An amino-rich polymer-coated magnetic nanomaterial for ultra-rapid separation of phosphorylated peptides in the serum of Parkinson's disease patients.

The elucidation of disease pathogenesis can be achieved by analyzing the low-abundance phosphopeptides in organisms. Herein, we developed a novel and easy-to-prepare polymer-coated nanomaterial. By improving the hydrophilicity and spatial conformation of the material, we effectively enhanced the adsorption of phosphopeptides and demonstrated excellent enrichment properties. The material was able to successfully enrich the phosphopeptides in only 1 min. Meanwhile, the material has high selectivity (1:2000), good loading capacity (100 µg/mg), excellent sensitivity (0.5 fmol), and great acid and alkali resistance. In addition, the material was applied to real samples, and 70 phosphopeptides were enriched from the serum of Parkinson's disease (PD) patients and 67 phosphopeptides were enriched from the serum of normal controls. Sequences Logo showed that PD is probably associated with threonine, glutamate, serine, and glutamine. Finally, gene ontology (GO) analysis was performed on phosphopeptides enriched in PD patients' serum. The results showed that PD patients expressed abnormal expression of the cholesterol metabolic process and cell-matrix adhesion in the biological process (BP), endoplasmic reticulum and lipoprotein in the cellular component (CC), and heparin-binding, lipid-binding, and receptor-binding in the molecular function (MF) as compared with normal individuals. All the experiments indicate that the nanomaterials have great potential in proteomics studies.

Self-assembly of hydrazide-linked porous organic polymers rich in titanium for efficient enrichment of glycopeptides and phosphopeptides from human serum.

In this work, titanium-rich hydrazide-linked porous organic polymers (hydrazide-POPs-Ti4+) were synthesized using hydrazine, 2,3-dihydroxyterephthalaldehyde (DHTA) and trimethyl 1,3,5-benzenetricarboxylate (TP) as the ligands. Hydrazide-POPs-Ti4+ combined with HILIC and IMAC can be used for simultaneous enrichment of glycopeptides and phosphopeptides. The detection limit of this protocol is 0.1 fmol µL-1 for glycopeptides and 0.005 fmol µL-1 for phosphopeptides, and the selectivities are 1 : 1000 and 1 : 2000 for glycopeptides and phosphopeptides, respectively. For practical bio-sample analysis, 201 glycopeptides associated with 129 glycoproteins and 26 phosphopeptides associated with 21 phosphoproteins were selectively captured from healthy human serum, and 186 glycopeptides associated with 117 glycoproteins and 60 phosphopeptides associated with 50 phosphoproteins were enriched in the serum of breast cancer patients. Gene Ontology analysis indicated that the identified glycoproteins and phosphoproteins were linked to breast cancer, including the binding of complement component C1q and low-density lipoprotein particles, protein oxidation and complement activation, suggesting that these connected pathways are probably engaged in the disease pathology of breast cancer.

Post-synthesis of a titanium-rich magnetic COF nanocomposite with flexible branched polymers for efficient enrichment of phosphopeptides from human saliva and serum.

A Ti4+-functionalized magnetic covalent organic framework material with flexible branched polymers (mCOF@ε-PL@THBA-Ti4+) built via an immobilized metal ion affinity chromatography (IMAC) enrichment strategy was proposed through post-synthesis modification. Hydrophilic ε-poly-L-lysine (ε-PL) rich in amino active groups was first introduced in the fabrication of the phosphopeptide enrichment material to increase the hydrophilicity while providing more functional modification pathways of the material. 2,3,4-Trihydroxy-benzaldehyde (THBA) provides abundant binding sites for the immobilization of numerous Ti4+, which is advantageous for the subsequent efficient phosphopeptide enrichment. The magnetic nanocomposite exhibited outstanding performance of phosphopeptide enrichment with good selectivity (1 : 5000), a low detection limit (2 fmol), and relatively high loading capacity (66.7 mg g-1). What's more, after treatment with mCOF@ε-PL@THBA-Ti4+, 16 endogenous phosphopeptides from fresh saliva of healthy people were recognized by MALDI-TOF MS, and 50 phosphopeptides belonging to 35 phosphoproteins from the serum of uremia patients were detected by nano-LC-MS/MS. Proteomics data analysis for the differential protein selection between uremia and normal controls was conducted using R software, and four down-regulated and three up-regulated proteins were obtained. The results suggested that the prepared material has potential applications in biomarker discovery.

