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.

A novel strategy for the effective enrichment of phosphopeptides based on magnetic hydro-xyapatite (HAp) clusters was developed in the current study. The structure of HAp ensures its probable separation capability, including cation exchange with P-sites (negatively charged pairs of crystal phosphates), calcium coordination, anion exchange with C-sites (positively charged pairs of crystal calcium ions). The prepared magnetic HAp clusters showed good performance on the efficient enrichment of phosphopeptides from the digestion mixture of β-casein and BSA. Compared to commercial HAp particles, the magnetic HAp clusters exhibited better selectivity toward phosphopeptides. In addition, the use of magnetic material greatly simplified the enrichment procedure, which avoided the tedious centrifugation steps in a typical phosphopeptides enrichment protocol. Finally, the material was successfully applied in the enrichment of phosphopeptides from human serum. Taken together, the efficient enrichment of the phosphopeptides by the easily prepared magnetic HAp clusters demonstrated a rapid and convenient strategy for the purification of phosphopeptides from complex samples, which may facilitate protein phosphorylation studies.

A novel carboxyl-bonded silica stationary phase was prepared by “thiol-ene” click chemistry. The resultant Thiol-Click-COOH phase was evaluated under hydrophilic interaction liquid chromatography (HILIC) mobile phase conditions. A comparison of the chromatographic performance of Thiol-Click-COOH and pure silica columns was performed according to the retention behaviors of analytes and the charged state of the stationary phases. The results indicated that the newly developed Thiol-Click-COOH column has a higher surface charge and stronger hydrophilicity than the pure silica column. Furthermore, the chromatographic behaviors of five nucleosides on the Thiol-Click-COOH phase were investigated in detail. Finally, a good separation of 13 nucleosides and bases, and four water-soluble vitamins was achieved.

As low abundance cis-diol biomolecules are of great significance in biological organisms, preparation of materials for the selective enrichment of such compounds is highly favorable for the development of the related proteomics and metabolomics. To this end, we have prepared monolithic borated titania by a non-aqueous sol-gel strategy as a new inorganic affinity material for the specific capture of nucleosides, glycopeptides and glycoproteins. Benefiting from the inorganic framework, this material prevented the hydrophobic interference, which was somewhat inevitable for the mainstream organic-based boronate affinity materials. The prepared material was carefully characterized by scanning electron microscope (SEM), X-ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS) and nitrogen-sorption experiments to investigate the morphology and elemental composition. The excellent performance of borated titania on enrichment of cis-diol biomolecules was demonstrated by extracting the glycopeptides from horseradish peroxidase (HRP) digestion, standard glycoproteins, and nucleosides from a human-urine matrix. This kind of inorganic affinity material offers a new option for selective enrichment or separation of cis-diol biomolecules.

A hyper-cross-linked polymer monolithic column, poly(methacrylatoethyl trimethyl ammonium-co-vinylbenzene chloride-co-divinylbenzene) (MATE-co-VBC-co-DVB) with phenyl and quaternary ammonium groups was successfully prepared in the current study. The prepared monolith possesses large specific surface area, narrow mesopore size distribution and high column efficiency. The poly(MATE-co-VBC-co-DVB) monolithic column was demonstrated to have strong anion exchange/reversed-phase (SAX/RP) mixed-mode retention for analytes on capillary liquid chromatography (cLC). By using this monolithic column, we developed a rapid and sensitive method for the detection of DNA methylation. Our results showed that six nucleobases (adenine, guanine, cytosine, thymine, uracil, and 5-methylcytosine (5-mC)) can be baseline separated within 15 min by electrostatic repulsion and hydrophobic interactions between nucleobases and the monolithic stationary phase. The limit of detection (LOD, signal/noise=3) of 5-mC is 0.014 pmol and endogenous 5-mC can be distinctly detected by using only 10 ng genomic DNA, which is comparable to that obtained by mass spectrometry analysis. Furthermore, by using the method developed here, we found that DNA methylation inhibitor 5-azacytidine (5-aza-C) and 5-aza-2′-deoxycytidine (5-aza-CdR) could induce a significant decrease of genome-wide DNA methylation in human lung carcinoma cells (A549) and cervical carcinoma cells (HeLa).

