Biobased Tannic Acid-Chitosan Composite Membranes as Reusable Adsorbents for Effective Enrichment of Phosphopeptides.

High-performance reusable materials from renewable resources are rare and urgently required in bioseparation. Herein, a series of tannic acid-chitosan composite membranes for the enrichment of phosphopeptides were fabricated by the freeze casting method. First, a tannic acid-chitosan composite membrane was acquired via the multiple hydrogen bonds between tannic acid and chitosan, which had a long-range aligned three-dimensional microstructure. Second, a covalent-hydrogen bond hybrid composite was also fabricated, with stable and aligned honeycomb-like microstructures that formed by the synergy of covalence and hydrogen bonding. Besides, a ternary composite membrane was "one-pot" synthesized by the copolymerization of tannic acid, chitosan, and Ti4+ ions, indicating the feasibility of involving metal ions in the composition of the polymer skeleton in place of additional modification steps. The as-prepared chitosan composite membranes exhibited excellent performance in the enrichment of phosphopeptides from ß-casein tryptic digest and human serum. Benefitting from the long-range aligned honeycomb-like structure coordinated by hydrogen bonds and covalent bonds, and a large number of pyrogallol functional groups provided by tannic acid, the covalent-hydrogen bond hybrid membrane showed excellent reusability and could be reused up to 16 times in phosphopeptide enrichment, as far as we know, which is the best reported result to date.

Selective adsorption and identification of penicillin G sodium in milk by molecularly imprinted polymer doped carbon dot.

A carbon dot (CD) was prepared by o-phenylenediamine and water, which showed bright yellow fluorescence under ultraviolet light irradiation (λ = 580 nm), and verified good fluorescence quenching effect on penicillin G sodium (Png-Na). Using methacrylic acid as a functional monomer, ethylene glycol dimethacrylate as a crosslinker, and Png-Na as a template, a kind of composite microsphere combining CD and molecularly imprinted polymer (MIP) was synthesized by surface-initiated atomic transfer radical polymerization (SI-ATRP). For reasons of comparison, we also prepared MIP without CD and non-imprinted polymers (NIPs). Through static and dynamic adsorption experiments, the maximum adsorption capacity was 47.05 mg g-1 and the equilibrium time was 30 min. High-performance liquid chromatography (HPLC) was utilized to determine the content of Png-Na in the spiked milk samples. A sensitive, rapid, and simple method for determination of Png-Na in food samples was developed. The utilized approach enabled the quantification of Png-Na within the concentration range 20-1000 µg L-1 (with a limit of detection of 5 µg L-1). The recoveries achieved were in the range 93.3-98.2%, with a relative standard deviation of 1.2-4.2%. The results demonstrated that CD@MIP possessed the capability of specific adsorption and fluorescence detection of Png-Na, enabling simultaneous detection and enrichment of Png-Na in real samples.

Facile Fabrication of Monodisperse Micron-Sized Dual Janus Silica Particles with Asymmetric Morphology and Chemical Environment.

Janus particles are a kind of materials with asymmetric morphology or surface chemical environment. But so far, the preparation of particles with dual asymmetry is still a challenging problem. Hence the cation surfactant hexadecyl trimethyl ammonium bromide and co-surfactant octadecylamine are applied to improve the Pickering emulsion stability, and the micron-sized silica particles are arranged in a single layer at the toluene-water interface through electrostatic interaction. Furthermore, organosilane reagents are added in the preparation process, resulting in the construction of asymmetric hydrophilic or hydrophobic mesoporous precisely onto the micron-sized silica particles surface. The cation surfactant-assisted Pickering emulsion method is simple, effective, and convenience, which can be applied in the synthesis of various dual Janus silica particles for specific applications.

Interface-Engineered Hollow Nanospheres with Titanium(IV) Binding Sites and Microwindows as Affinity Probes for Ultrafast and Enhanced Phosphopeptides Enrichment.

