Preparation and characterization of a biomimetic honeycomb cross-linked chitosan membrane and its application in the serum of gastric cancer patients.

Chitosan, as a biological macromolecule with excellent biocompatibility, has great potential for application in immobilized metal affinity chromatography (IMAC) strategies. In-depth analysis of low-abundance phosphopeptides in organisms can help reveal the pathological mechanisms of diseases. Here, we developed an IMAC material based on a biomimetic honeycomb chitosan membrane. The material demonstrates excellent biocompatibility, good hydrophilicity, and strong metal chelating capacity, which collectively confer outstanding enrichment properties. The material has high sensitivity (0.05 fmol), great selectivity (1:2000), excellent cycling stability (at least 10 cycles) and acid-base stability. In addition, the material was employed in human serum, successfully enriching 129 phosphopeptides from the serum of gastric cancer patients and 146 phosphopeptides from healthy controls. Sequence logo suggests a potential association between gastric cancer and glutamine. Ultimately, an in-depth gene ontology analysis was carried out on the phosphopeptides that were enriched in the serum samples. Compared to normal controls, our results demonstrated dysregulated expression of biological process, cellular component, and molecular function in gastric cancer patients. This suggests that the disease involves, such as blood coagulation pathways, cholesterol metabolism, and heparin binding. All experimental outcomes converge to demonstrate the substantial promise of the material for applications within proteomics research.

Rapid quantification of disaccharide isomers by derivatization in combination with ion mobility spectrometry in beer and milk.

The subtle structural variations among carbohydrate isomers pose significant challenges for their identification and quantification. Here, we propose a strategy for rapid identification and quantification of isomeric disaccharides via derivatization with 4-(3-methyl-5-oxo-pyrazolin-1-yl) benzoic acid (CPMP) and analysing by ion mobility spectrometry (IMS). After derivatization, the ionization efficiency of disaccharides was significantly improved. The disaccharide isomers were distinguished by determining the different ion mobilities of CPMP-labelled disaccharides. Among them, [M + 2CPMP + H]+ was separated with a resolution of 1.484, almost achieving baseline separation. Subsequently, [M + CPMP + Na]+ was used for the relative quantification of lactose and maltose, showing a good linear relationship with R2 > 0.990. Finally, the method was successfully applied to the identification of lactose and maltose in beer and milk. The method is fast, accurate and effective for the identification of disaccharide isomers in complex samples.

Hydrophilic materials based on hydrogel polymers for enrichment of glycopeptides from serum of colorectal cancer patients.

In this work, a composite hydrogel material consisting of chitosan-based composite hydrogel was prepared by a simple and rapid synthetic method and will be named three-dimensional (3D)-IL-COF-1@CS hydrogel. Possessing a stable 3D network structure and outstanding hydrophilicity, the novel hydrogel is capable of capturing glycopeptides. The 3D-IL-COF-1@CS hydrogel showed good sensitivity (0.1 fmol/µL) and selectivity (1:2000). In addition, 19 glycopeptides were captured in standard samples. In the analysis of human serum, 148 glycopeptides assigned to 72 glycoproteins were assayed in the serum of normal individuals, and 245 glycopeptides corresponding to 100 glycoproteins were found in the serum of colorectal cancer (CRC) patients. More importantly, several functional programs based on Gene Ontology analysis supported molecular biological processes that may be relevant to the pathogenesis of CRC, including aging, fibrinogen complex, and arylesterase activity. The low cost, simplicity, rapid synthesis, and good enrichment performance have a great future in glycoproteomics analysis and related diseases.

Engineered Bimetallic MOF-Crafted Bullet Aids in Penetrating Serum Metabolic Traits of Chronic Obstructive Pulmonary Disease.

Metabolomics analysis based on body fluids, combined with high-throughput laser desorption and ionization mass spectrometry (LDI-MS), holds great potential and promising prospects for disease diagnosis and screening. On the other hand, chronic obstructive pulmonary disease (COPD) currently lacks innovative and powerful diagnostic and screening methods. In this work, CoFeNMOF-D, a metal-organic framework (MOF)-derived metal oxide nanomaterial, was synthesized and utilized as a matrix to assist LDI-MS for extracting serum metabolic fingerprints of COPD patients and healthy controls (HC). Through machine learning algorithms, successful discrimination between the COPD and HC was achieved. Furthermore, four potential biomarkers significantly downregulated in COPD were screened out. The disease diagnostic models based on the biomarkers demonstrated excellent diagnostic performance across different algorithms, with area under the curve (AUC) values reaching 0.931 and 0.978 in the training and validation sets, respectively. Finally, the potential metabolic pathways and disease mechanisms associated with the identified markers were explored. This work advances the application of LDI-based molecular diagnostics in clinical settings.