A novel hydrophilic polymer-coated magnetic nanomaterial based on the HILIC strategy for fast separation of glycopeptides and glycosylated exosomes.

Novel hydrophilic poly(N, N-methylenebisacrylamide/1,2-epoxy-5-hexene) coated magnetic nanospheres functionalized with 2-aminopurine (denoted as Fe3O4@poly(MBA/EH)@2AP) for enriching glycopeptides and glycosylated exosomes were successfully obtained using a simple and green method on the basis of the HILIC (hydrophilic interaction liquid chromatography) enrichment strategy. The high density of polar groups endows the material with amazing hydrophilicity, enabling the nanomaterial to successfully capture glycopeptides and glycosylated exosomes within 1 min. Meanwhile, the materials demonstrated great sensitivity (0.01 fmol/µL), good loading capability (125 µg/mg), high selectivity (BSA:HRP = 1000:1), and repeatability (more than 10 times). Besides, the material was applied in the analysis of bio-samples, a total of 290 glycosylated peptides and 184 glycosylation sites mapping to 185 glycoproteins were identified in the serum of uremic patients. Besides, 42 glycopeptides were enriched from the saliva of healthy people. At the same time, it was verified by TEM and western blot that the complete glycosylated exosomes were successfully captured from the serum of the uremic patients. All experiments have demonstrated that Fe3O4@poly(MBA/EH)@2AP has a promising future in practical applications.

Fabrication of a polymer brush-functionalized porphyrin-based covalent organic framework for enrichment of N-glycopeptides.

A surface-initiated atom transfer radical polymerization method combining click chemistry was employed to prepare a novel porphyrin-based covalent organic framework composite grafted with polymer brushes (TAPBB@GMA@AMA@Cys) for the specific enrichment of N-glycopeptides. The material successfully realized the high efficiency enrichment of N-glycopeptides with good selectivity (1:1000), low detection limit (0.2 fmol/µL), and high loading capacity (133.3 mg·g-1). The TAPBB@GMA@AMA@Cys was successfully applied to actual sample analysis; 235 N-glycopeptides related to 125 glycoproteins and 210 N-glycopeptides related to 121 glycoproteins were recognized from the serum of normal individuals and Alzheimer's disease patients, respectively. Gene ontology studies of molecular functions, cellular components, and biological processes have revealed that identified glycoproteins are strongly associated with neurodegenerative diseases involving innate immune responses, basement membranes, calcium binding, and receptor binding. The above results confirm the surprising potential of materials in glycoproteomics research and practical sample applications.

Magnetic guanidyl-functionalized covalent organic framework composite: a platform for specific capture and isolation of phosphopeptides and exosomes.

A guanidine-functionalized (GF) covalent organic framework (COF) nanocomposite has been developed by a post-synthetic approach for specific capture and separation of phosphopeptides and exosomes. The abundant binding sites on COF can immobilize a large number of gold nanoparticles (AuNPs), which can be used to react with amino groups to graft polyethyleneimine (PEI). Finally, Fe3O4@COF@Au@PEI-GF is obtained through the reaction of PEI and guanidyl group for phosphopeptides and exosomes detection. This composite shows a low detection limit (0.02 fmol), size exclusion effect (ß-casein digests:Albumin from bovine serum protein = 1:10,000), good reusability (10 cycles), and high selectivity (ß-casein digests:Albumin from bovine serum digests = 1:10,000). For complex biological sample, 4 phosphopeptides can be successfully identified from human serum. Furthermore, for the first time, we used guanidyl-functionalized probe to capture exosomes in human serum, providing a new method for enriching exosomes. The above experiments showed that Fe3O4@COF@Au@PEI-GF not only effectively enrich phosphopeptides and remove macromolecular proteins, but also successfully separate and capture exosomes. This demonstrates the great potential of this composite for the specific enrichment of phosphopeptides and isolation of exosomes.

Hydrophilic carrageenan functionalized magnetic carbon-based framework linked by silane coupling agent for the enrichment of N-glycopeptides from human saliva.