Enrichment and identification of phosphopeptides in real biological samples are of great significance in many aspects. Herein, Ti4+-immobilized silica hollow nanospheres were tailored via chelating with phosphonic acid groups produced from dealkylation of phosphonate ester functionalized silica hollow nanospheres, which were synthesized through a single micelle templated method with diethylphosphatoethyltriethoxysilane (DPTES) and tetramethoxysilane (TMOS) as silane precursors under neutral conditions. The characterization results of transmission electron microscopy (TEM), nitrogen sorption isotherms, FT-IR, and energy-dispersive X-ray (EDX) spectroscopy confirmed the successful preparation of Ti4+-immobilized silica hollow nanospheres (SHS-Ti; approximately 17 nm particle size), which possessed a 10 nm hollow cavity with 1.6 nm micropores on the thin shell (about 3.5 nm). Attributed to the immobilized Ti4+ and high specific area (396 m2/g), SHS-Ti was applied as a Ti4+-immobilized metal affinity chromatography (Ti-IMAC) material and showed good specificity, a low limit of detection (5 fmol), high selectivity (tryptic digestion mixture of bovine serum albumin/ß-casein, 1000:1 molar ratio), high binding capacity (120 mg/g for pyridoxal 5'-phosphate), and a high binding constant (1.30 × 103 L/mg). Particularly, benefiting from the unique hollow structure with microwindows on the thin shell, a short transport path, and small mass transfer resistance, SHS-Ti exhibited excellent enrichment speed in which both phosphopeptide loading and elution could be completed in 1 min. The 5298 unique phosphopeptides from 1618 unique phosphoproteins were identified after enrichment by SHS-Ti from 100 µg Jurkat cell lysates within three independent replicates. The results showed that SHS-Ti could be utilized as a novel and promising enrichment probe for phosphopeptide characterization in MS-based phosphoproteomics and related fields.

Preparation of core-shell microporous organic polymer-coated silica microspheres for chromatographic separation and N-glycopeptides enrichment.

Through a "one-pot" strategy, a layer of microporous organic polymer was coated onto the surface of monodisperse amino-functionalized silica microsphere via amino-aldehyde condensation reaction with core-shell structure. The change in chemical structure of material before and after modification was determined by Fourier-transform infrared spectroscopy and X-ray photoelectron spectroscopy. Due to existence of a large number of amino and aldehyde groups in microporous organic polymer shell, the water contact angle decreased from 56.8° (silica microspheres) to 34.7° (microporous organic polymer-coated silica microspheres). Based on these properties, microporous organic polymer-coated silica microspheres were employed as the stationary phase for capillary liquid chromatography and successfully offered baseline separation of polar small molecules. Additionally, the material could also be served as the sorbent of hydrophilic interaction chromatography to enrich glycopeptides from human serum digest. A total of 470 unique N-glycopeptides and 342 N-glycosylation sites mapped to 112 N-glycosylated proteins were unambiguously identified from 2 µL of human serum, exhibiting a promising application prospect of microporous organic polymer-coated silica microspheres in the pretreatment of proteomics samples.

Monodisperse Ti4+-immobilized macroporous adsorbent resins with polymer brush for improved multi-phosphopeptides enrichment in milk.

Enrichment of phosphopeptides before mass spectrometry (MS) analysis is essential due to the limitations of low abundance and poor ionization efficiency in complex biological samples. Immobilized metal affinity chromatography (IMAC), especially titanium ion (Ti4+)-IMAC, has become a popular strategy for enrichment of phosphopeptides due to high selectivity and sensitivity. Conventional Ti4+-immobilized macroporous adsorption resin (MAR) fabricated by monolayer modification can preferentially capture mono-phosphopeptide over multi-phosphopeptides, which takes on more functions in the regulation of cell behaviors of organism. In this paper, a kind of monodisperse MAR microsphere with functional polymer brush (Ti4+-Brush@MAR) was prepared and modified via surface-initiated atom transfer radical polymerization (SI-ATRP). Compared with common Ti4+-MAR without polymer brush, Ti4+-Brush@MAR exhibited high enrichment specificity not only for mono-phosphopeptides but also for multi-phosphopeptides in ß-casein or milk digest samples. As a result, a total of 93 unique phosphopeptides mapped to 18 phosphoproteins were identified from 5 µL milk, and the limit of detection is 10 fmol. It is expected that Ti4+-Brush@MAR would be utilized to enrich both multi-phosphopeptides and mono-phosphopeptides in additional biological or food samples.

