Facile Preparation of Hydrophilic Mesoporous Metal-Organic Framework via Synergistic Etching and Surface Functionalization for Glycopeptides Analysis.

In view of the size and hydrophilicity of glycopeptides, materials having suitable channels (size-exclusion) and strong hydrophilic surface (hydrophilic interaction) are preferred to enrich the glycopeptides in biological samples. Metal-organic frameworks (MOFs) are good candidates. However, their smaller microporous channels and low chemical stability have limited the application. Herein, a facile strategy was established to construct hydrophilic mesoporous MOF via synergistic etching and surface functionalization by using phytic acid (PA). Besides, polyvinylpyrrolidone (PVP) was added during MOF synthesis to enhance the water stability of the MOF. Owing to the expanded hydrophilic mesoporous channels, the PA-modified Ce-MOF effectively and selectively captured 422 glycopeptides from 155 glycoproteins in tryptic digests of human serum (2 µL). The present work sheds light on the easy fabrication of hydrophilic mesoporous materials, and this established material holds unique advantages for glycopeptides analysis in biological samples.

Hydrophilic Nanocomposite Functionalized by Carrageenan for the Specific Enrichment of Glycopeptides.

A hydrophilic nanocomposite was synthesized by an easy route to improve glycopeptides enrichment efficiency. This new composite, prepared with a method based on electrostatic interaction, was demonstrated to be efficient for immobilization of carrageenan on graphene oxide/poly(ethylenimine) support (denoted as GO-PEI-Carr). Carrageenan, which has a large number of hydroxyl groups and is fully negatively charged, is a new modified phase of hydrophilic materials in glycoproteomics. The introduction of carrageenan provided the composite not only a perfect surface charge but also a greater ability to enrich glycosylated peptides. Thirty-four glycopeptides from human serum immunoglobulin G (IgG) tryptic digests were obviously observed with greatly improved signal-to-noise (S/N) ratio. A good selectivity was still kept even when the molar ratio of IgG and bovine serum albumin (BSA) tryptic digest mixtures reached to 1:500. Meanwhile, 76 glycopeptides derived from 56 glycoproteins with 83 N-glycosylation sites were identified from human serum and 149 glycopeptides derived from 129 glycoproteins with 157 N-glycosylation sites were identified from mouse liver tissues, which showed the ability to enrich glycopeptides from complex biological samples. In addition, GO-PEI-Carr exhibited a unique repeatability and stability even after enrichment of glycopeptides for 20 times. It also performed a higher sensitivity (1 fmol/µL IgG), a better enrichment capacity (up to ∼300 mg/g), and an ideal enrichment recovery (90.8% and 109.5%) for glycopeptides enrichment, indicating a great potential for the application of glycoproteomic research.

Preparation of a hydrophilic interaction liquid chromatography material by sequential electrostatic deposition of layers of polyethyleneimine and hyaluronic acid for enrichment of glycopeptides.

A hydrophilic interaction liquid chromatography (HILIC) material with application in glycoproteomics was obtained by sequential deposition of polyethyleneimine (PEI) and hyaluronic acid (HA) on a negatively charged substrate by means of electrostatic self-assembly. This kind of surface modification endows the material with excellent hydrophilicity and warrants efficient glycopeptides enrichment. The feasibility of this enrichment was verified by using dendritic mesoporous silica nanoparticles (DMSNs) and magnetic graphene oxide (MagG) as negatively charged substrates for PEI and HA adhesion. The two final products (DMSNs@PEI@HA and MagG@PEI@HA) exhibit high enrichment selectivity (molar ratios of IgG and BSA digests = 1:500 and 1:1000), sensitivity (detection limit, 2 fmol/µL), recovery (>90%) and enrichment capacity (300 mg/g). When using DMSNs@PEI@HA, 419 N-glycopeptides derived from 105 glycoproteins were identified. When using MagG@PEI@HA, 376 N-glycopeptides derived from 102 glycoproteins were identified, both from a 2 µL serum sample. This is better than by methods described in previous reports. Graphical abstract Schematic representation of hydrophilic modification of negatively charged nanomaterial substrates by electrostatic self-assembly techniques to obtain hydrophilic interaction liquid chromatography (HILIC) materials for enrichment of N-glycopeptides.

Hydrophilic Phytic Acid-Coated Magnetic Graphene for Titanium(IV) Immobilization as a Novel Hydrophilic Interaction Liquid Chromatography-Immobilized Metal Affinity Chromatography Platform for Glyco- and Phosphopeptide Enrichment with Controllable Selectivity.