In this work, a magnetic graphene material coated with mesoporous silica was selected as the substrate, 3-glycidoxypropyltrimethoxysilane and polyethyleneimine were sequentially bonded through chemical reactions, and then carrageenan was successfully introduced by electrostatic interaction; finally, hydrophilic nanocomposite material was prepared. Due to the large number of hydrophilic groups, and polyethyleneimine was connected by means of chemical bonds, this material exhibits good hydrophilicity and stability for glycopeptide enrichment. In the actual enrichment process, nanomaterial exhibits high selectivity (1:500), high sensitivity (2 fmol), and good repeatability (five cycles). In addition, the synthesized material also shows a good enrichment effect in the face of actual complex biological samples, which captured 40 N-glycopeptides from human saliva, indicating the application potential for enrichment of N-glycopeptides.

Efficient separation of phosphopeptides employing a Ti/Nb-functionalized core-shell structure solid-phase extraction nanosphere.

A strategy for effectively enriching global phosphopeptides was successfully developed by using ammonia methyl phosphate (APA) as a novel chelating ligand and Ti4+ and Nb5+ as double functional ions (referred to as Fe3O4@mSiO2@APA@Ti4+/Nb5+). With the advantage of large specific surface area (151.1 m2/g), preeminent immobilized ability for metal ions (about 8% of total atoms), and unbiased enrichment towards phosphopeptides, Fe3O4@mSiO2@APA@Ti4+/Nb5+ displays high selectivity (maximum mass ratio ß-casein to BSA is 1:1500), low limit of detection (LOD, as low as 0.05 fmol), good relative standard deviation (RSD, lower than 7%), recovery rate of 87% (18O isotope labeling method), outstanding phosphopeptide loading capacity (330 µg/mg), and at least five times re-use abilities. In the examination of the actual sample, 24 phosphopeptides were successfully detected in saliva and 4 phosphopeptides were also selectively extracted from human serum. All experiments have shown that Fe3O4@mSiO2@APA@Ti4+/Nb5+ exhibits exciting potential in view of the challenge of low abundance of phosphopeptides. Graphical abstract.

Post-synthesis of boric acid-functionalized magnetic covalent organic framework as an affinity probe for the enrichment of N-glycopeptides.

A novel type of boric acid-functionalized magnetic covalent organic framework (mCOF) with polyethyleneimine (PEI) as a linker (denoted as mCOF@PEI@B(OH)2) has been prepared through a post-synthesis strategy, which points out an achievable path for the construction of boronic acid-functionalized COFs. Based on the boric acid chemistry, the obtained core-shell structured mCOF@PEI@B(OH)2 can selectively isolate glycopeptides through the modified boronic acid groups. The mCOF@PEI@B(OH)2 exhibits excellent performance with good reusability (ten cycles), low detection limit (0.5 fmol·µL-1), size-exclusion effect, and relatively high loading capacity (80 µg·mg-1), recovery yield (94.9 ± 2.8%), and selectivity (HRP digests:BSA digests = 1:500). Detection is done by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). In addition, 37 endogenous glycopeptides are captured from human saliva with mCOF@PEI@B(OH)2, providing effective proofs for its capability to capture low-abundance glycopeptides from actual biological samples.

In situ synthesis of a novel metal oxide affinity chromatography affinity probe for the selective enrichment of low-abundance phosphopeptides.

RATIONALE: Due to the dynamic nature of phosphorylation states and the low stoichiometry of phosphopeptides, it is still a challenge to efficiently capture phosphopeptides from complex biological samples before mass spectrometry analysis. Among the enrichment strategies, metal oxide affinity chromatography (MOAC) is one of the most widely used and the one with the most potential. It is based on reversible Lewis acid-base interactions between the metal oxides and the negatively charged phosphate groups to achieve the specific selection of phosphopeptides. METHODS: A novel MOAC affinity probe, denoted as G@PDA@ZrO2 , was successfully synthesized by in situ grafting ZrO2 onto the surface of graphene (G) modified with polydopamine (PDA). The novel MOAC probe thus obtained was used for phosphopeptide enrichment. RESULTS: This novel MOAC affinity probe when used to selectively enrich phosphopeptides from standard protein digest solutions exhibited a high selectivity (ß-casein:bovine serum albumin = 1:1000), a low detection limit (4 fmol), and a high loading capacity (400 mg/g). At the same time, the experimental results proved that G@PDA@ZrO2 had great recyclability (five cycles), stability, and reproducibility. Subsequently, G@PDA@ZrO2 was applied to enrich phosphopeptides from human saliva and human serum, in which 25 and 4 phosphopeptide peaks, respectively, were detected. CONCLUSIONS: This novel MOAC affinity probe (G@PDA@ZrO2 ) showed good performance in enriching phosphopeptides. Thus, G@PDA@ZrO2 has good potential in phosphopeptidomics analysis.

