High-throughput proteomic analysis of extracellular vesicles from saliva by chemical probe-based array.

Extracellular vesicles (EVs) are cell-released, nucleus-free particles with a double-membrane structure that effectively prevents degradation of internal components by a variety of salivary enzymes. Saliva is an easily accessible biofluid that contains a wealth of valuable information for disease diagnosis and monitoring and especially reflect respiratory and digestive tract diseases. However, the lack of efficient and high-throughput methods for proteomic analysis of salivary biomarkers poses a significant challenge. Herein, we designed a salivary EV amphiphile-dendrimer supramolecular probe (SEASP) array which enables efficient enrichment and in situ detection of EVs protein biomarkers. Detergent Tween-20 washing of SEASP arrays removes high abundance of heteroproteins from saliva well. This array shows good analytical performance in the linear range of 10 µL-150 µL (LOD = 0.4 µg protein, or 10 µL saliva), exhibiting a good recovery (80.0 %). Compared to ultracentrifugation (UC), this procedure provides simple and convenient access to high-purity EVs (1.3 × 109 particles per mg protein) with good physiological status and structure. Coupling with mass spectrometry based proteomic analysis, differentially expressed proteins as selected asthma biomarkers have been screened. Then, we validated the proteomics primary screening results through clinical samples (100 µL each) using the SEASP array. Utilizing the dual antibody fluorescence technology, SEASP enables the simultaneous high-throughput detection of two proteins. Therefore, the EVs marker protein CD81 could be used as an internal standard to normalize the number of EVs, which was stably expressed in EVs. Proteomics and array results suggested that HNRNPU (P = 4.9 * 10-6) and MUC5B (P = 4.7 * 10-11) are promising protein biomarkers for infantile asthma. HNRNPU and MUC5B may be associated with disease onset and subtypes. The SEASP arrays provide a significant advancement in the field of salivary biomarker. The array enables high-throughput in situ protein detection for highly viscous and complex biological samples. It provides a rapid, low-cost, highly specific screening procedure and experimental basis for early disease screening and diagnosis in the field of liquid biopsy.

Glycan-Imprinted Nanoparticle as Artificial Neutralizing Antibody for Efficient HIV-1 Recognition and Inhibition.

The HIV-1 envelope is a heavily glycosylated class 1 trimeric fusion protein responsible for viral entry into CD4+ immune cells. Developing neutralizing antibodies against the specific envelope glycans is an alternative method for antiviral therapies. This work presents the first-ever development and characterization of artificial neutralizing antibodies using molecular imprinting technology to recognize and bind to the envelope protein of HIV-1. The prepared envelope glycan-imprinted nanoparticles (GINPs) can successfully prevent HIV-1 from infecting target cells by shielding the glycans on the envelope protein. In vitro experiments showed that GINPs have strong affinity toward HIV-1 (Kd = 36.7 ± 2.2 nM) and possess high anti-interference and specificity. GINPs demonstrate broad inhibition activity against both tier 1 and tier 2 HIV-1 strains with a pM-level IC50 and exhibit a significant inhibitory effect on long-term viral replication by more than 95%. The strategy provides a promising method for the inhibition and therapy of HIV-1 infection.

The occurrence and probabilistic risk assessment of 3-MCPDEs and GEs in infant formulas from Chinese market employing Monte Carlo simulation technique.

3-monochloropropane-1,2-diol esters (3-MCPDEs) and glycidyl esters (GEs) are food contaminants and have arisen continuous attention due to their toxicity, especially towards infants. Current risk assessment of these contaminants was mostly employing deterministic approaches, lacking quantitative characterization of the likelihood, incidence, or severity of the risks involved. Herein, 3-MCPDE and GE levels in 46 representative infant formulas (IFs) from Chinese market were determined by GC-MS/MS. Then, combining the occurrence data and consumption data from China National Food Consumption Survey, the Monte Carlo simulation-based probabilistic model for risk assessment of 3-MCPDEs and GEs in IFs from Chinese market was established. The results showed that all P90 (90th percentiles) hazard quotient values were below 1, demonstrating 3-MCPDEs didn't pose health risks to most populations aged 0-36 months old. However, for 0-12 months old groups, P10 (10th percentiles) margin of exposure values were all below 25000, indicating GEs may pose potential risks to 10% of this group. Uncertainty analysis revealed that the probabilistic model had considered uncertainties of model input and distribution, and realized refined assessment. This study is the first report on probabilistic assessment of 3-MCPDEs and GEs in IFs, which also provided references for the formulation of related regulatory limits in China.