One-step fabrication of dipeptide-based bifunctional polymer for individual enrichment of glycopeptides and phosphopeptides from serum.

A dipeptide-based bifunctional material immobilized with Ti4+ (denoted as APE-MBA-VPA-Ti4+) was developed using precipitation polymerization. This polymer combines hydrophilic interaction liquid chromatography (HILIC) and immobilized metal affinity chromatography (IMAC) enrichment strategies, allowing for the individual and simultaneous enrichment of glycopeptides and phosphopeptides. It demonstrated high sensitivity (0.1 fmol µL-1 for glycopeptides, 0.005 fmol µL-1 for phosphopeptides), strong selectivity (molar ratio HRP: BSA = 1:1000, ß-casein: BSA = 1:2500), consistent reusability (10 cycles) and satisfactory recovery rate (93.5 ± 1.8 % for glycopeptides, 91.6 ± 0.6 % for phosphopeptides) in the individual enrichment. Utilizing nano LC-MS/MS technology, the serum of liver cancer patients was analyzed after enrichment individually, resulting in the successful capture of 333 glycopeptides covering 262 glycosylation sites, corresponding to 131 glycoproteins, as well as 67 phosphopeptides covering 57 phosphorylation sites, related to 48 phosphoproteins. In comparison, the serum of normal healthy individuals yielded a total of 283 glycopeptides covering 244 glycosylation sites corresponding to 126 glycoproteins, as well as 66 phosphopeptides covering 56 phosphorylation sites related to 37 phosphoproteins. Label-free quantification identified 10 differentially expressed glycoproteins and 8 differentially expressed phosphoproteins in the serum of liver cancer patients. Among them, glycoproteins (HP, BCHE, AGT, C3, and PROC) and phosphoproteins (ZYX, GOLM1, GP1BB, CLU, and TNXB) showed upregulation and displayed potential as biomarkers for liver cancer.

Exploitation of porphyrin-based titanium-rich porous organic polymers for targeted phosphopeptide enrichment from the serum of colorectal cancer individuals.

A porphyrin-based titanium-rich porous organic polymer (Th-PPOPs@Ti4+) was designed based on immobilized metal ion affinity chromatography technique and successfully applied to phosphopeptide enrichment with 5,10,15,20-tetrakis(4-carboxyphenyl) porphine tetramethyl ester (TCPTE), 2,3-dihydroxyterephthalaldehyde (DHTA), and 2,3,4-trihydroxybenzaldehyde (THBA) as raw materials. Th-PPOPs@Ti4+ exhibited remarkable sensitivity (0.5 fmol), high selectivity (ß-casein: BSA = 1:2000, molar ratio), outstanding recovery (95.0 ± 1.9%), reusability (10 times), and superior loading capacity (143 mg·g-1). In addition, Th-PPOPs@Ti4+ exhibited excellent ability to specifically capture phosphopeptides from the serum of colorectal cancer (CRC) individuals and normal subjects. Sixty phosphopeptides assigned to 35 phosphoproteins were obtained from the serum of CRC individuals, and 43 phosphopeptides allocated to 28 phosphoproteins were extracted in the serum of healthy individuals via nano-LC-MS/MS. Gene ontology assays revealed that the detected phosphoproteins may be inextricably tied to CRC-associated events, including response to estrogen, inflammatory response, and heparin binding, suggesting that it is possible that these correlative pathways may be implicated in the pathogenesis of CRC.

Ti4+ functionalized zirconium metal-organic frameworks with polymer brushes for specific identification of phosphopeptides in human serum and skimmed milk.