Design and fabrication of reusable core-shell composite microspheres based on nanodiamond for selective enrichment of phosphopeptides.

A kind of core-shell composite microsphere (CM) with nano-on-micro structure was synthesized via grafting amine-modified nanodiamonds onto the surface of monodisperse nonporous polymeric microsphere. In this way, the agglomeration of nanodiamond particles in the solution was avoided. After modification with pyrogallol groups, CM could chelate titanium ions (Ti4+) and thus be utilized as immobilized metal affinity chromatography (IMAC) sorbent to enrich phosphopeptides from biological samples. The resulting Ti4+-CM exhibited high enrichment efficiency and specificity to phosphopeptides. A total of 106 of unique phosphopeptides mapped to 29 phosphoproteins were clearly identified from 5 µL of a milk digest after enrichment. Owing to the strong chelation between Ti4+ and pyrogallol ligands, the Ti4+ is not released from the sorbent after completion of the enrichment process. As a result, the Ti4+-CM sorbent could be reused, and no significant loss of enrichment efficiency occurred even on the fourth run employing a ß-casein digest as the sample. The strategy adopted presents a new way to prepare a high-performance reusable IMAC sorbent.

Fabrication of hydrophilic zwitterionic microspheres via inverse suspension polymerization for the enrichment of N-glycopeptides.

A kind of zwitterionic microsphere was prepared via one-step inverse suspension polymerization employing 3-[N,N-dimethyl-[2-(2-methylpropyl-2-enyloxy) ethyl] ammonium] propane-1-sulfonate (MSA) and N,N-methylene bisacrylamide (BIS) as the precursors. The preparation conditions were carefully investigated and optimized by regulating the content of total monomers, ratio of MSA to BIS, ratio of water to oil, and content of stabilizer. The properties of microspheres were characterized by helium ion microscopy (HIM), Fourier transform infrared spectroscopy (FT-IR), X-ray photoelectron spectroscopy (XPS), N2 adsorption/desorption measurement, and water contact angle measurement. The particle size of resulting polydisperse microspheres ranged from 15-25 µm, exhibiting high specific surface area of 138 m2 g-1. Owing to great hydrophilicity, the resulting zwitterionic microspheres could be directly used as hydrophilic interaction chromatography (HILIC) sorbent to enrich glycopeptides from biosamples without any chemical modification. A total of 19 N-glycopeptides was enriched from 10 µg of IgG digest. Besides, up to 383 N-glycopeptides and 224 N-glycosylation sites were unambiguously identified from 2 µL of human serum digest by cLC-MS/MS after enrichment with zwitterionic microspheres, indicating their great enrichment performance to N-glycopeptides. The approach of preparing hydrophilic zwitterionic microspheres contains only one synthesis reaction and is suitable for large-scale preparation.

Integrated Microstructured Photonic Fiber as a Bifunctional Robust Frit and Efficient Electrospray Emitter of a Packed Column for Capillary Liquid Chromatography-Tandem Mass Spectrometry Analysis of Complex Biological Samples.

Although capillary liquid chromatography married with tandem mass spectrometry (cLC-MS/MS) has become a powerful technique for proteomics and metabolomics research, it is still a great challenge to fabricate durable capillary-based analytical columns coupling continuous nanoflow (<1 000 nL/min) electrospray ionization (ESI) with MS, owing to the issue of clogging and fragile of emitters. Here, we proposed a simple approach to integrate microstructured photonic fibers (MPFs) into wide bore capillaries with 150 µm i.d., serving as an integral bifunctional frit or/and ESI emitter of packed columns. Two kinds of MPFs containing 126 homogeneous microchannels with different inner diameter, 3.2 µm for MPF-1 and 2.6 µm for MPF-2, were explored for preparation. The octadecylsilicate (ODS) silica-packed column using MPF-1 as a frit exhibited the lowest plate heights of 14.2-19.7 µm for five alkylbenzenes at the velocity of 1.5 mm/s, which were slightly lower than those of packed column with porous polymer monolith (PPM)-based frit by cLC coupling with ultraviolet (UV) detection. Additionally, the packed columns with integral MPF frit-emitters were further applied in analysis of a complex biological sample of digest of Hela cells by cLC-MS. An average of 7109 unique peptides could be identified in a single analysis by using MPF-1 emitter, and 7110 unique peptides were identified by using the MPF-2 emitter, which were superior to the identified result of packed column with an integral tapered tip emitter (6894 peptides). It is obvious that this novel integral MPF-based frit-emitter does not easily suffer from the issues of cracking owing to the silica cladding around independent microchannels (>100), which always encumbers both independent and integral tapered tip emitters for cLC-MS.

