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.

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.

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.

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.

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.

Application of Microscopic Highly Hydrophilic Silica-Based Nanocomposites with High Surface Exposure in the Efficient Identification of Intact N-Glycopeptides.

Glycosylation of proteins regulates the life activities of organisms, while abnormalities of glycosylation sites and glycan structures occur in various serious diseases such as cancer. A separation and enrichment procedure is necessary to realize the analysis of the glycoproteins/peptides by mass spectrometry, for which the surface hydrophilicity of the material is an important factor for the separation and enrichment performance. In the present work, under the premise of an obvious increase of the surface silicon exposure (79.6%), the amount of surface polar silanol is remarkably generated accompanying the introduction of the active amino groups on the surface of silica. The microscopic hydrophilicity, which is determined with water physical-adsorption measurements and can directly reflect the interaction of water molecules and the intrinsic surface of the material, maximally increases by 44%. This microscopically highly hydrophilic material shows excellent enrichment ability for glycopeptides, such as extremely low detection limits (0.01 fmol µL-1), remarkable selectivity (1:8000), and size exclusion effects (1:8000). A total of 677 quantifiable intact N-glycopeptides were identified from the serum of patients with cervical cancer, and the glycosylation site and glycan structure were analyzed in depth, indicating that this novel material can show a broad practical application in cervical cancer diagnosis.

One-step preparation of boronic acid-rich hydrothermal spheres for N-glycopeptide analysis from preeclampsia serum.

In this study, we developed a green, one-step hydrothermal carbonization (HTC) method that used water as the sole solvent to create boronic acid group-rich carbonaceous spheres (BCS). When the abundant boronic acid groups on the carbonaceous spheres react with the hydroxyl groups on the glycans in an alkaline environment, the glycopeptides are specifically captured. The results showed that BCS had excellent detection limits (0.1 fmol µL-1), selectivity (1 : 1000), and stability (10 cycles). In addition, the BCS also demonstrated excellent glycopeptide enrichment capabilities in complex biological samples; 219 glycopeptides attributed to 167 glycoproteins and 235 glycopeptides related to 166 glycoproteins in PE patient and normal pregnancy control serum were identified by nano LC-MS/MS, respectively. In addition, the molecular function of heparin binding and biological process of complement activation, positive regulation of immune response, and positive regulation of tumor necrosis factor production were significantly different between PE patients and healthy pregnant women according to gene ontology analysis, indicating that these may be associated with the development of PE.

Post-synthesis of covalent organic frameworks as a hydrophilic platform for specific detection of egg ovalbumin under physiological pH.

Ovalbumin (OVA) is an important protein source in our daily life. Unfortunately, the food safety problem has become more and more serious, such as protein allergy and contaminated protein. Therefore, it is necessary to detect vital proteins efficiently and rapidly. Mass spectrometry (MS) is a powerful tool for the detection of proteins. Herein, dual amino acids functionalized covalent organic frameworks containing disulfide covalent bonds (COF@SS@GC, where G is glutathione and C is cysteine) were facilely prepared for OVA enrichment through hydrophilic interaction liquid chromatography (HILIC) under physiological pH. The results showed that COF@SS@GC had displayed sensitive detection (0.1 fmol), good selectivity (OVA: BSA = 1:100), adsorption capacity (311 mg/g), stability, reproducibility, linearity, LOQ level (42 µg/mL) and recovery ratio (64.83 %) for OVA. COF@SS@GC also demonstrated satisfactory purification ability in the enrichment of egg white, indicating that COF@SS@GC had great potential in the enrichment of protein from complex samples.

A Ti/Nb-functionalized COF material based on IMAC strategy for efficient separation of phosphopeptides and phosphorylated exosomes.

In this work, on the basis of an immobilized metal ion affinity chromatography enrichment strategy, a new kind of covalent organic framework (COF) material for enrichment of phosphorylated peptides and exosomes was successfully prepared in a facile method, and Ti4+ and Nb5+ were used as dual-functional ions (denoted as COF-S-S-COOH-Ti4+/Nb5+). With the advantage of unbiased enrichment towards phosphopeptides, COF-S-S-COOH-Ti4+/Nb5+ shows ultra-high selectivity (maximum molar ratio of ß-casein: BSA is 1:20,000) and low limit of detection (0.2 fmol). In addition, the material has an excellent phosphopeptide loading capacity (100 µg/mg) and reusability (at least seven times). Furthermore, applying the material to the actual sample, 4 phosphopeptides were selectively extracted from the serum of renal carcinoma patients. At the same time, exosomes with an intact structure in the serum of renal carcinoma patients were successfully isolated rapidly using this strategy. All experiments have shown that COF-S-S-COOH-Ti4+/Nb5+ exhibits exciting potential in practical applications.