In the present study, click chemistry and Schiff base reactions were simultaneously applied to prepare polymer brush (PEG)-functionalized MOF materials (UiO-66-NH2) and immobilized with Ti4+ (MOF-Brush-THBA-Ti4+) for phosphopeptide analysis. The material has a detection limit of 0.5 fmol, a selectivity of 2000:1, and a loading capacity of 133 mg/g for phosphopeptides. It also demonstrated great repeatability (10 cycles) and recovery rate (96.7 ± 1.4%). During the analysis of bio-samples, 4 specific phosphopeptides were identified in endogenous breast cancer serum, while 11 phosphopeptides were identified in skimmed milk. Moreover, 47 phosphopeptides correlated with 29 phosphorylated proteins were selectively identified from normal control serum, and 66 phosphopeptides correlated with 26 phosphorylated proteins were identified from breast cancer serum. Further analysis of gene ontology (GO) revealed that the detected phosphorylated proteins associated with breast cancer included positive regulation of receptor-mediated endocytosis, proteolysis, extracellular exosome, heparin binding, and chaperone binding. These findings suggest that these associated pathways might contribute to the etiology of breast cancer. Overall, this application exhibits enormous potential in the identification of phosphorylated peptides within bio-samples.

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.

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.

A non-targeting magnetic metal-organic framework probe for highly specific peptide-mediated precise disease monitoring.

Alzheimer's disease (AD) is a universal neurodegenerative disease in older adults with incurable and progressive properties, urging for precise monitoring to perform timely treatment to delay its progression. Herein, we introduced a non-targeting magnetic metal-organic framework probe coupled with high-throughput mass spectrometry, creating a rapid screening strategy for highly specific peptides associated with AD. Notably, an elution-free extraction process was proposed, significantly reducing sample preprocessing time while simultaneously ensuring the efficient detection of captured peptides. Using this elution-free extraction process, high-quality peptide profiles were rapidly extracted from the hundreds of samples from both diseased and healthy individuals. By integrating machine learning algorithms, LC-MS/MS, and Uniprot database searching, we identified three specific serum endogenous peptides (m/z = 4215.41, 2884.77 and 2704.61) closely associated with AD. Remarkably, with the use of any single specific peptide, the AUC (Area Under the Curve) values can reach approximately 0.9 during monitoring AD. Moreover, integrating three specific biomarkers provides a robust basis for machine learning algorithms to build monitoring models, with AUC value up to 1.000. This work represents a substantial advancement in the development of peptide-specific precise monitoring approaches for complex diseases, serving as a catalyst for increased dedication to the molecular detection field.

Combination of click chemistry and Schiff base reaction: Post-synthesis of covalent organic frameworks as an immobilized metal ion affinity chromatography platform for efficient capture of global phosphopeptides in serum with chronic obstructive pulmonary disease.

Reasonable design and construction of functionalized materials are of great importance for the enrichment of global phosphopeptides. In this work, Ti4+ functionalized hydrophilic covalent organic frameworks by introducing glutathione (GSH) and 2,3,4-trihydroxy benzaldehyde (THBA) via click chemistry and Schiff base reaction (COF-V@GSH-THBA-Ti4+ ) was constructed and applied for selective enrichment of phosphopeptides in serum. Benefit from the high surface area, excellent hydrophilicity as well as regular mesoporous structure, COF-V@GSH-THBA-Ti4+ displayed high selectivity (molar ratio of 2000:1), low limit of detection (0.5 fmol), high load capacity (100.0 mg/g) and excellent size-exclusion effect (1:10000) for enrichment of phosphopeptides. For actual bio-sample analysis, 15 phosphopeptides assigned to 10 phosphoproteins with 16 phosphorylated sites and 33 phosphopeptides assigned to 25 phosphoproteins with 34 phosphorylated sites were detected from the serum of patients with chronic obstructive pulmonary disease (COPD), and normal controls. Biological processes and molecular functions analysis further disclosed the difference of serums with phosphoproteomics between COPD and normal controls.

Designed Directional Growth of Ti-Metal-Organic Frameworks for Decoding Alzheimer's Disease-Specific Exosome Metabolites.