Facile Fabrication of Biomimetic Chitosan Membrane with Honeycomb-Like Structure for Enrichment of Glycosylated Peptides.

In the study of glycoproteomics with mass spectrometry, certain pretreatments of samples are required for eliminating the interference of nonglycopeptides and improving the efficiency of glycopeptides detection. Although hydrophilic interaction chromatography (HILIC) has been developed for enrichment of glycosylated peptides, a plethora of hydrophilic materials always suffered from large steric hindrance, great cost, and difficulty with modifications of high-density hydrophilic groups. In this work, a 1 mm thick biomimetic honeycomb chitosan membrane (BHCM) with honeycomb-like accessible macropores was directly prepared by the freeze-casting method as an adsorbent for HILIC. The N-glycopeptides from trypsin digests of immunoglobulin G (IgG), mixture of IgG and bovine serum albumin (BSA), and serum proteins were enriched using this material and compared with a commercial material ZIC-HILIC. The biomimetic membrane could identify as many as 32 N-glycopeptides from the IgG digest, exhibiting high sensitivity (about 50 fmol) and a wide scope for glycopeptide enrichment. A molar ratio of IgG trypsin digest to bovine serum albumin trypsin digest as low as 1/500 verified the outstanding specificity and efficiency for glycopeptide enrichment. In addition, 270 unique N-glycosylation sites of 400 unique glycopeptides from 146 glycosylated proteins were identified from the triplicate analysis of 2 µL human serum. Furthermore, 48 unique O-glycosylation sites of 278 unique O-glycopeptides were identified from the triplicate analysis of 30 µg deglycosylated fetuin digest. These results indicated that the chitosan-based membrane prepared in this work had great potential for pretreatment of samples in glycoproteomics.

Fast fabrication of a hybrid monolithic column containing cyclic and aliphatic hydrophobic ligands via photo-initiated thiol-ene polymerization.

Three monomers, octakis (3-mercaptopropyl) octasilsesquioxane, 1,2,4-trivinylcyclohexane and isophytol were employed to synthesize a novel monolithic stationary phase via photo-initiated thiol-ene click polymerization for reversed-phase liquid chromatography. Several factors such as porogenic system, reaction time and the molar ratio of functional groups were investigated in detail. The resulting poly(POSS-co-TVCH-co-isophytol) monolithic column exhibited suitable permeability for fast separation and outstanding thermal stability. Five alkylbenzenes were employed to evaluate the ability of chromatographic separation of the resulting monolithic columns at different flow rates, and showed the highest column efficiencies of 90,200-93,100 N/m (corresponding to 10.4-10.6 µm of plate height) at a velocity of 0.41 mm/s. The baseline separations of five anilines and eight phenols further proved the applicability of poly(POSS-co-TVCH-co-isophytol) monolithic column in the separation of small molecules.

Facile Preparation of Titanium(IV)-Immobilized Hierarchically Porous Hybrid Monoliths.