Post-synthesis of covalent organic frameworks with dual-hydrophilic groups for specific capture of serum exosomes.

Exosomes can reflect the physiological state of parent cells and are potential disease biomarkers. In this study, we developed an innovative hydrophilic material by post-synthesis of covalent organic frameworks with dual hydrophilic groups of glutathione and cysteine (denoted as COF-S@Au@GC) to detect glycosylated exosomes in human serum. COF-S@Au@GC enriched glycosylated exosomes in human serum due to glutathione and cysteine (GC) hydrophilicity. Our results show that COF-S@Au@GC has a detection limit of 5 amol µL-1, selectivity of 1:2000, size-exclusion effect of 1:10,000, repeatability of 10 cycles, recovery of 98.3 ± 0.5%, and loading capacity of 50 mg g-1 for glycopeptides. In addition, 182 glycopeptides were detected after enrichment with COF-S@Au@GC from renal carcinoma serum, demonstrating the feasibility of enriching glycopeptides from complex biological samples. Furthermore, COF-S@Au@GC successfully captured glycosylated exosomes in the serum of renal cancer patients, with their 161 glycopeptides detected by nano liquid chromatography with tandem mass spectrometry (LC-MS/MS). This study provides a new heuristic strategy for isolating exosomes and contributes to further functional analysis of exosomes.

Construction of boric acid-functionalized metal-organic frameworks for glycopeptide recognition in the serum of cervical cancer patients.

RATIONALE: Cervical cancer is one of the most common malignant tumors in women, and it is essential to explore potential biomarkers such as glycopeptides closely related to cancer in physiological samples of cervical cancer patients. Sample pretreatment is required before direct detection using mass spectrometry because there are certain limitations. Meanwhile, it is still highly desired to promote the functionalization and application of metal-organic framework (MOF)-derived materials. METHODS: Using a post-synthesis modification method, a novel type of boric acid-functionalized MOF probe (designated as UiO-66@PEI@Au@B(OH)2 ) is prepared for recognition of glycopeptides. The results are obtained using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and nano-liquid chromarography-tandem mass spectrometry. RESULTS: The UiO-66@PEI@Au@B(OH)2 probe exhibits a low detection limit (0.6 fmol µL-1 ), an excellent recovery rate, comparatively good reusability and selectivity (HRP digests:BSA digests = 1:500). When UiO-66@PEI@Au@B(OH)2 is used to selectively capture glycopeptides from the serum of a healthy person and a cervical cancer patient, 101 glycopeptides corresponding to 54 glycoproteins and 108 glycopeptides corresponding to 57 glycoproteins are detected, respectively. CONCLUSIONS: The successful preparation of UiO-66@PEI@Au@B(OH)2 provides a path for the investigation of the functionalization of MOF-derived materials. The excellent performance of UiO-66@PEI@Au@B(OH)2 not only demonstrates the huge potential of functionalized MOFs in the glycoproteome, but also opens up new phases of the application of MOF-based materials.

Systematic construction and external validation of an immune-related prognostic model for nasopharyngeal carcinoma.

BACKGROUND: We attempted to identify the most reliable immune-related index for predicting nasopharyngeal carcinoma (NPC) prognosis and to reveal its precise and integrated relationship with NPC progression. METHOD: One thousand seven hundred and six patients with newly diagnosed NPC (1320 from the primary cohort and 386 from the validated cohort) from January 2010 to March 2014 were enrolled. Clinical features and 12 immune-related variables were analyzed. RESULTS: A high absolute lymphocyte count (ALC; >3.2 × 109 /L) correlated with a poor prognosis of patients with NPC. Significant OS differences were discovered between patients with high ALC and no ALC elevation (p < 0.05, in primary cohort), showing similar prognostic risk to patients with advanced NPC (p > 0.05, in validated cohort). ALC improved the predictive performance of the basic tumor-node-metastasis prognostic model (p = 0.025), which was reliably validated in the external independent cohort. CONCLUSION: High ALC is a surrogate marker for improved prognostic risk stratification in NPC.

Bi-amino acid functionalized biomimetic honeycomb chitosan membrane as a multifunctional hydrophilic probe for specific capture of N-linked glycopeptides in nasopharyngeal carcinoma's disease patient's serum.