Proteomic Discovery and Array-Based Validation of Biomarkers from Urinary Exosome by Supramolecular Probe.

Exosomes are nanoscale, membrane-enclosed vesicles with contents similar to their parent cells, which are rich in potential biomarkers. Urine, as a noninvasive sampling body fluid, has the advantages of being simple to collect, stable in protein, diverse and not regulated by homeostatic mechanisms of the body, making it a favorable target for studying tumor biomarkers. In this report, the urinary exosomal proteome was analyzed and high-throughput downstream validation was performed using a supramolecular probe-based capture and in situ detection. The technology demonstrated the efficient enrichment of exosomes with a high concentration (5.5 × 1010 particles/mL) and a high purity (2.607 × 1010 particles/mg) of exosomes from urine samples. Proteomic analysis of urine samples from patients with hepatocellular carcinoma and healthy individuals combined with proteomic screening techniques revealed that 68 proteins were up-regulated in patients with hepatocellular carcinoma. As a proof-of-principle study, three of these differentially expressed proteins, including OLFM4, HDGF and GDF15, were validated using the supramolecular probe-based array (48 samples per batch). These findings demonstrate the great potential of this approach toward a liquid biopsy for the discovery and validation of biomarkers from urinary exosomes, and it can be extended to various biological samples with lower content of exosomes.

Epitope Imprinting of Phospholipids by Oriented Assembly at the Oil/Water Interface for the Selective Recognition of Plasma Membranes.

Phospholipids, as fundamental building blocks of the cell membrane, play important roles for molecule transportation, cell recognition, etc. However, due to the structural diversity and amphipathic nature, there are few methods for the specific recognition of lipids as compared to other biomolecules such as proteins and glycans. Herein, we developed a molecular imprinting strategy for controllable imprinting toward the polar head of phospholipid exposed on the surface of cellular membranes for recognition. Phosphatidylserine, as unique lipid on the outer membrane leaflet of exosome and also hallmark for cell apoptosis, was imprinted with the developed method. The phosphatidylserine imprinted materials showed high efficiency and specific targeting capability not only for apoptotic cell imaging but also for the isolation of exosomes. Collectively, the synthesized molecularly imprinted materials have great potential for selective plasma membrane recognition for targeted drug delivery and biomarker discovery.

Proteomic and phosphoproteomic landscape of salivary extracellular vesicles to assess OSCC therapeutical outcomes.

Circulating extracellular vesicles (EVs) have emerged as an appealing source for surrogates to evaluate the disease status. Herein, we present a novel proteomic strategy to identify proteins and phosphoproteins from salivary EVs to distinguish oral squamous cell carcinoma (OSCC) patients from healthy individuals and explore the feasibility to evaluate therapeutical outcomes. Bi-functionalized magnetic beads (BiMBs) with Ti (IV) ions and a lipid analog, 1,2-Distearoyl-3-sn-glycerophosphoethanolamine (DSPE) are developed to efficiently isolate EVs from small volume of saliva. In the discovery stage, label-free proteomics and phosphoproteomics quantification showed 315 upregulated proteins and 132 upregulated phosphoproteins in OSCC patients among more than 2500 EV proteins and 1000 EV phosphoproteins, respectively. We further applied targeted proteomics by coupling parallel reaction monitoring with parallel accumulation-serial fragmentation (prm-PASEF) to measure panels of proteins and phosphoproteins from salivary EVs collected before and after surgical resection. A panel of three total proteins and three phosphoproteins, most of which have previously been associated with OSCC and other cancer types, show sensitive response to the therapy in individual patients. Our study presents a novel strategy to the discovery of effective biomarkers for non-invasive assessment of OSCC surgical outcomes with small amount of saliva.

Isoform-specific recognition of phosphopeptides by molecular imprinting nanoparticles with double-binding mode.