Postsynthesis of zwitterionic hydrophilic composites for enhanced enrichment of N-linked glycopeptides from human serum.

RATIONALE: Glycosylation of proteins plays an important role in life activities, but the concentration of naturally occurring glycopeptides is usually relatively low, and glycosylation has microfacies heterogeneity, so direct mass spectrometry is not feasible. Therefore, selective enrichment of glycopeptides before mass spectrometry has turned into an urgent problem to be resolved. METHODS: Herein, the zwitterionic L-cysteine functionalized hydrophilic graphene oxide composite (GO@PDA@MIL-125-NH2 @Au@L-Cys) was prepared via a postsynthetic method. The obtained material was used for glycopeptide enrichment. The enriched peptides were then detected using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOFMS) to demonstrate the enrichment performance of the material. RESULTS: In the actual enrichment process, GO@PDA@MIL-125-NH2 @Au@L-Cys nanomaterials exhibited high selectivity (1:1000), outstanding sensitivity (0.5 fmol), and excellent repeatability for the enrichment of glycopeptides. In addition, the proposed material showed good performance in the enrichment of glycopeptides from complex biosamples; 56 glycopeptides were detected from 2 µL of human serum using MALDI-TOFMS. CONCLUSIONS: The experimental results showed that GO@PDA@MIL-125-NH2 @Au@L-Cys exhibited excellent performance on glycopeptide analysis. It has great potential in the enrichment of glycopeptides and provides new ideas for synthetic materials with better enrichment properties in the future.

Binary magnetic metal-organic frameworks composites: a promising affinity probe for highly selective and rapid enrichment of mono- and multi-phosphopeptides.

A binary magnetic metal-organic framework (MOF)-functionalized material (magG@PDA@Ni-MOF@Fe-MOF) was prepared through grafting Ni-MOF and Fe-MOF on magnetic (Fe3O4) graphene with polydopamine (PDA) as a middle layer. Compared with single MOFs functionalized materials (magG@PDA@Ni-MOF and magG@PDA@Fe-MOF) under the same conditions, magG@PDA@Ni-MOF@Fe-MOF not only displays lower detection limits (4 fmol) and selectivity (1:1000), but also has better enrichment efficiency for both multi- and monophosphopeptides. Other than this, magG@PDA@Ni-MOF@Fe-MOF exhibits fine reusability (five cycles) and rapid enrichment property (1 min), and 24 phosphopeptides were detected when it was applied to the analysis of human saliva. Graphical abstractThe strategy of preparing a binary magnetic metal-organic framework (MOF)-functionalized material (magG@PDA@Ni-MOF@Fe-MOF) through grafting Ni-MOF and Fe-MOF on magnetic graphene with polydopamine (PDA) as a middle layer.

A highly sensitive and selective fluorescent biosensor for breast cancer derived exosomes using click reaction of azide-CD63 aptamer and alkyne-polymer dots.

Exosomes have gained recognition as valuable reservoirs of biomarkers, holding immense potential for early cancer detection. Consequently, there is a pressing need for the development of an economical and highly sensitive exosome detection methodology. In this work, we present a fluorescence method for breast cancer-derived exosome detection based on Cu-triggered click reaction of azide-modified CD63 aptamer and alkyne functionalized Pdots. The detection threshold for the exosomes obtained from the breast cancer serum was determined to be 6.09 × 107 particles per µL, while the measurable range spanned from 6.50 × 107 to 1.30 × 109 particles per µL. The employed methodology achieved notable success in accurately distinguishing breast cancer patients from healthy individuals through serum analysis. The application of this method showcases the significant potential for early exosome analysis in the clinical diagnosis of breast cancer patients.