In this work, a novel porous bifunctionalized composite material was synthesized via a simple method. Gold nanoparticles are uniformly dispersed on the surface of the biomimetic honeycomb chitosan membrane through the interaction between amino and Au, and then cysteine and glutathione are successfully grafted onto the surface of the Au by the Au-S bond. The modification of cysteine and glutathione makes this bifunctionalized composite material have significant advantages of superhydrophilicity and small steric hindrance simultaneously. This material manifests excellent property in glycopeptides enrichment, with high selectivity (1:5000), low detection limit (0.1 fmol·µL-1 ), high recovery rate (99.4 ± 0.5%), and good repeatability. In addition, with the help of nano-flow liquid chromatography tandem mass spectrometry, this composite achieved excellent performance in efficiently enriching glycopeptides in the serum of healthy people and nasopharyngeal carcinoma's disease patient. More excitingly, further gene ontology analysis of molecular function and biological process indicated that 41 original glycoproteins of the identified glycopeptides from serum of nasopharyngeal carcinoma's disease patient significantly partake in numerous cancer-associated events, including protease binding, calcium ion binding, enzyme binding, extracellular matrix organization, cellular response to tumor necrosis factor, and inflammatory response.

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.

Graphene functionalized with structurally complementary amino acids for sensitive recognition of N-linked glycopeptides.

Herein, a hydrophilic graphene composite functionalized with glutathione (GSH) and L(+)-Cysteine (Cys) was prepared via a simple and fast synthesis route, which was named G@S@Au@GC. The combination attack with two different zwitterionic polymers resulted in enhanced adsorption sites for glycopeptides. The obtained G@S@Au@GC exhibited excellent performance on a low limit of detection (0.2 fmol), a high selectivity (HRP: bovine serum albumin = 1:1500), a good load capacity (250 µg•mg-1) and recovery rate (93%), which was also evaluated with IgG. Subsequently, 60 glycopeptides from complex biological sample (human saliva) were identified by Nano-LC-MS/MS. The advantages of combination attack, low-cost, simple and fast synthesis, and superior enrichment performance make G@S@Au@GC composite a bright future on glycoproteomics analysis and related diseases.

Gold nanoparticle-glutathione functionalized MOFs as hydrophilic materials for the selective enrichment of glycopeptides.

Herein, a novel zwitterionic hydrophilic metal-organic framework (MOF)-functionalized material was synthesized through grafting l-glutathione (GSH) onto the Au which acts as the intermediate layer to modify the base material (PEI-ZIF-8) by the sulfhydryl group provided by GSH and the affinity provided by Au (denoted as PEI-ZIF-8@Au@GSH). The obtained product was employed to capture glycopeptides. Benefit from its excellent hydrophilic properties, abundant amphoteric ions, and unique large specific surface area, this material demonstrated amazing ability in the enrichment and identification of glycopeptides. As a result, the PEI-ZIF-8@Au@GSH displayed high sensitivity (as low as 2 fmol), excellent binding capacity (500 mg/g), outstanding enrichment selectivity (maximum mass ratio HRP to BSA is 1:1000) toward glycopeptides, and the ability to recycle at least five times. Furthermore, 35 and 51 glycopeptides were successfully detected from 5 µL human saliva and human serum respectively in the examination of the actual sample by MALDI-TOF MS. The above results indicated that the PEI-ZIF-8@Au@GSH had a satisfactory potential in the field of glycoproteomics.

Mass filter with phase modulation of radio frequency voltage.

In this work, the parametric quadrupole resonance caused by the phase modulation of waveform potential is studied. Based on analytical and numerical description of the ion motion in the quadrupole mass filter with the phase modulation, a stability island is found with good ion optical properties such as high ion transmission efficiency (16%), high mass resolution (peak width measured at 10% of peak height, R0.1 = 6000), the required separation time (100-150 radio frequency [RF] cycles), and good peak shape of trapezoid form. Furthermore, the analysis of the frequency spectrum of the applied potential and the quadrupole mass filter (QMF) acceptance are also presented; a resonance frequency is found from this spectrum. Finally, a suitable stability X-islands with relative modulation frequency, ν = 2 ± ß , ß = 1 P , 10 ≤ P ≤ 40 , is established and studied in detail. Here, ß is the imaginary part of the characteristic exponent of stable solutions to the Mathieu equation.

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.

Characterization of the interaction between 8-bromoadenosine with human serum albumin and its analytical application.

This study was designed to examine the interaction of 8-bromoadenosine with human serum albumin (HSA) by fluorescence spectroscopy in combination with molecular modeling under simulative physiological conditions. The results of fluorescence measurements indicate that 8-bromoadenosine has a strong ability to quench the intrinsic fluorescence of HSA through static quenching procedure. The binding constants (K) at different temperatures and thermodynamic parameters, enthalpy changes (DeltaH) and entropy changes (DeltaS) were calculated according to the fluorescence data. The results showed that the hydrophobic force played the major role in the binding of 8-bromoadenosine to HSA. The fluorescence experimental results were in agreement with the results obtained by molecular modeling study. The effects of some normal positive and negative ions on the binding constants were also discussed. Moreover, the synchronous fluorescence technique was used to characterize the interaction of 8-bromoadenosine to HSA and successfully applied to determine the total proteins in human serum, urine and saliva samples at room temperature under the optimum conditions with a wide linear range and satisfactory results.