Phosphorylation is one of the most important post-translational modifications of proteins, but due to the low abundance of phosphopeptides, enrichment is an essential step before mass spectrometric analysis. Although there are a number of enrichment methods developed targeting different forms of proteins phosphorylations, there are few reports on specific recognition and capture of single phosphopeptide. Herein, based on the advantages of dual affinity of TiO2 and urea to a phosphate group and molecular imprinting towards the peptide sequence, the precise recognition of intact phosphorylated peptides was successfully achieved. The same peptide sequence with different phosphorylation forms (c.a. Ser, Thr and Tyr) were used as templates for proof-of-principle study, and the imprinted particles were successfully synthesized, characterized, and have the capacity to specifically recognize the targeted unique phosphorylation excluding even its isoforms. In addition, the produced molecularly imprinted nanoparticles have numerous important advantages, including strong affinity, high specificity toward single phosphopeptides, tolerance to interferences, fast binding kinetics, substantial binding capacity, excellent stability and reusability, making them an ideal sorbent for specific enrichment of unique phosphopeptides. Finally, different phosphorylation forms were specifically enriched from both standard peptides' mixture and casein/milk digests.

Molecular imprinting of glycoproteins: From preparation to cancer theranostics.

Glycoprotein imprinted polymers have rapidly grown as excellent receptors for cancer targeting, diagnostics, inhibition, and nanomedicines as they specifically target glycans and glycosites overexpressed in various tumors. Compared to natural antibodies, they are easy to synthesize, stable, and cost-efficient. Currently, no study specifically discusses glycoproteins imprinting strategies for cancer theranostics. In this review, firstly we explored various factors involved in designing and synthesis of glycoprotein imprinted materials, including, the characteristics and choice of monomers for imprinting, types of templates and their interactions involved, and the imprinting methods. Secondly, the integration of these MIPs with different probes that have been applied for in vitro and in vivo targeting for cancer diagnostics including biosensing and bioimaging, and image-guided therapeutic applications as nanomedicines. These Glycoprotein imprinted polymers have been found to specifically target the glycoprotein biomarkers and glycosylated cell receptors overexpressed in different cancers and have been reported as excellent diagnostic tools. As nanomedicines, they have been potentially employed in various modes of cancer therapy such as targeted drug delivery, photodynamic therapy, photothermal therapy, and nanoMIPs themselves as therapeutics for locally killing tumor cells. Although the research is still in its early stages and no real-world clinical trials on humans have been conducted, nanoMIPs have a promising future in this field. We believe these findings will pave the way for MIPs in advanced diagnostics, antibody treatment, and immunotherapy as future nanomedicine for real-world cancer theranostics.

Analysis of the evolution of potential and free furfural compounds in the production chain of infant formula and risk assessment.

Furfural compounds are produced during infant formula production when heat treatment is involved. In this study, a robust method was established for determining potential and free furfural compounds (furfural, 5-methyl-2-furfural, 2-acetylfuran and 5-hydroxymethyl-2-furfural) using a modified QuEChERS technique coupled with GC-MS/MS. Further, 36 samples of two batches, covering the whole infant formula production chain were analyzed by the method to investigate how furfurals evolved during process. Then risk assessment was conducted using the Toxtree and T.E.S.T. software and evaluated by hazard quotient. Results showed the contents of bound and free 5-hydroxymethyl-2-furfural demonstrated largest increase during spray-drying (6-11 times) and homogenization stages (12-33 times), respectively. As the sum up of bound and free 5-hydroxymethyl-2-furfural, potential 5-hydroxymethyl-2-furfural was found can cause safety risks in the production chain due to the high hazard quotient value (3.11), indicating it should be controlled in homogenization and spray-drying stages.

Development of a high-throughput method for the comprehensive lipid analysis in milk using ultra-high performance supercritical fluid chromatography combined with quadrupole time-of-flight mass spectrometry.