In this work, multifunctional Ti4+-immobilized phytic acid-modified magnetic graphene (denoted as MagG@PEI@PA-Ti4+) nanocomposites were fabricated through a facile route for simultaneous/respective enrichment of N-glyco- and phosphopeptides. Phytic acid (PA), with six phosphate groups, possesses excellent hydrophilicity and metal ion coordination ability, which endowed the MagG@PEI@PA-Ti4+ with combined properties of immobilized metal ion affinity chromatography (IMAC)- and hydrophilic interaction liquid chromatography (HILIC)-based materials. On the basis of the different binding ability of N-glyco- and phosphopeptides on MagG@PEI@PA-Ti4+, the MagG@PEI@PA-Ti4+ nanocomposites could enrich N-glyco- and phosphopeptides simultaneously or respectively by using different enrichment conditions, achieving controllable selective enrichment of N-glyco- and phosphopeptides. The proposed nanocomposites demonstrated an outstanding performance for selective enrichment of N-glycopeptides (selectivity, 1:1000 molar ratios of IgG/BSA; sensitivity, 0.5 fmol/µL IgG; loading capacity, 300 mg g-1; recovery, >90%) and phosphopeptides (selectivity, 1:5000 molar ratios of α-casein/BSA; sensitivity, 0.1 fmol/µL α-casein; loading capacity, 100 mg g-1; recovery, >90%). Taking advantage of these merits, a total of 393 N-glycopeptides derived from 259 glycoproteins and 574 phosphopeptides derived from 341 phosphoproteins were identified from 200 µg of HeLa cell extracts through a single-step enrichment using MagG@PEI@PA-Ti4+.

Dendritic Mesoporous Silica Nanoparticles with Abundant Ti4+ for Phosphopeptide Enrichment from Cancer Cells with 96% Specificity.

Selective enrichment and sensitive detection of phosphopeptides are of great significance in many bioapplications. In this work, dendritic mesoporous silica nanoparticles modified with polydopamine and chelated Ti4+ (denoted DMSNs@PDA-Ti4+) were developed to improve the enrichment selectivity of phosphopeptides. The unique central-radial pore structures endowed DMSNs@PDA-Ti4+ with a high surface area (362 m2 g-1), a large pore volume (1.37 cm3 g-1), and a high amount of chelated Ti4+ (75 µg mg-1). Compared with conventional mesoporous silica-based materials with the same functionalization (denoted mSiO2@PDA-Ti4+) and commercial TiO2, DMSNs@PDA-Ti4+ showed better selectivity and a lower detection limit (0.2 fmol/µL). Moreover, 2422 unique phosphopeptides were identified from HeLa cell extracts with a high specificity (>95%) enabled by DMSNs@PDA-Ti4+, better than those in previous reports.

pH-Sensitive DOX-Loaded PAA-PF127-PAA Micelles Combined with Cryotherapy for Treating Walker 256 Carcinosarcoma in a Rat Model.

In this work, a novel combination of drug therapy and cryotherapy for tumor treatment was developed. Pluronic F127 modified with poly(acrylic acid) at two terminals (denoted as PAA-PF127-PAA) was used as carriers for doxorubicin (DOX) delivery. Due to the pH-sensitive property endowed by PAA, the DOX-loaded PAA-PF127-PAA micelles released DOX faster in the slightly acidic physiological environment of the tumor site than that in the neutral physiological environment. Three-group rats bearing Walker 256 carcinosarcoma were respectively treated with cryotherapy, combination of DOX-loaded micelles and cryotherapy, and saline injection to investigate the effect of the combined therapy. The apoptosis rate of TdT-mediated dUTP nick-end labeling (TUNEL) slices indicated that compared with cryotherapy alone, the DOX-loaded PAA-PF127-PAA micelles combining with cryotherapy showed increased apoptosis. Thus, the combination of drug therapy and cryotherapy can be used as an effective technique for tumor treatment.

Elution-free ultra-sensitive enrichment for glycopeptides analyses: Using a degradable, post-modified Ce-metal-organic framework.

In this work, we presented a facile elution-free method for ultrasensitive enrichment of glycopeptides using two kinds of novel Ce-metal-organic frameworks (Ce-MOF) post-modified with hyaluronic acid (Ce-MOF@HA) and glutamic acid (Ce-MOF@Glu). Both of the synthesized materials remained stable in the loading buffer to enrich glycopeptides selectively and degrade in the eluent to release captured glycopeptides. Due to the dissolution of materials, the elution step of the enrichment process is omitted, resulting in an extremely high sensitivity (detection limit, 0.5 fmol/µL). Meanwhile, Ce-MOF@HA and Ce-MOF@Glu also possessed excellent selectivity with molar ratios of IgG and BSA digests being 1:1000 and 1:500, respectively. Noticeably, the practical applicability of the obtained materials was inspected by analyzing the glycopeptides enriched from human serum (2 µL) by nano-LC-MS, in which 434 N-glycopeptides from 182 N-glycoproteins (by Ce-MOF@HA) and 328 N-glycopeptides from 135 N-glycoproteins (by Ce-MOF@Glu) were detected, respectively. This work provides a new method to simplify the process of glycopeptides enrichment and also paves a novel way for the enrichment of trace targets from complex matrices.

