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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.

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.

Highly sensitive and selective enrichment of endogenous peptides or proteins from complex bio-system takes a significant important place to the proteomic. In this work, a unique Fe 3 O 4 @2SiO 2 @mSiO 2 -C nanomaterial was synthesized, contributing to the separation and enrichment of low concentration peptides from complex mixture. The highly ordered mesoporous carbon structure render the nanospheres with unique properties of strongly connected pore channels, strong hydrophobic properties, high specific surface area (254.90 m 2 /g), uniform pore size (3.61 nm). Which made it a promising candidates for the efficient enrichment of peptides through hydrophobic-hydrophobic interaction with low detection limit (0.2 fmol), superb size-exclusion of high molecular weight proteins, highly selectivity for BSA digest (molar ratio of BSA tryptic digests/BSA, 1:400), ideal peptides recovery (about 87.5%), wonderful repeatability (RSD less than 25%). Moreover, the as-prepared Fe 3 O 4 @2SiO 2 @mSiO 2 -C nanoparticles were successfully enriched 2198 endogenous peptides from human serum, which fully indicated that the mesoporous carbon nanoparticles was a promising candidate for isolating proteins or peptides from complex biologicals.

The composite materials consist of Covalent Organic Frameworks (COFs) and silica have been regarded as a kind of promising stationary phases due to combination of the large specific surface area and good mechanical strength of porous silica microspheres and the porous structure and the excellent stability of COFs. Herein, a novel COFs-silica composite (SiO2@rLZU1, reduced Lan Zhou University-1) was prepared via an in-situ growth strategy with a 32 nm-thick COFs layer on the surface of silica and a 2.2 nm-thick COFs layer on the inner surface of the mesopores of spherical silica (5 μm, 120 A). With secondary amine and phenyl groups, the novel stationary phase provided hydrophilic, hydrophobic and π-π interactions when used in HPLC, showing different selectivity from typical reversed-phase stationary phases. Probe molecules with aromatic moieties varying in polarity, including acidic (phenol, pyrocatechol, and pyrogallol), basic (aniline, 4-chloroaniline, and 4-nitroaniline) and neutral (benzene homologues) compounds, were all baseline separated on the SiO2@rLZU1 column, indicating its excellent separation performance. Besides, the SiO2@rLZU1 column also exhibited great repeatability with intraday RSDs of the retention time of three anilines less than 0.31% (n = 6) and peak area less than 1.63% (n = 6). On the SiO2@rLZU1 column, satisfied results were achieved in the separation of real samples such as fullerenes and coking wastewater, suggesting the great potential of the as-synthesized stationary phase in HPLC applications.

Exosomal proteins play an important role in screening disease biomarker and clinic diagnosis. However, low concentration, complex composition and difficulty in extraction hindered the study of their structure and properties. In this study, a new protein pre-fractionation system, discontinuous monolithic immobilized pH gradient capillary isoelectric focusing (DM-IPG-CIEF), was constructed by reassembling pieces of monolithic IPG-CIEF column to create small cavity with well-defined pH range. The monolithic IPG-CIEF column exhibited good linearity (R2 = 0.9997) and preparation and operation reproducibility. Then removal of high-abundant proteins and enrichment of low-abundant proteins were performed by the DM-IPG-CIEF system successfully. The system was applied to pre-fractionation and enrichment of exosomal proteins extracted from HeLa cells. The exosomal proteins with pI in the range of 6.0–9.0 were focused in enrichment cavity and collected. According to nano-HPLC-MS/MS analysis, 130 proteins in this range were identified, indicating great potential of the DM-IPG-CIEF system in pretreatment of exosomal proteins.

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 (β-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.

A near infrared fluorescent probe YSP for sulfite was synthesized, in which a julolidine fused with a pyran-2-one was employed as the fluorophore and the vinyl activated by an indole salt as the receptor. The introduction of julolidine and indole salt strengthens the electron push-pull effect of the probe and allows it to absorb (597 nm) and emit (681 nm) in red wavelength region. The addition of sulfite to the C˭C bond led to prominent blue-shifts in both absorption (171 nm) and emission (165 nm) spectra, which made it possible for colorimetric and ratiometric fluorescent detection of sulfite. NMR titration results illustrated that the determination of sulfite is a two-step process: nucleophilic addition of sulfite to the unsaturated carbon of C˭N in indole ring followed by intramolecular rearrangement through a four-membered ring to form adduct-B with shorter absorption wavelength. In addition, the cationic feature of YSP enables the probe to be specifically localized in mitochondria, and it could ratiometric bioimaging sulfite in living HepG-2 and L929 cells.

A novel enzymatic reactor was prepared by incorporating SBA-15 nanoparticles into hybrid organic–inorganic monolith and immobilizing trypsin with glutaraldehyde as bridging reagent. Preparation and operation conditions including nanoparticles percentage and residence time were optimized to improve the digestion efficiency. The digestion products were characterized by Matrix Assisted Laser Desorption Ionization Time of Flight Mass Spectrometry (MALDI-TOF-MS) with sequence coverage of 50%, 93% and 71% for bovine serum albumin, myoglobin and cytochrome C, while consuming only about 19 s in dynamic mode. Compared with enzymatic reactor without nanoparticles incorporated, the enzymatic reactor with SBA-15 nanoparticles embedded achieved higher digestion efficiency by introducing more trypsin, which was originated from combination of SBA-15 nanoparticles and hybrid organic–inorganic monolith.