Milk lipids are one of the most complex materials in nature and are associated with many physiological functions, hence it is important to comprehensively characterize lipids profiles to evaluate the nutritional value of milk. A quick method was developed by ultra-high performance supercritical fluid chromatography coupled to electrospray ionization quadrupole time-of-flight mass spectrometry (UHPSFC-ESI-QTOF-MS) to analyze the non-polar and polar lipids profiles of cow, goat, buffalo, human milk, and infant formulas in 7 min. All chromatographic conditions were carefully optimized and their effect on the chromatographic behavior of lipid classes and species was discussed. Under optimized conditions, 12 lipid classes (triacylglycerols, diacylglycerols, monoglyceride, fatty acids, phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, phosphatidylglycerol, sphingomyelin, lyso-phosphatidylcholine, and lyso-phosphatidylethanolamine) were separated and each class was further separated in single analysis to facilitate the identification. 250 lipid species in real samples were characterized and quantified. This result demonstrates the applicability of the UHPSFC-ESI-QTOF-MS method in the high-throughput and comprehensive lipid analysis of milk, and will hopefully help to provide nutritionists with the lipid distribution in different types of milk, as well as help in the design of more suitable infant formula for babies.

Flexible and hierarchical metal-organic framework composite as solid-phase media for facile affinity-tip fabrication to selectively enrich glycopeptides and phosphopeptides.

Micro-tip-based solid-phase microextraction is considered as one of the green and powerful analytical sample preparation techniques, but its efficiency is severely hampered by some basic issues such as tedious fabrication, instability of sorbent bed, and blocking of the tip, especially for biological samples due to low permeability. These issues are tackled by introducing a flexible and hierarchical substrate in the microtip, having good mechanical strength and specific functionality to capture the desired biomolecules. Considering the well-ordered and flexible structure of melamine foam, it was used as a substrate and for hydrophilic interaction chromatography (HILIC). Metal-organic framework, due to its excellent characteristics, was grafted on its surface anchored by self-assembling polydopamine. The resulting material was characterized and packed in the tip by just pressing the material in the conical structure of the tip. This affinity tip established good and tunable permeability and was used to selectively enrich glycopeptides as well as phosphopeptides. The affinity tip demonstrated excellent performance to enrich glycopeptides and phosphopeptides with a low limit of detection up to 0.5 fmol µL-1 from tryptic digests of horseradish peroxidase and ß-Casein, respectively, and was stable up to 5 rounds of enrichment. Moreover, this affinity-tip also exhibited high selectivity up to up to 1:1000 (HRP digest to BSA digest) for glycopeptides and 1:200 (ß-Casein digest to BSA digest) for phosphopeptides and demonstrated several other fascinating characteristics such as; excellent size exclusion effect for the omission of large-sized proteins, modest backpressure, reproducibility, reusability, smooth enrichment, and successfully applied to a human saliva sample.

A risk classification strategy for migrants of food contact material combined with three (Q)SAR tools in silico.

The chemical constituents in food contact materials (FCMs) may transfer into food during the contact, which may pose potential risk to humans. So, it is important to evaluate the safety of FCMs. Due to the advantages of cost-effectiveness and high throughput, (Q)SAR tools have been gradually used for risk assessment. In this work, a risk classification strategy for migrants of food contact materials combined with three (Q)SAR tools was developed based on a single endpoint (Mutagenicity) assessment and risk matrix approach, respectively. 419 migrants existing in a self-built toxicology database beneficial from Python crawler technology were evaluated. 5 toxic hazard ranks and 4 risk ranks were obtained for single endpoint assessment and risk matrix respectively, with 21 substances assigned as Toxic hazard Class I and 43 substances assigned as RISK Ⅰ which need the highest safety concern. Besides, for the Toxic hazard Class I substances assessed by the single endpoint, 19 of them were confirmed experimentally, and all of them were overlapped in the RISK Ⅰ substances, which suggests the effectiveness and reliability of this strategy.

Boronoisophthalic acid as a novel affinity ligand for the selective capture and release of glycoproteins near physiological pH.