Hydrophilic phytic acid-functionalized magnetic dendritic mesoporous silica nanospheres with immobilized Ti4+: A dual-purpose affinity material for highly efficient enrichment of glycopeptides/phosphopeptides.

In this work, magnetic mesoporous silica microspheres (Mag-MSMs) with ordered radial mesochannels were fabricated by a self-assembly synthesis in chlorobenzene-water mixed system. Then, the obtained Mag-MSMs were modified with polyethyleneimine (PEI), phytic acid (PA) and Ti4+ (denoted as Mag-MSMs@PEI-PA-Ti4+) via layer by layer (LbL) assembly. Due to the excellent hydrophilicity of PEI and PA and the large amount of Ti4+, the Mag-MSMs@PEI-PA-Ti4+ possessed combined properties of hydrophilic interaction liquid chromatography (HILIC)- and immobilized metal ion affinity chromatography (IMAC)-based materials, which could be used as a dual-purpose material for N-glycopeptides or phosphopeptides enrichment. The proposed Mag-MSMs@PEI-PA-Ti4+ exhibited an outstanding performance for N-glycopeptides enrichment (selectivity: IgG/BSA = 1:1000; sensitivity: 0.5 fmol/µL IgG) and phosphopeptides enrichment (selectivity: α-casein/BSA=1:5000; sensitivity: 0.2 fmol/µL α-casein). Furthermore, after enrichment with Mag-MSMs@PEI-PA-Ti4+, a total of 276 N-glycopeptides assigned to 132 glycoproteins were identified from 2 µL human serum and 1645 phosphopeptides corresponding to 704 phosphoproteins were identified from 200 µg HeLa cell extracts.

Highly efficient enrichment of phosphopeptides from HeLa cells using hollow magnetic macro/mesoporous TiO2 nanoparticles.

In this work, hollow magnetic macro/mesoporous TiO2 nanoparticles (denoted as Fe3O4@H-fTiO2) were synthesized by a facile "hydrothermal etching assisted crystallization" route to improve the phosphopeptide enrichment efficiency. The porous nanostructure of TiO2 shell and large hollow space endowed the Fe3O4@H-fTiO2 with a high surface area (144.71 m2 g-1) and a large pore volume (0.52 cm3 g-1), which could provide more affinity sites for phosphopeptide enrichment. Besides, the large pore size of TiO2 nanosheets and large hollow space could effectively prevent the "shadow effect", thereby facilitating the diffusion and release of phosphopeptides. Compared with the hollow magnetic mesoporous TiO2 with small and deep pores (denoted as Fe3O4@H-mTiO2) and solid magnetic macro/mesoporous TiO2, the Fe3O4@H-fTiO2 nanoparticles showed a better selectivity (molar ratio of α-casein/BSA up to 1:10000) and a higher sensitivity (0.2 fmol/µL α-casein) for phosphopeptide enrichment. Furthermore, 1485 unique phosphopeptides derived from 660 phosphoproteins were identified from HeLa cell extracts after enrichment with Fe3O4@H-fTiO2 nanoparticles, further demonstrating that the Fe3O4@H-fTiO2 nanoparticles had a high-efficiency performance for phosphopeptide enrichment. Taken together, the Fe3O4@H-fTiO2 nanoparticles will have unique advantages in phosphoproteomics analysis.

Yolk-shell magnetic mesoporous TiO2 microspheres with flowerlike NiO nanosheets for highly selective enrichment of phosphopeptides.

In this work, we fabricated a yolk-shell magnetic composite that contains mesoporous TiO2 as the inner shell and flowerlike NiO as the outer shell (denoted as Fe3O4@H-TiO2@f-NiO) to reduce the limitations of single-component metal oxides in phosphopeptide enrichment. The NiO nanosheets play a synergistic role in phosphopeptide enrichment. And the unique flowerlike structure of NiO with sufficient space can facilitate the reversible insertion/extraction of peptides, which will have less impact on the enrichment process of the inner TiO2 shell. The yolk-shell structure and two types of porous nanostructures endowed this composite with a high surface area (156.58 m2 g-1) and a large pore volume (0.37 cm3 g-1). Owing to the high surface area and combined properties of TiO2 and NiO, the Fe3O4@H-TiO2@f-NiO microspheres showed a better performance for phosphopeptide enrichment than the same material without NiO nanosheets (Fe3O4@H-TiO2). According to the LC-MS/MS results, 972 unique phosphopeptides were identified from HeLa cell extracts with a high selectivity (91.9%) by Fe3O4@H-TiO2@f-NiO relative to 837 phosphopeptides (selectivity: 60.2%) by Fe3O4@H-TiO2. The results demonstrated that, compared with single-component metal oxides, composite metal oxides could enhance the selectivity and sensitivity for phosphopeptide enrichment.