Exosomes, a growing focus for liquid biopsies, contain diverse molecular cargos. In particular, exosome metabolites with valuable information have exhibited great potential for improving the efficiency of liquid biopsies for addressing complex medical conditions. In this work, we design the directional growth of Ti-metal-organic frameworks on polar-functionalized magnetic particles. This design facilitates the rapid synergistic capture of exosomes with the assistance of an external magnetic field and additionally synergistically enhances the ionization of their metabolites during mass spectrometry detection. Benefiting from this dual synergistic effect, we identified three high-performance exosome metabolites through the differential comparison of a large number of serum samples from individuals with Alzheimer's disease (AD) and normal cognition. Notably, the accuracy of AD identification ranges from 93.18 to 100% using a single exosome metabolite and reaches a flawless 100% with three metabolites. These findings emphasize the transformative potential of this work to enhance the precision and reliability of AD diagnosis, ushering in a new era of improved diagnostic accuracy.

Identification and quantification of bipyridyl dicarboxylic acid isomers by ion mobility spectrometry.

The identification of positional isomers is of interest because different isomers have different chemical or biological functions and applications. The analysis of positional isomers is sometimes challenging since they have similar chemical structures and properties. For example, the analysis of mass cannot identify different positional isomers because they have identical mass-to-charge ratios and show a single mass peak in mass spectrometry. In this study, an efficient and simple qualitative and quantitative analytical method for differentiating 2,2'-bipyridine-3,3'-dicarboxylic acid (3,3'-BDA), 2,2'-bipyridine-4,4'-dicarboxylic acid (4,4'-BDA), and 2,2'-bipyridine-5,5'-dicarboxylic acid (5,5'-BDA) was developed by using ion mobility spectrometry (IMS). The results revealed that the three BDA isomers formed non-covalent complexes with cyclodextrins (CDs) and Mg2+ ions in the gas phase: [ß-CD+3,3'/4,4'/5,5'-BDA+Mg]2+ and [γ-CD+3,3'/4,4'/5,5'-BDA+Mg]2+, which were distinguished by measuring the mobility of the complexes because of their spatial conformational differences. The peak-to-peak resolution (Rp-p) values of the three isomers of [γ-CD+3,3'/4,4'/5,5'-BDA+Mg]2+ reached 2.983 and 2.892, respectively. The conformations of the ternary complexes simulated by the theoretical calculations revealed the different interactions and shapes of the stereoisomers, and the predicted results agreed with the experimental results. Simultaneously, further studies on the collisional dissociation of the ternary complexes revealed that the dissociation energies of the different complex ions varied were different owing to the diverse different conformations. Finally, the relative quantitative analysis of the different isomers in mixed samples was performed and satisfactory linearity results (R2 > 0.99) were obtained. Thus, an effective analytical method was proposed for the identification and quantification of BDA isomers without chemical derivatization, offering a promising approach for the identification of similar derivatives or positional isomers that could be applied in various fields including chemicals and pharmaceuticals.

Facile preparation of nitrogen/titanium-rich porous organic polymers for specific enrichment of N-glycopeptides and phosphopeptides.

The comprehensive investigation of protein phosphorylation and glycosylation aids in the discovery of novel biomarkers as well as the understanding of the pathophysiology of illness. In this work, a nitrogen/titanium-rich porous organic polymer was developed by copolymerizing carbohydrazide (CH) and 2,3-dihydroxyterephthalaldehyde (2,3-Dha) and modifying with Ti4+ (CH-Dha-Ti4+). The adequate nitrogen contributes to the enrichment of glycopeptides via HILIC, while titanium benefits from capturing phosphopeptides through IMAC. The proposed method exhibits excellent selectivity (1 : 1000, both for glycopeptides and phosphopeptides), LOD (for glycopeptides: 0.05 fmol µL-1, for phosphopeptides: 0.2 fmol), loading capacity (for glycopeptides: 100 mg g-1, for phosphopeptides: 125 mg g-1) and size-exclusion effect (1 : 10 000, both for glycopeptides and phosphopeptides). Furthermore, CH-Dha-Ti4+ was applied to capture glycopeptides and phosphopeptides from human serum; 205 glycopeptides and 45 phosphopeptides were detected in the serum of normal controls; and 294 glycopeptides and 63 phosphopeptides were found in the serum of uremia patients after being analyzed by nano LC-MS/MS. The discovered glycopeptides and phosphopeptides were involved in several molecular biological processes and activities, according to a gene ontology study.

