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.

Selective enrichment of glycopeptides using ground eggshell materials.

The current research of protein glycosylation is focused on develop various functionalized hydrophilic materials that can effectively enrich glycopeptides. However, most of these materials require complex synthesis steps, plenty of chemical reagents, and high cost. In this study, we employed the natural eggshell for glycopeptides enrichment for the first time. Using horseradish peroxidase (HRP) tryptic digest as a standard sample, eggshell exhibited excellent sensitivity (0.05 fmol µL-1), good selectivity [HRP tryptic digest:bovine serum albumin (BSA) tryptic digest = 1:1000], excellent size-exclusion effect (HRP tryptic digest:BSA protein = 1:10,000), good loading capacity (75 mg g-1), and recovery (97.6 ± 0.3%). In addition, 153 and 114 glycopeptides were captured by eggshell from the serum tryptic digests of normal humans and diabetic patients, respectively. Benefiting from the singular porous structure and abundant biomass, eggshell performed excellently in the capture and separation of glycopeptides. These results demonstrated the potential of environmentally friendly eggshell in glycosylation proteomics analysis.

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.

Advances in proteomics sample preparation and enrichment for phosphorylation and glycosylation analysis.

As the common and significant chemical modifications, post-translational modifications (PTMs) play a key role in the functional proteome. Affected by the signal interference, low concentration, and insufficient ionization efficiency of impurities, the direct detection of PTMs by mass spectrometry (MS) still faces many challenges. Therefore, sample preparation and enrichment are an indispensable link before MS analysis of PTMs in proteomics. The rapid development of functionalized materials with diverse morphologies and compositions provides an avenue for sample preparation and enrichment for PTMs analysis. In this review, we summarize recent advances in the application of novel functionalized materials in sample preparation for phosphoproteomes and glycoproteomes analysis. In addition, this review specifically discusses the design and preparation of functionalized materials based on different enrichment mechanisms, and proposes research directions and potential challenges for proteomic PTMs research.

Hydrophilic, dual amino acid-functionalized zinc sulfide quantum dot for specific identification of N-glycopeptides from biological samples.

RATIONALE: Glycosylation of proteins is one of the most significant and complex post-translational modifications, and N-glycosylation plays a crucial role in life activities. Mass spectrometry (MS) has been a powerful technique in the analysis of protein glycosylation. However, the direct detection of glycoproteins in biological samples based on MS still suffers from huge challenges. Therefore, enrichment and purification of samples before MS analysis is an essential prerequisite. METHODS: Hydrophilic interaction liquid chromatography (HILIC) has significantly developed for selective enrichment of glycopeptides due to its simple operation process and unbiased enrichment. Herein, hydrophilic, dual amino acid-functionalized zinc sulfide quantum dots (ZnS QDs) were prepared to enrich glycopeptides using an easy procedure. The enriched glycopeptides were detected using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). RESULTS: The obtained material exhibited high selectivity (1:2000), low detection limit (0.1 fmol/µl), good repeatability (10 times), and excellent recovery (89.8%) in glycopeptide enrichment. In the actual application in biological samples, 71 N-glycopeptides and 161 N-glycopeptides were detected from human saliva and serum, respectively. CONCLUSIONS: ZnS-Au-GC was successfully prepared using an easy method. The results showed that the obtained material exhibited excellent performance in glycopeptide enrichment. Furthermore, it had showed great potential for glycopeptide enrichment in complex biological 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.

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.