A new fluorescent probe 7-(diethylamino)-3-((1e,3e)-5-oxo-5-phenylpenta-1,3-dien-1-yl)-2H-chromen-2-one (SPH), based on Michael addition mechanism, was designed and synthesized for selective detection of sulfite. The probe was constructed by incorporating an α,β-unsaturated ketone conjugated with a C˭C bond into the coumarin fluorophore as a specifical reaction site for sulfite utilizing its nucleophilic property. The extra conjugated C˭C bond induced obvious red-shifts in both absorption and emission maxima, and remarkably promoted the nucleophilic addition rate. Once treated with sulfite, the solution's color changed from orange to yellow companied with a strong blue-green fluorescence. The ratio of fluorescence intensity at 488nm and 630nm (I488/I630) was linear with sulfite concentration over the range of 0-80μM with a detection limit of 0.23μM. High selectivity and good competition of the probe toward sulfite over other anions and thiols enable us to monitor sulfite levels in realistic samples as well as in living HeLa cells.

The rod-shaped SBA–15–C18 mesoporous nanoparticles were incorporated into hybrid organic–inorganic monolithic column with aminopropyl moiety to develop novel stationary phases with mixing mechanism of reverse phase and ion exchange. Experimental conditions including dispersion pattern, nanoparticles percentage were optimized for simple and stable column preparation. The monolithic columns were evaluated with mixture of organic acids in capillary electrochromatography (CEC) mode and the column efficiency reaches 280, 000 plates/m. The results indicate that the column containing nanoparticles enhances both selectivity and column efficiency due to high specific surface area of nanoparticles and mixing separation mechanism.

A chemical potential driven micro-membrane sampler for enrichment of trace gaseous carbonyl compounds has been developed. The sampler is composed of exposed parts with membrane and analysis parts with polypropylene tube. The membrane acts as a barrier, through which the analytes dynamically diffuse and transfer from absorbents present outside to extract solvent inside through the difference of chemical potential. Formaldehyde and acetic acid were selected as verification samples. Quantification is achieved through high performance liquid chromatography (HPLC) analysis. The mass of analytes determined shows a linear correlation with concentration of the gaseous analytes. The limits of detection of formaldehyde and acetic acid after 8 h sampling were 3.32 and 0.76 μg m−3.

Based on combination of chiral recognition ability of beta-cyclodextrin (beta-CD) derivatives and flexibility of monolithic material, a series of chiral stationary phases (CSPs) were prepared by the immobilization of beta-CD and three of its derivatives to the epoxy-activated poly(glycidyl methacrylate-co-ethylene dimethacrylate) monolith under mild condition. Immobilization condition for the connection reaction by different functional groups and bonding ways was studied to obtain good enantiomer selectivity. Prepared CSPs were evaluated by separating racemic mixtures of eight amino acids and two chiral drugs with capillary electrochromatography (CEC).

Monolith with immobilized pH gradient (M-IPG) is a novel separation matrix for amphoteric substances, such as peptides and proteins. To improve the properties of the newly designed column, efforts were made to optimize the preparation procedure, including the concentrations of the monomers, glycidyl methacrylate (GMA) and ethylene glycol dimethacrylate (EDMA), as well as the kinds of porogens, which led to a monolith with improved permeability, uniformity and continuity. In addition, different diamines, including ethylenediamine, 1,3-diaminopropane, 1,4-diaminobutane and 1,5-diaminopentane, were subjected to aminate the polymer, and the last two exhibited excellent reactivity with epoxy on the polymer surface, which could obviously make the immobilization of pH gradient facilitated. Under the optimal conditions, a simple method to prepare M-IPG column with narrow pH gradient was developed with commercial carrier ampholytes (CAs) solution. Such columns were applied into the analysis of proteins and peptides, and showed the improved resolution compared to the traditional one with a wide pH distribution.

Novel open-tubular capillary electrochromatography (OT-CEC) systems with core/shell magnetic nanoparticles modified by amino or C18 groups as stationary phase were constructed by immobilizing nanoparticles in the capillary with permanent magnets. Influence of preparation method of OT-CEC column with series stationary phases (continuous two-dimension) on column performance and effect of dispersant on capability of OT-CEC column prepared by stationary phases with mixed functionalities (mixed stationary phases) were investigated in details to achieve stable preparation. Organic acids were used to evaluate the OT-CEC systems, and the relative column efficiency of salicylic acid was 420,000 plates/m for series stationary phases, while that of benzoic acid reached 480,000 plates/m for mixed stationary phases. The excellent within-column and between-column repeatability (n=5) testified with the RSDs of retention time were less than 0.44% and 10.20% for series stationary phases and 1.65% and 4.29% for mixed stationary phases. The two OT-CEC systems were further applied to separation of the aqueous extract of Rhizoma gastrodiae. Comparing with normal OT capillary column, the new systems show extra high column efficiency due to large surface areas of nanoparticles and multiple separation mechanisms, and they have great potential in the method development for the analysis of complicated samples.

In this study, a 38 mL monolith with homogeneous porous structure was produced by a single polymerization from glycidyl methacrylate (GMA) and ethylene dimethacrylate (EDMA) in the presence of porogens and an initiator. The uniform temperature distribution within the reaction system was achieved by adding reactant mixture continuously and enhancing the heat transfer ability of the polymerization system. Homogeneous porous structure in the monolith was proved by SEM and the pore size distribution profiles measured by mercury intrusion porosimetry. Experimental results from proteins separation indicated that the dynamic capacity and resolution of radial flow monolithic column were independent of flow rates. Furthermore, the pressure drop on the column was linearly dependent on the flow rate and did not exceed 1.7 MPa even at a flow rate of 50 mL/min, which proved that the prepared monolith could be used in the quick separation and preparation of biopolymers.