Hierarchically porous materials have become a key feature of biological materials and have been widely applied for adsorption or catalysis. Herein, we presented a new approach to directly prepare a phosphate-functionalized hierarchically porous hybrid monolith (HPHM), which simultaneously contained mesopores and macropores. The design was based on the copolymerization of polyhedral oligomeric vinylsilsesquioxanes (vinylPOSS) and vinylphosphonic acid (VPA) by adding degradable polycaprolactone (PCL) additive. The phosphate groups could be directly introduced into the hybrid monoliths. This approach was simple and time-saving, and overcame the defect of a rigorous, complex process for preparing traditional Ti4+-immobilized metal ion affinity chromatography (IMAC) materials. The specific surface area of an optimal hybrid monolith could reach 502 m2/g obtained by nitrogen adsorption/desorption measurements, which originated from the degradation of PCL. Meanwhile, the characterization of scanning electron microscopy (SEM) and mercury intrusion porosimetry (MIP) also suggested that the macropores existed in the hybrid monoliths. The size of macropores could be controlled by the content of PCL in the polymerization mixture. The prepared Ti4+-IMAC HPHMs exhibited high adsorption capacity (63.6 mg/g for pyridocal 5'-phosphatemonohydrate), and excellent enrichment specificity (tryptic digest of ß-casein/BSA at a molar ratio of 1:1000) and sensitivity (tryptic digest of 5 fmol of ß-casein). Moreover, the Ti4+-IMAC HPHMs provided effective enrichment ability of low-abundance phosphopeptides from human serum and HeLa cell digests.

Preparation of cyclodextrin-modified monolithic hybrid columns for the fast enantioseparation of hydroxy acids in capillary liquid chromatography.

Cyclodextrins and their derivatives are one of the most common and successful chiral selectors. However, there have been few publications about the use of cyclodextrin-modified monoliths. In this study, organic hybrid monoliths were prepared by the immobilization of derivatized ß-cyclodextrin alone or with l-2-allylglycine hydrochloride to the polyhedral oligomeric silsesquioxane methacryl substituted monolith. The main topic of this study is a combined system with dual chiral selectors (l-2-allylglycine hydrochloride and ß-cyclodextrin) as monolithic chiral stationary phase. The effect of l-2-allylglycine hydrochloride concentration on enantioseparation was investigated. The enantioseparation of the four acidic compounds with resolutions up to 2.87 was achieved within 2.5 min on the prepared chiral monolithic column in capillary liquid chromatography. Moreover, the possible mechanism of enantioseparation was discussed.

Synthesis of a stationary phase based on silica modified with branched octadecyl groups by Michael addition and photoinduced thiol-yne click chemistry for the separation of basic compounds.

A novel silica-based stationary phase with branched octadecyl groups was prepared by the sequential employment of the Michael addition reaction and photoinduced thiol-yne click chemistry with 3-aminopropyl-functionalized silica microspheres as the initial material. The resulting stationary phase denoted as SiO2 -N(C18)4 was characterized by elemental analysis, FTIR spectroscopy and Raman spectroscopy, demonstrating the existence of branched octadecyl groups in silica microspheres. The separations of benzene homologous compounds, acid compounds and amine analogues were conducted, demonstrating mixed-mode separation mechanism on SiO2 -N(C18)4 . Baseline separation of basic drugs mixture was acquired with the mobile phase of acetonitrile/H2 O (5%, v/v). SiO2 -N(C18)4 was further applied to separate Corydalis yanhusuo Wang water extracts, and more baseline separation peaks were obtained for SiO2 -N(C18)4 than those on Atlantis dC18 column. It can be expected that this new silica-based stationary phase will exhibit great potential in the analysis of basic compounds.

Preparation of hybrid monolithic columns via "one-pot" photoinitiated thiol-acrylate polymerization for retention-independent performance in capillary liquid chromatography.