Protein phosphorylation is a common posttranslational modification, and involved in many cellular processes. Like endogenous peptides, endogenous phosphopeptides contain many biomarkers of preclinical screening and disease diagnosis. In this work, titanium-containing magnetic mesoporous silica spheres were synthesized and applied for effective enrichment of peptides from both tryptic digests of standard proteins and human serum. Besides, the enriched peptides can be further separated into nonphosphopeptides and phosphopeptides by a simple elution. First, titanium-containing magnetic mesoporous silica spheres were synthesized by a sol–gel method and found to have high surface area, narrow pore size distribution, and useful magnetic responsivity. Then, as the prepared material was used for selective capturing of phosphopeptides, it demonstrated to have higher selectivity than commercial titanium dioxide. Moreover, via combination of size-exclusion mechanism, hydrophobic interaction, and affinity chromatography, titanium-containing magnetic mesoporous silica spheres were successfully applied to simultaneously extract and separate nonphosphopeptides and phosphopeptides from standard protein digestion and human serum.

A novel hydrophilic polymer-coated silica sorbent has been prepared using the radical “grafting from” polymerization method through surface-bound azo initiators for hydrophilic-interaction chromatography (HILIC). The azo groups were introduced to the surface of silica gel through the reaction with amino groups on the surface of silica gel with 4,4′-azobis(4-cyanopentanoic acid chloride) (ACVC). The resultant azo-immobilized silica gel served as surface initiator to polymerize hydrophilic triol acrylamide monomer N-acryloyltris(hydroxymethyl) aminomethane (NA) in methanol to get hydrophilic polymer-coated silica sorbent. The obtained poly(NA)-coated silica (pNA-sil) was characterized by Fourier transform infrared spectroscopy (FT-IR), elemental analysis (EA), and nitrogen sorption porosimetry (NSP). Then the pNA-sil was packed into the stainless-steel column and evaluated in high-performance liquid chromatography (HPLC). Good chromatographic performance for the separation of peptides and nucleosides was obtained under HILIC mode. The results indicated that the pNA-sil stationary phase behaved as mixed-mode retention mechanisms of hydrophilic and ionic interactions. Furthermore, the pNA-sil phase was used to separate tryptic digest of β-casein and our results showed that more than 12 peptides peaks were resolved and well distributed within the elution window. Finally, the pNA-sil stationary phase was demonstrated to possess remarkable reproducibility and stability. Taken together, the pNA-sil stationary phase prepared in the current study offers a potential application in proteomics study.

In this work, a novel method for the fabrication of magnetic carbon nanotubes based on ‘aggregation wrap’ was proposed. When carbon nanotubes and magnetic nanoparticles were vortically mixed in a solvent, the magnetic nanoparticles were wrapped into the carbon nanotube bundles that formed during the aggregation process, leading to the formation of magnetic carbon nanotubes. Thus, the resultant material can be separated from the solvent rapidly and conveniently by a magnet. Our investigation demonstrated that the ‘aggregation wrap’ mechanism for the preparation of magnetic composite is also applicable to other self-aggregated micro/nanomaterials, including graphene, graphite, C₆₀, etc. To testify the feasibility of the magnetic composites in sample preparation, the resultant magnetic carbon nanotubes were applied as sorbents for magnetic solid-phase extraction (MSPE) of estrogens in milk samples. Under optimized conditions, a rapid, convenient and efficient method for the determination of estrogens in milk samples was established by the combination of MSPE with high-performance liquid chromatography with fluorescence detector. The linearity range of the proposed method was 5–2000 μg/L with correlation coefficients (R) of 0.9983–0.9994. The limit of detection (LOD) for three estrogens ranged from 1.21 to 2.35 μg/L. The intra- and inter-day relative standard deviations (RSDs) were <9.3%. The reproducibility of the MSPE with different batches of magnetic carbon nanotubes was acceptable with RSD values <3.6%.

A novel magnetic material Fe₃O₄/SiO₂/P(MAA-co-VBC-co-DVB) was prepared via the hypercrosslinking of its precursor which was produced via precipitation polymerization of methacrylic acid (MAA), vinylbenzyl chloride (VBC), and divinylbenzene (DVB) in the presence of Fe₃O₄/SiO₂ submicrospheres with the surface containing abundant reactive double bonds. The resultant sorbent was characterized by scan electron microscopy, N₂ adsorption, and Fourier transform infrared spectroscopy. It was found that this material had remarkable features such as large surface area (500 m²/g) and pore volume (0.32 cm³/g), as well as desirable chemical composition (including hydrophobic and ion-exchange moieties). Taking advantages of the Fe₃O₄/SiO₂/P(MAA-co-VBC-co-DVB), a magnetic SPE (MSPE) coupled with capillary electrophoresis (CE) method was developed for the determination of illegal drugs in urine samples. The extraction time could be clearly shortened up to 3 min. The recoveries of these drug compounds were in the range of 84.0–123% with relative standard deviations ranging between 1.7 and 10.5%; the limit of detection was in the range of 4.0–6.0 μg/L. The proposed method is simple, effective, and low-cost, and provides an accurate and sensitive detection platform for abused drug analysis.

