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.

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.

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.

A homogeneous binary matrix assisted laser desorption/ionization time-of-flight mass spectrometry assay for determination of artificial sweeteners in beverages.

Artificial sweeteners have been widely used as additives in various beverages. Due to the safety risks associated with artificial sweeteners, it is essential to develop a simple, rapid, and high-throughput method for the analysis of artificial sweeteners. Here, we report a homogeneous binary matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) assay for the simultaneous analysis of sweeteners including aspartame (ASP), neotame (NEO), and advantame (ADV) with a simple dilution step. The combination of nanodiamonds with 2,5-dihydroxybenzoic acid effectively improved the signal response of sweeteners, decreased the background noise, and improved the "spot-to-spot" repeatability. After the optimization, the method exhibits low limits of detection (ASP: 20 nΜ; NEO: 10 nΜ; ADV: 5 nΜ), good linearity (r > 0.995), satisfactory accuracy (96.2-103.0%), and lower RSDs (1.5-5.8%). Finally, the target sweeteners in 17 soft beverages were successfully determined with this method, showing the potential for the routine analysis of artificial sweeteners.

A Simple and Rapid Quantitative Assay for Gossypol via Reactive Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry.

The development of simple and rapid analytical tools for gossypol (GSP) is important to the food industry and medical field. Here, we report a matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) method for the detection of GSP by using a reactive matrix 4-hydrazinoquinazoline (4-HQ). The two aldehyde groups of GSP react with the 4-HQ and therefore improve the detection sensitivity and selectivity of GSP. Moreover, GSP forms homogeneous crystals with the 4-HQ matrix, allowing the quantification of the GSP by the proposed method. With the optimized experimental conditions, GSP could be detected at concentrations as low as 0.1 µM and quantified in a wide linear range (1-500 µM). After a brief extraction with an organic solvent, the GSP contents in cottonseeds and cottonseed kernels from different provinces of China were determined successfully. The spiked recovery of GSP in cottonseed/cottonseed kernel samples was obtained as 97.88-105.80%, showing the reliability of the assay for GSP determination in real samples.

Differentiation of Cis/trans-geometrical isomers in long-chain unsaturated fatty acids based on ion mobility and theoretical calculations.

In recent years, fatty acids containing conjugated CCs have attracted extensive research attention due to their biological activities against human diseases. However, their differentiation is challenging. This study developed a comprehensive analytical solution to accurately differentiate cis/trans-fatty acid isomers using ion mobility mass spectrometry (IM-MS) and theoretical calculations. Cis/trans-fatty acids were mobility-differentiated via simple complexation with 1,5,9-triazacyclododecane (9C3N) or 1,4,8,11-tetraazacyclotetradecane (10C4N) and metal ions, obtaining baseline separation with a peak-to-peak resolution of 0.35-0.92. Moreover, the conformation of the complexes was optimized theoretically, revealing different binding modes between the cis/trans-fatty acid-9C3N/10C4N-metal ion systems, yielding in-depth structural data on the complexes and elucidating the principles of mobility separation. Furthermore, the proposed method was assessed in terms of quantification, accuracy, and precision repeatability. Finally, the method was applied to analyze oil samples. Given its simplicity, speed, and lack of chemical derivatization or chromatographic separation, this technique has potential applications in food analysis.

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.

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.

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 simple strategy for d/l-carnitine analysis in food samples using ion mobility spectrometry and theoretical calculations.

This work presents a straightforward approach to the separation d/l-carnitine (d/l-Carn) using ion mobility-mass spectrometry (IM-MS) and theoretical calculations. Natamycin (Nat) was used as separation reagent to interact with the Carn, metal ions (G) were employed as ligand, the resultant ternary complexes [d/l-Carn + Nat + G]+ were observed experimentally. IM-MS results revealed that d/l-Carn could be baseline separated via complex formation using Li+, Na+, K+, Rb+, and Cs+, with a maximum peak separation resolution (Rp-p) of 2.91; Theoretical calculations were performed to determine the optimal conformations of [d/l-Carn + Nat + Li/K]+, and the predicted collisional cross section values were consistent with the experimental values. Conformational analysis was used to elucidate the enantiomeric separation of d/l-Carn at the molecular level via the formation of ternary complexes. Furthermore, quantitative analyses for the determination of the enantiomers were established with effective linearity and acceptable sensitivity. Finally, the proposed method was successfully applied in the determination of d/l-Carn in food samples.

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.

Development of dual-photoionization ion trap mass spectrometry and its application for direct analysis of VOCs in fruit aroma.

Photoionization-ion trap mass spectrometry (PI-ITMS) is one of the major directions of mass spectrometer miniaturization because of its great potential for rapid on-site VOCs detection in many cases. Traditionally, PI has always been investigated separately and is restrained by ion transmission structure, so a new structure needs to be designed and investigated for simplifying and improving the ion transmission efficiency. Interestingly, our preliminary experiments found that the signal intensity and mass range can be effectively improved by combing atmospheric pressure photoionization (APPI) and low-pressure photoionization (LPPI). Therefore, in this paper, a new dual photoionization - ion trap mass spectrometry (DPI-ITMS) was developed, explored and used to directly analyze complex VOCs. Compared with traditional single PI configuration, it presents two obvious merits: (1) simplified ion transmission structure, eliminating the need to use deflection electrode to repel ions and avoiding breakdown risk. (2) some missing/weak low m/z ion mass spectral peaks in APPI and some high m/z ion mass spectral peaks in LPPI were improved in DPI detection mode. In addition, by combining multivariate statistical analysis, we preliminary achieved in differentiating fruit types and maturity level. In summary, we concluded that the developed DPI-ITMS has moderate detection sensitivity (limited by the homemade ITMS, 0.1-1 ppmv with RSD of 6.36 %), and the DPI-ITMS configuration can be referenced by future PI-MS, and this study also provides a high-throughput, simple, noninvasive and no chemical contamination solution for analyzing main VOCs in fruit aroma.