Facile preparation of porphyrin-based porous organic polymers for specific enrichment and isolation of phosphopeptides and phosphorylated exosomes.

Exosomes, which can be used to investigate various disease processes, are novel disease markers that have been extensively studied in recent years. In this work, zirconium-rich porphyrin-based porous organic polymers (Imi-Pops-Zr) were synthesized by a facile and low-cost strategy for specific enrichment and isolation of phosphorylated peptides and exosomes. The proposed material demonstrates a low detection limit (0.5 fmol), a high selectivity (bovine serum albumin (BSA): ß-casein = 1000:1), and a loading capability of 100 mg/g for phosphopeptides. For complex practical samples, after enrichment with Imi-Pops-Zr, 4 characteristic phosphopeptides from human serum, 20 and 12 phosphopeptides from human saliva and defatted milk were detected, respectively. Besides, 74 phosphorylated peptides with 67 phosphorylation sites belonging to 61 phosphoproteins and 67 phosphorylated peptides with 63 phosphorylation sites belonging to 65 phosphoproteins were detected from the serum of normal controls and uremic patients, respectively. Biological processes, cellular components and molecular functions revealed that interleukin-6, tumor necrosis factor, high density lipoprotein and proteases binding may be associated with uremia. Furthermore, Imi-Pops-Zr was successfully used to enrich and isolate exosomes from human serum. The experimental results show that Imi-Pops-Zr has promising application in the specific enrichment of phosphorylated peptides and exosomes in complex bio-samples.

Post-synthesis modification of covalent organic frameworks for ultrahigh enrichment of low-abundance glycopeptides from human saliva and serum.

In this study, a novel type of covalent organic framework (COF) material rich in boronic acid sites was prepared through post-synthesis modification (TbBD@PEI@Au@4-MPBA). The surface of COF material had abundant carboxylic acid groups, which could bind a large amount of polyethyleneimine (PEI) through electrostatic interaction. At the same time, the amino groups on the PEI can be grafted with Au nanoparticles (Au NPs) in situ, and then 4-mercaptophenylboronic acid (4-MPBA) was modified by the reaction of Au and sulfhydryl groups. The massive grafting of boronic acid groups made the material's enrichment effect on glycopeptides expected. The results of experiments indicated that the composite material has high sensitivity (5 amol µL-1) and selectivity (1:1000). In addition, the material has outstanding stability and reusability, with a load capacity of about 100 mg g-1 and a recovery of 99.3 ± 2.2%. What's more, after enriched by TbBD@PEI@Au@4-MPBA, 56 endogenous glycopeptides from fresh human saliva were detected by MALDI-TOF MS, 56 unique glycopeptides corresponding to 31 glycoproteins from human saliva and 513 unique glycopeptides corresponding to 208 glycoproteins from serum of throat cancer patient were detected by nano-LC-MS/MS, respectively, which was expected to be applied to glycoproteomics research.

One-step preparation of carbonaceous spheres rich in phosphate groups via hydrothermal carbonization for effective phosphopeptides enrichment.

A green strategy was developed to prepare carbonaceous spheres rich in phosphoric acid groups on the surface with D-Glucose 6-phosphate sodium salt (called G6PNa2) as a sole carbon source through one-step hydrothermal carbonization method. The method is simple and facile and meets the standards of green chemistry as water is the sole solvent employed. Following the hydrothermal carbonization synthesis, the carbonaceous spheres were further functionalized with Ti4+. The main factors including reaction temperature, reaction time, and concentration of G6PNa2 were systematically studied in order to obtain the desirable morphology and the optimum phosphopeptides enrichment, for the resulting Ti4+ functionalized carbonaceous spheres (CS-Ti4+). The performance evaluation of the CS-Ti4+ prepared under the optimum conditions demonstrated excellent selectivity (1:1000), low detection limit (1 fmol) and high recovery rate (85%) towards phosphopeptides. Furthermore, 24 low-abundance phosphopeptides were captured from human saliva using CS-Ti4+, indicating its great potential in mass spectrometry-based phosphoproteome studies.