In our current work, we describe how open tubular-immobilized metal-ion affinity chromatography (OT-IMAC) capillary columns connected to a solid phase microextraction (in-tube SPME) device can be used for the enrichment of phosphopeptides. A phosphonate modified silica nanoparticle (NP)-deposited capillary was prepared by liquid phase deposition (LPD), and used for the immobilization of Fe³⁺, Zr⁴⁺ or Ti⁴⁺. The enrichment capacities of three different OT-IMAC capillary columns were compared by using tryptically digested α-casein as sample. The improved extraction efficiency in our technique was demonstrated by comparing to a directly modified capillary, and a comparison of phosphopeptide extraction from simple and complex samples was tested for both modes. Our results show that the NP-IMAC-Zr⁴⁺ capillary column can be used to selectively isolate phosphopeptides from real samples, and can enrich for β-casein phosphopeptides from concentrations as low as 1.7×10⁻⁹ M.

Ceria-zirconia composites at different molar ratios were synthesized. Several methods were used to characterize these composites, including X-ray photoelectron spectroscopy, surface area and surface acid-base property detection. A one-step method for isolation and identification of phosphopeptides from peptide mixture was created using these ceria-zirconia composites. Using tryptic digest of standard phosphorylated protein, we have shown that these enrichment and dephosphorylation activities are effective. The adsorption capacity and catalytic property of ceria-zirconia composites at different molar ratios and calcinated temperatures were studied. In combination with MALDI-TOF-based peptide mass finger printing technique, we have established a method to utilize the enrichment/dephosphorylation dual properties of these ceria-zirconia composites for the analysis of phosphoprotein in nonfat milk successfully.

A poly(N-isopropylacrylamide-co-ethylene dimethacrylate) (poly(NIPAAm-co-EDMA)) monolith was in situ prepared in the capillary and was investigated for in-tube solid-phase microextraction (SPME). NIPAAm, an acrylamide monomer with isopropyl group, was crosslinked with EDMA. PEG of 400–20 000 Da molecular weight and methanol were selected as the binary porogens. The porous structures of the resulting monoliths have been assessed by SEM, nitrogen adsorption–desorption, and pressure drop measurements. To investigate the extraction mechanism, several groups of model analytes (including neutral, acidic, and basic) were examined. The result showed that this monolithic material exhibited high extraction efficiencies for compounds under highly acidic and basic conditions, which was due to the hydrophobic interactions and excellent pH stability of the monolith. The equilibrium extraction time profiles were also monitored for model compounds to evaluate the extraction capacity of monolithic capillary. Finally, the developed monolith in-tube SPME–HPLC method was applied to the determination of three tricyclic antidepressants from urine samples.

An analytical method based on online combination of polymer monolith microextraction (PMME) technique with hydrophilic interaction LC (HILIC)/MS is presented. The extraction was performed with a poly(methacrylic acid-co-ethylene glycol dimethacrylate) monolithic column while the subsequent separation was carried out on a Luna silica column by HILIC. After 1:1 v/v dilution with 20 mM phosphate solution at pH 7.0 and centrifugation, urine sample was directly used for extraction. After optimization, 85% ACN (containing 0.3% formic acid v/v) was used for rapid online elution, which was also the mobile phase in HILIC to avoid band broadening during separation or carry-over that was usually observed in PMME-RP LC system. Online automation of extraction and separation procedures was realized under the control of a program in this study. The developed method was applied to rapid and sensitive monitoring of three β₂-agonist traces in human urine. The LODs (S/N = 3) of the method were found to be 0.05–0.09 ng/mL of β₂-agonists in urine. The recoveries of three β₂-agonists spiked in five different urine samples ranged from 79.8 to 119.8%, with RSDs less than 18.0%.