. On the basis of the size-exclusion effect of the mesopores and mixed-mode hydrophobic/ion-exchange interactions, the OMSCFs were applied for rapid enrichment of endogenous peptides by using a miniaturized solid-phase extraction format. The adsorption mechanism was studied, and the eluting solution was optimized with standard peptide/protein solutions and protein digests. Employing a successive three-step elution strategy, followed by LC-MS/MS analysis, led to excellent performance with this approach in the extraction and prefractionation of peptides from human serum.