Nanostructure stable hydrophilic hierarchical porous metal-organic frameworks for highly efficient enrichment of glycopeptides.

Protein glycosylation plays a vital role in many physiological activities in organisms. Due to the low abundance of glycopeptides and the interference of numerous non-glycopeptides in biological samples, selective enrichment of glycopeptides is of great significance for their successful identification. Metal organic frameworks (MOFs) materials are appropriate for glycopeptides enrichment by virtue of their large specific surface area and outstanding hydrophilic properties. However, the instability of hydrophilic MOFs in acidic solutions have severely limited their applications. In this work, a rational facile strategy was established to synthesize a stable hydrophilic hierarchical porous MOF (denoted as HP-MOF-Arg@mSiO2). This new material improved the selectivity and sensitivity of enrichment for glycopeptides via modification of arginine groups. More importantly, the mesoporous silica layer was introduced to enhance the stability of MOFs in aqueous solution and achieve the size exclusion effect of large-size proteins in complex samples. Overall, owing to the unique hierarchical porous and the hydrophilic modification, the synthesized HP-MOF-Arg@mSiO2 materials showed excellent hydrophilicity and hydrolytic stability, resulting in outstanding specific separation capacity in glycopeptides enrichment. A total of 521 and 342 glycopeptides were respectively captured from 2 µL human serum digests and mouse testis tissue digests, revealing the potential of the materials in the study of glycoproteomics in complex biological samples.

Hydrophilic arginine-functionalized mesoporous polydopamine-graphene oxide composites for glycopeptides analysis.

Considering the importance of glycopeptides in the clinical diagnosis of cancer and some serious diseases, the identification of glycopeptides from complex biological samples has attracted considerable attention. Effective pre-enrichment before mass spectrometry analysis plays an important role. In this work, a kind of hydrophilic two-dimensional composites (denoted as GO@MPDA@Arg) based on mesoporous polydopamine-graphene oxide were used to selectively enrich glycopeptides in biological samples. The mesoporous polydopamine (MPDA) layer self-assembled with template Pluronic F127 provided more binding sites to load arginine, and bound arginine enhanced the hydrophilicity of the material. As a result, GO@MPDA@Arg composites exhibited excellent enrichment performance for glycopeptides, containing good selectivity (IgG digests : BSA digests = 1:50, molar ratio), low detection limit for IgG digests (10 fmol µL-1), high loading capacity for IgG digests (200 µg mg-1), and good size exclusion (IgG digests : IgG : BSA = 1:100:100, mass ratio). In addition, mouse brain tissue was selected as the actual biological sample to further study the enrichment effect of GO@MPDA@Arg composites. In three parallel experiments, a total of 401 glycopeptides belonging to 233 glycoproteins were enriched from 200 µg digestion of mouse brain extract. The enrichment results demonstrate that GO@MPDA@Arg composites have application potential for glycopeptides enrichment in protein post-translational modification research.

Targeted immobilization of titanium (IV) on magnetic mesoporous nanomaterials derived from metal-organic frameworks for high-efficiency phosphopeptide enrichment in biological samples.

A selectively modified porous metal/carbon nanocomposite was fabricated to enhance the enrichment of low-abundance phosphopeptides from biological samples. The carbon matrix derived from the metal-organic framework provides a suitable pore size to allow the diffusion of peptides, while the deliberately modified metal nanoparticles within the pores enhance their interaction with the phosphopeptides. This nanocomposite shows extremely high enrichment selectivity for phosphopeptides in the MALDI-TOF MS detection, even when the molar ratio of α-casein digests versus bovine serum albumin digests was up to about 1:20,000. By combining such nanocomposite with nano-LC-MS/MS, 4556 unique phosphopeptides were identified with high selectivity (95.2%) from HeLa cell extracts. Furthermore, phosphopeptides from prostate tissue digests were also determined. A total of 277 and 1242 phosphopeptides were identified from normal and tumor tissues of a patient with prostate cancer, respectively. This indicates that phosphorylation and prostate cancer can be related to each other.

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.

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-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.

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-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+.

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.