Highly sensitive quantitative detection of glycans on exosomes in renal disease serums using fluorescence signal amplification strategies.

Exosomal glycoproteins play a significant role in many physiological and pathological processes. However, the detection of exosome surface glycans is currently challenged by the complexity of biological samples or the sensitivity of the methods. Herein, we prepared a novel fluorescent probe of biotin-functionalized nanocrystals (denoted as CdTe@cys-biotin) and applied it for the first time for the detection of the expression of exosomal surface glycans using a fluorescence amplification strategy. First, the dual affinity of TiO2 and CD63 aptamers of Fe3O4@TiO2-CD63 was utilized to rapidly and efficiently capture exosomes within 25 min. In this design, interference from other vesicles and soluble impurities can be avoided due to the dual recognition strategy. The chemical oxidation of NaIO4 oxidized the hydroxyl sites of exosomal surface glycans to aldehydes, which were then labeled with aniline-catalyzed biotin hydrazide. Using the high affinity between streptavidin and biotin, streptavidin-FITC and probes were successively anchored to the glycans on the exosomes. The fluorescent probe achieved the dual function of specific recognition and fluorescent labeling by modifying biotin on the surface of nanocrystals. This method showed excellent specificity and sensitivity for exosomes at concentrations ranging from 3.30 × 102 to 3.30 × 106 particles/mL, with a detection limit of 121.48 particles/mL. The fluorescent probe not only quantified exosomal surface glycans but also distinguished with high accuracy between serum exosomes from normal individuals and patients with kidney disease. In general, this method provides a powerful platform for sensitive detection of exosomes in cancer diagnosis.

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.

In situ grown magnetic COF@MOF with a phosphoserine anchor for in-depth N-glycopeptide analysis in serum.

A hydrophilic phosphoserine-functionalized magnetic organic framework composite (termed Fe3O4@COF@MOF-PS) was synthesized by an in situ growth strategy for effective capture of N-glycopeptides. Fe3O4@COF@MOF-PS exhibited high sensitivity (0.2 fmol µL-1), outstanding exclusion of size capability (1 : 10 000), good selectivity (1 : 2000), and reusability (at least 10 times). It also exhibited remarkable performance in the N-glycopeptide analysis in complex biological samples. Via nano-LC-MS/MS analysis, a total of 223 N-glycopeptides with 161 glycosylation sites assigned to 91 glycoproteins and 331 N-glycopeptides with 243 glycosylation sites assigned to 134 glycoproteins were identified in sera from cervical cancer patients and normal controls, respectively. Biological processes and molecular functional analyses indicate that the captured glycoproteins are of significant relevance to cervical cancer, for example, gene coverage or expression of cell adhesion and extracellular matrix structural constituents. Thus, Fe3O4@COF@MOF-PS not only efficiently captures N-glycopeptides, but also has the possibility of screening potential disease markers and elucidating the process of cervical cancer development.

Ti4+ functionalized ß-cyclodextrin covalent organic framework as a new immobilized metal ion affinity chromatography platform for selective capture of phosphorylated peptides and exosomes.

A Ti4+ functionalized ß-cyclodextrin covalent organic framework nanoparticle (named as ß-CD-COF@Ti4+) was synthesized using a one-pot method successfully realizing the enrichment of phosphorylated peptides and exosomes based on the immobilized metal ion affinity chromatography strategy. The functionalized ß-CD-COF@Ti4+ exhibited superior performance on the enrichment of phosphopeptides, including high selectivity (1:1000), low detection limit (0.5 fmol), and loading capacity for phosphopeptides (100 mg·g-1). After treatment with ß-CD-COF@Ti4+, 9 phosphopeptides from defatted milk, 29 phosphopeptides related to 23 phosphoproteins from normal group serum, and 24 phosphopeptides related to 22 phosphoproteins from the serum of uremia patients were captured. Through the analysis of Gene Ontology, the captured phosphoprotein is closely related to kidney disease, including lipoprotein metabolism, very-low-density lipoprotein particle, high-density lipoprotein particle, and lipid binding activity process. Furthermore, western blot verification showed that this nanoparticle could successfully capture exosomes from human serum. This study demonstrates great prospects for the enrichment of phosphopeptides and exosomes from actual bio-samples.

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.