A novel "one-pot" approach was developed for ultrarapid preparation of various hybrid monolithic columns in UV-transparent fused-silica capillaries via photoinitiated thiol-acrylate polymerization of an acrylopropyl polyhedral oligomertic silsesquioxane (acryl-POSS) and a monothiol monomer (1-octadecanethiol or sodium 3-mercapto-1-propanesulfonate) within 5 min, in which the acrylate not only homopolymerizes, but also couples with the thiol. This unique combination of two types of free-radical reaction mechanisms offers a simple way to fabricate various acrylate-based hybrid monoliths. The physical characterization, including scanning electron microscopy (SEM), Fourier transform infrared (FT-IR) spectroscopy, and thermal gravimetric analysis was performed. The results indicated that the monothiol monomers were successfully incorporated into acryl-POSS-based hybrid monoliths. The column efficiencies for alkylbenzenes on the C18-functionalized hybrid monolithic column reached to 60 000-73 500 plates/m at the velocity of 0.33 mm/s in capillary liquid chromatography, which was far higher than that of previously reported POSS-based columns prepared via thermal-initiated free-radical polymerization without adding any thiol monomers. By plotting the plate height (H) of the alkylbenzenes versus the linear velocity (u) of the mobile phase, the results revealed a retention-independent efficient performance of small molecules in the isocratic elution. These results indicated that more homogeneous hybrid monoliths formed via photoinitiated thiol-acrylate polymerization; particularly, the use of the multifunctional cross-linker possibly prevented the generation of gel-like micropores, reducing mass transfer resistance (C-term). Another sulfonate-containing hybrid monolithic column also exhibited hydrophobicity and ion-exchange mechanism, and the dynamic binding capacity was calculated as 71.1 ng/cm (75 µm i.d.).

Thiol-epoxy click polymerization for preparation of polymeric monoliths with well-defined 3D framework for capillary liquid chromatography.

A facile approach was developed for direct preparation of organic monoliths via the alkaline-catalyzed thiol-epoxy click polymerization. Two organic monoliths were prepared by using tetraphenylolethane glycidyl ether as a multiepoxy monomer, and trimethylolpropane tris(3-mercaptopropionate) and pentaerythritol tetrakis(3-mercaptopropionate) as the multithiol monomer, respectively, in the presence of a ternary porogenic system (DMSO/PEG200/H2O). The obtained organic monoliths showed high thermal, mechanical and chemical stabilites. Benefiting from the step-growth polymerization process, two organic monoliths possessed well-defined 3D framework microstructure, and exhibited high permeabilities and column efficiencies in capillary liquid chromatography. A series of neutral, basic and acidic small molecules were used to comprehensively evaluate the separation abilities of these monoliths, and satisfactory chromatographic performance with column efficiencies ranged from 35,500 to 132,200 N/m was achieved, demonstrating good separation abilities of these organic monoliths prepared via thiol-epoxy click polymerization approach. Besides, multiple retention mechanisms, including hydrophobic, hydrophilic and π-π conjugate interactions were observed during the separation of analytes on these monoliths, which would make them promising for more extensive applications in capillary liquid chromatography.

Recent development of hybrid organic-silica monolithic columns in CEC and capillary LC.

As an attractive alternative to organic and silica monoliths, hybrid organic-inorganic monolith somewhat combines the advantages of them, such as high surface area, excellent mechanical strength, and thermal stability. We have reviewed the preparation and application of hybrid monoliths in 2011 and 2013. The preparation approaches have been mainly summarized into three categories: (1) common sol-gel process using trialkoxysilanes and tetraalkoxysilanes as precursors; (2) "one-pot" process of alkoxysilanes and organic monomers simultaneously via sol-gel chemistry and free radical polymerization; and (3) other polymerization of silane-containing monomers. Herein, we would focus on the recent progress and development of preparation approaches, mainly covering the literatures since July of 2012. First, the direct synthesis approach of hybrid monoliths via sol-gel chemistry and following postmodification was an important route to fabricate various monolithic stationary phases, particularly, to modify the hybrid monoliths containing amino, epoxy, vinyl, and other groups. Second, "one-pot" process, as a novel preparation approach of hybrid monoliths, was further developed in the past 2 years, in which various organic functional monomers, not only water-soluble monomers, but also hydrophobic monomers could be added in the preparation system. Other polymerization techniques in the preparation of organic monolithic materials, particularly, free radical polymerization and ring-opening polymerization, were successfully transferred to fabricate the hybrid monoliths by using silane-containing monomers including POSS monomers or other self-synthesized monomers.

Preparation of monolithic polymer columns with homogeneous structure via photoinitiated thiol-yne click polymerization and their application in separation of small molecules.