Post-synthesis of biomimetic chitosan with honeycomb-like structure for sensitive recognition of phosphorylated peptides.

Chemical modification of biological materials is indispensable for enrichment of phosphorylated peptides. In this work, we synthesized a biomimetic honeycombed affinity chromatography (IMAC) adsorbent by preparing Crosslinked Chitosan, chelating aminomethyl phosphate decorated with Ti (IV) cation. The as-prepared CTSM@AMPA-Ti4+ composites with stable structure, low steric hindrance, and high Ti4+ loading amount were used as a promising adsorbent for enrichment of phosphopeptides. CTSM@AMPA-Ti4+ showed extremely high sensitivity (0.4 fmol) and selectivity at a low composition molar ratio of ß-casein/BSA (1:1000). What's more, it can keep its performance in the case that used to capture phosphorylated peptides from standard protein ten times or soaking in the acid/base solution for a long time. In addition, CTSM@AMPA-Ti4+ successfully captured 35 phosphorylated peptides from human saliva. This study offers a way about diversiform functionalization of CTSM in phosphoproteome analysis and disease research.

Facile preparation of polymer-grafted ZIF-8-modified magnetic nanospheres for effective identification and capture of phosphorylated and glycosylated peptides.

As a member of MOFs, Zn-MOFs (ZIF-8) are seldom used in phosphopeptide enrichment because ZIF-8 is soluble in acid solutions. Therefore, properly designing a novel strategy to overcome the defect of ZIF-8 is necessary. In this study, a novel multifunctional nanoprobe was designed by uniting magnetic core, titania shell and hydrophilic metal-organic frameworks (named as Fe3O4@PDA@mTiO2@PEI-g-ZIF-8). Integrating the strategies of hydrophilic interaction affinity chromatography (HILIC), immobilized metal ion affinity chromatography (IMAC) and metal oxide affinity chromatography (MOAC), the Fe3O4@PDA@mTiO2@PEI-g-ZIF-8 mesoporous microspheres can enrich phosphorylated peptides and glycosylated peptides simultaneously. Fe3O4@PDA@mTiO2@PEI-g-ZIF-8 has high selectivity (maximum molar ratio ß-casein/HRP : BSA = 1 : 1000), low detection limit (2 fmol) towards phosphopeptides and glycopeptides. Besides, the Fe3O4@PDA@mTiO2@PEI-g-ZIF-8 also exhibited a fine performance in the actual sample detection. In the experiment, taking saliva as a sample, 16 phosphorylated peptides were identified, and from a human serum sample, 4 phosphorylated peptides were selectively identified. All in all, the materials show great potential in the future study of phosphoproteomics and glycoproteomics.

Poly (methacrylic acid-co-diethenyl-benzene) monolithic microextraction column and its application to simultaneous enrichment and analysis of mycotoxins.

A poly (methacrylic acid-co-divinyl-benzene) [poly (MAA-co-DVB)] monolithic column was specially prepared according to the chemical structures of the three mycotoxins of aflatoxin B1, zearalenone and sterigmatocystin, and used for in-tube solid-phase microextraction (in-tube SPME) of the selected mycotoxins. The poly (MAA-co-DVB) monolithic column was characterized in detail, and exhibited effective hydrophobic, π-π, and hydrogen bonding interactions towards the target analytes. By coupling the poly (MAA-co-DVB) monolithic column-based in-tube SPME with high-performance liquid chromatography, a simple, sensitive and matrix effect-free method for determination of mycotoxins was developed. Some important parameters related to extraction efficiency were optimized systematically. Under the optimal conditions, the enrichment factors for the three mycotoxins ranged from 71.9 to 98.7. Recoveries of mycotoxins in rice grains with three spiked concentration levels were in the range of 78.0-102.8%, with satisfactory relative standard deviations (n = 4) of 2.96-4.74%. Good linearity was obtained with correlation coefficients larger than 0.99, and the detection limits (S/N = 3) of the three mycotoxins were between 0.69 and 2.03µgkg-1. The developed poly (MAA-co-DVB) monolithic column based in-tube SPME-HPLC method was successfully used to sensitively determine trace mycotoxins in practical samples of rice grain without interference peaks, indicating that it is promising for the detection of trace mycotoxins in complex grain samples.