Boronic acid-based affinity materials have gained tremendous attention for the selective separation and recognition of cis-diol containing biomolecules. But often, these boronate affinity materials are stuck to some serious issues like high binding pH and weak affinity, especially in the case of glycoproteins. Here in this study, we used 5-boronoisophthlic acid as a novel affinity ligand for the selective capture and release of glycoproteins. The pKa value of 5-boronoisophthalic acid is investigated to be 7.8 which is just closed to physiological pH and is ideally suitable for the fast binding and elution kinetics of glycoproteins to avoid their degradation and deactivation. The affinity ligand is attached to the surface of polymer support using branched polyethyleneimine (PEI) which enhances the binding strength as it has multiple amine groups available for the attachment of 5-boronoisophthalic for synergistic interactions. The resulting affinity material is characterized and packed in a micropipette-tip using hydrophilic melamine foam as a frit to make the separation process smooth, simple, reliable, and robust. This boronic acid-based affinity tip exhibits binding constants for model glycoproteins in the range of 10-6-10-7 M, binding capacities in the range of 0.662 µM/g, and selectivity up to 1:1000 (HRP to BSA) under optimized extraction conditions. Finally, the boronic-based affinity tip is successfully applied to selectively capture the glycoproteins from the human milk sample, especially lactoferrin which is highly important in dairy manufacture.

Melamine foam assisted in-tip packed amine-functionalized titanium metal-organic framework for the selective enrichment of endogenous glycopeptides.

Endogenous glycopeptides are significantly important in diverse pathological and physiological systems, but their direct analysis is severely hampered by their low abundance and presence of interfering species in biological fluids. In this study, we synthesized the amine-functionalized titanium metal-organic framework (NH2-MIL-125(Ti)) by a simple hydrothermal method, characterized and used for glycopeptides enrichment. The designed separation media is highly hydrophilic and stable which is suitable for hydrophilic interaction chromatography (HILIC). To make the process smooth, simple, reliable, and robust, NH2-MIL-125(Ti) crystals were packed in pipette-tip using hydrophilic melamine foam, as supporting frit. Free amine groups, present in the structure imparted hydrophilicity and a unique pattern of porosity, contributing to the size exclusion effect that excluded the large-sized proteins up to 1:700 peptide to protein ratio. The prepared MOF particles possessed regular porosity, high surface area, good hydrophilicity, and offered an in-tip flow-based set-up enhanced the enrichment performance for N-linked glycopeptides. The affinity material showed a detection limit of 1 fmol.µL-1 and selectivity up to 1:1000 (HRP digest to BSA digest). Moreover, repeatability and reusability were evaluated up to five rounds of enrichment using the same affinity tip, and scanning electron microscopic images revealed no structural changes in the MOF crystals. Finally, the MOF packed in pipette tip was applied to selectively capture the N-linked endogenous glycopeptides from a healthy saliva sample and 64 unique endogenous glycopeptides were identified. These results demonstrated the excellent potential of NH2-MIL-125(Ti) based affinity tip for glycopeptides which can be used to enrich trace glycopeptide biomarkers from the biological fluids.

On-site rapid screening of benzodiazepines in dietary supplements using pipette-tip micro-solid phase extraction coupled to ion mobility spectrometry.

In this study, a reliable and novel coupling of pipette-tip micro-solid phase extraction (PT-µSPE) with corona discharge ionization-ion mobility spectrometry (CD-IMS) was developed for on-site fast detection of benzodiazepines (BZDs) in dietary supplements. The poly (styrene-co-divinylbenzene) (St-co-DVB) monoliths fabricated in pipette-tips through an in-situ polycondensation reaction. The extraction procedure of PT-µSPE for aqueous sample was performed for an appropriate number of aspirating/dispensing cycles by using a manual micropipettor. Then analytes retained in the polymeric monoliths were eluted with elution solvents by repeating the aspirating/dispensing cycles, and the eluates were analyzed by CD-IMS. In order to find the best extraction conditions, several parameters such as the number of aspirating/dispensing cycles for extraction and desorption, pH, elution solvent, amount of polymer and ionic strength were investigated. Under the optimal conditions, the LOD of this method was in the range of 5-15 ng mL-1. The spiked recovery values in real sample were obtained by PT-µSPE-IMS in the range of 84.2-112.1% and validated by ultraperformance liquid chromatography tandem mass spectrometry (UPLC-MS/MS). PT-µSPE as an on-site sampling method can be suitably combined to IMS which has great potential to achieve high throughput detection by using a multichannel micropipette. The satisfactory analytical results reveal that the developed method can be effectively applied for on-site screening BZDs in health products.