Xiao-Mei He

Electrospun polystyrene/oxidized carbon nanotubes film as both sorbent for thin film microextraction and matrix for matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

In the current study, polystyrene/oxidized carbon nanotubes (PS/OCNTs) film was prepared and applied as both an adsorbent of thin film microextraction (TFME) and matrix for matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) for the first time. The uniform size of PS/OCNTs film with OCNTs evenly and firmly immobilized in PS was obtained by electrospinning. And a novel TFME device was developed using the prepared PS/OCNTs film to enrich benzo[a]pyrene (BaP) from water, and also BaP and 1-hydroxypyrene (1-OHP) from urine sample. Then the extracted analytes on the PS/OCNTs film were directly applied to MALDI-MS analysis with PS/OCNTs film as the MALDI matrix. Our results show that PS/OCNTs film is a good TFME adsorbent toward the analytes and an excellent matrix for the sensitive determination of BaP and 1-OHP using MALDI-TOF-MS. The employment of PS/OCNTs as the matrix for MALDI can effectively avoid the large variation of signal intensity normally resulting from heterogeneous distribution of the adsorbed analyte on matrix layer, which therefore significantly improve spot-to-spot reproducibility. The introduction of PS in the film can prevent OCNTs from flying out of MALDI plate to damage the equipment. In addition, PS/OCNTs film also largely extended the duration of ion signal of target analyte compared to OCNTs matrix. The developed method was further successfully used to quantitatively determine BaP in environmental water and 1-OHP in urine samples. The results show that BaP and 1-OHP could be easily detected at concentrations of 50pgmL(-1) and 500pgmL(-1), respectively, indicating the high detection sensitivity of this method. For BaP analysis, the linear range was 0.1-20ngmL(-1) with a correlation coefficient of 0.9970 and the recoveries were in the range of 81.3 to 123.4% with the RSD≤8.5% (n=3); for urinary 1-OHP analysis, the linear range was 0.5-20ngmL(-1) with a correlation coefficient of 0.9937 and the recoveries were in the range of 79.2 to 103.4% with the RSD≤7.6% (n=3). Taken together, the developed method provides a simple, rapid, cost-effective and high-throughput approach for the analysis of BaP in environmental water and endogenous 1-OHP in urine samples.

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 (β-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.

Preparation of mesoporous silica embedded pipette tips for rapid enrichment of endogenous peptides

Mesoporous silica embedded pipette tips (mSiO2-Tips) were successfully prepared by a simple method and applied to rapid enrichment of endogenous peptides for the first time. The prepared mSiO2-Tips showed low back pressure when solvent was pipetted up and down. As a result, mSiO2-Tip could selectively trap peptides and exclude high-MW proteins simultaneously based on size-exclusion mechanism due to the uniform mesopore structure of the sorbent bed. The in-pipette-tip SPE approach was proved to be easy-operation, sensitive and fast (less than 2 min) for the analysis of peptides, which was further successfully applied in the efficient enrichment of peptides from human plasma.

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.

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.

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.

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.

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.

A micro-solid phase extraction in glass pipette packed with amino-functionalized silica for rapid analysis of petroleum acids in crude oils

In this work, a micro-solid phase extraction (micro-SPE) method was developed for the analysis of petroleum acids in crude oils. Plastic vessels were found to be unsuitable as the micro-SPE vessel, because of the plasticizer interferences and the serious adhesion of oil samples on vessel surfaces. To reduce or eliminate these influences, a glass pipette was selected as the micro-SPE vessel. With the glass pipette micro-SPE format, amino-functionalized silica was chosen as the sorbent for extraction of petroleum acids based on mix-mode hydrogen bonding/ion-exchange interactions. The conditions of the micro-SPE were carefully optimized. Under the optimized conditions, no evaporation was needed, which can save time and reduce losses of analytes. By coupling with gas chromatography-mass spectrometry (GC-MS), the proposed micro-SPE method for analysis of petroleum acids was proved to be easily operated, fast (the micro-SPE can be accomplished within 5 min), solvent-saving (3 mL of solvent), reproducible and sensitive (the limits of detection range from 2 ng g−1 to 6 ng g−1).

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.