Two monolithic polymer columns were directly prepared in the UV-transparent fused-silica capillaries via photoinitiated thiol-yne click polymerization of 1,7-octadiyne (ODY) with a dithiol (1,6-hexanedithiol, 2SH) or a tetrathiol (pentaerythriol tetrakis(3-mercaptopropionate), 4SH) within 15 min. The rapid polymerization provided a time-saving approach to optimize preparation conditions. Then, two porogenic systems of diethylene glycol diethyl ether (DEGDE)/tetrahydrofuran (THF) and DEGDE/poly(ethylene glycol) (PEG, Mn = 200) were found to effectively control the porous structure of two kinds of polymeric monoliths (O2SH and O4SH), respectively. The almost disappearance of thiol and alkynyl vibrations (2560 and 2115 cm(-1), respectively) in infrared spectra and Raman spectra indicated a high conversion of the thiol-yne polymerization reaction. The thiol-yne polymerization was further proved by analyzing the energy-dispersive X-ray spectrum (EDS), MALDI-TOF mass spectrum, and elemental data. Scanning electron microscopy (SEM) images showed the monolithic polymer columns with homogeneous porous structure and macropore size of 0.5-1.0 µm, which facilitated the minimum plate heights of 10.0-12.0 µm for alkylbenzenes in reversed-phase liquid chromatography (RPLC). The low values of the A and C terms (<1.0 µm and <15.5 ms, respectively) in the van Deemter equation were similar to those obtained by some monolithic silica columns. The BSA tryptic digest was also separated on the monolithic polymer column by cLC-MS/MS. The result with 85% protein coverage was better than those given by some hybrid monolithic columns. The monolithic polymer columns were further applied for separation of phenols, natural products, and standard proteins and demonstrated satisfactory separation ability.

Synthesis of novel perphenylcarbamated ß-cyclodextrin based chiral stationary phases via thiol-ene click chemistry.

Novel cyclodextrin (CD) chiral stationary phases (CD-CSPs) with well-defined structure have been successfully synthesized by immobilization of mono/di(10-undecenoyl)-perphenylaminocarbonyl ß-CD on the 3-mercaptopropyl functionalized silica gel via thiol-ene click chemistry. The phenyl carbamate groups on the rims of CD extended the cavity of CD-CSPs, which facilitated the formation of inclusion complex with various types of racemic compounds under RP mode, and also improved the π-π stacking interaction, dipole-dipole interaction, and hydrogen bonding interaction with racemic compounds under normal phase mode. Fifteen racemic compounds were successfully separated on this CD-CSP with HPLC, and the chromatographic results also demonstrated that thiol-ene click chemistry affords a facile approach for preparation of CSPs.

Preparation of hydrazine functionalized polymer brushes hybrid magnetic nanoparticles for highly specific enrichment of glycopeptides.

Hydrazide chemistry is a powerful technique in glycopeptides enrichment. However, the low density of the monolayer hydrazine groups on the conventional hydrazine-functionalized magnetic nanoparticles limits the efficiency of glycopeptides enrichment. Herein, a novel magnetic nanoparticle grafted with poly(glycidyl methacrylate) (GMA) brushes was fabricated via reversible addition-fragmentation chain transfer (RAFT) polymerization, and a large amount of hydrazine groups were further introduced to the GMA brushes by ring-opening the epoxy groups with hydrazine hydrate. The resulting magnetic nanoparticles (denoted as Fe3O4@SiO2@GMA-NHNH2) demonstrated the high specificity of capturing glycopeptides from a tryptic digest of the sample comprising a standard non-glycosylated protein bovine serum albumin (BSA) and four standard glycoproteins with a weight ratio of 50 : 1, and the detection limit was as low as 130 fmol. In the analysis of a real complex biological sample, the tryptic digest of hepatocellular carcinoma, 179 glycosites were identified by the Fe3O4@SiO2@GMA-NHNH2 nanoparticles, surpassing that of 68 glycosites by Fe3O4@SiO2-single-NHNH2 (with monolayer hydrazine groups on the surface). It can be expected that the magnetic nanoparticles modified with hydrazine functionalized polymer brushes via RAFT technique will improve the specificity and the binding capacity of glycopeptides from complex samples, and show great potential in the analysis of protein glycosylation in biological samples.