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Importantly, the sensor enabled the measurement of HA in real samples obtained from fish, thus demonstrating its practical potential as a HA analytical detector.Protein N-glycosylation plays an essential role on cancers and other pathological processes. Its structural and functional studies rely on complete qualitative and quantitative information. Thus, it is important to quantify differentially expressed intact N-glycopeptides (DEGPs) at their molecular level. Here we report our application of stable isotopic diethyl labeling (SIDE) of amino groups for relative quantitation of intact N-glycopeptides. The amino groups from both the N-terminal and lysine residues of N-glycopeptides were diethylated with XH3XHO (X = 13C or C) and NaBH3CN. A linear quantitation dynamic range up to 50-fold was obtained with R2 = 0.9985 with intact N-glycopeptides from standard glycoprotein ribonuclease B (RNase B). In proof-of-principle comparative N-glycoproteomics study of aberrant N-glycosylation of gastric cancer tissues vs. Ko143 in vitro adjacent tissues using SIDE, 644 DEGPs (≥1.5-fold change, p less then 0.05, p was calculated using t-test) were discovered. With its accuracy and big dynamic range, SIDE can be applied to quantitative study of any aberrant glycosylation at the intact glycopeptide level.Chemical derivatization of glycans is a common strategy to increase the analytical performance of MALDI-MS-based glycan profiling techniques. Hydrazide, one of the most popular tags, offers important advantages including allowing purification-free procedures. Several hydrazides have thus been used for glycomics combined with an on-target strategy to further simplify the analytical procedures. Usually, gentle heating and mildly acidic conditions with somewhat long reaction times are needed for these hydrazide derivatizations to reach a high reaction efficiency, which makes the current hydrazide tags not yet perfectly conducive to high-throughput analysis. To further optimize these hydrazide tags for high-throughput analysis, based on the structure of a reported hydrazide and the theoretical calculations, a new cationic hydrazide tag, 4-(hydrazinecarbonyl)-N,N,N-trimethylbenzenaminium (HTMBA), was designed, synthesized and tested in this work. HTMBA could completely derivatize glycans at room temperature in several seconds under very mildly acidic conditions ( less then 3% acetic acid). A 19-fold enhancement in the signal intensity was obtained without interference from alkali adduct ions in the MALDI-MS detection of HTMBA-labeled maltoheptaose. To broaden the applicability of HTMBA, an HTMBA on-target derivatization (HOD) strategy was developed and fully validated with maltoheptaose and RNase B, and the method showed a good repeatability and stability. Finally, the HOD strategy was successfully applied to serum samples, 44 glycans in human serum were detected, and the O-acetylation information of sialic acid in horse serum was preserved. These results showed that the HOD strategy was suitable for the MS-based rapid analysis of all glycoforms in complex biological samples.As a typical kind of endogenous reactive nitrogen species, peroxynitrite (ONOO-) is believed heavily involved in the pathogenesis of many diseases such as inflammation, neurodegenerative conditions, and cardiovascular disorders. Precisely estimating ONOO- level in cell compartments is crucial for unraveling the biological relevance of ONOO- and enabling effective control of ONOO--associated pathogenicity but suffers from serious difficulty owing to the daunting elusive features of ONOO-, namely nanomolar level physiological concentration and millisecond level biological half-life. A new fluorescent probe capable of detecting ONOO- with limit of detection down to 1.2 nM, response time less than 1s, and high recognition specificity over other similarly interfering species was developed in this work. For the probe constructed by conjugating an isatin moiety with an electron-withdrawing tricyanofuran (TCF) moiety, the former enabled a highly selective ONOO--mediated oxidative decarbonylation reaction while the latter significantly improved the electrophilicity of the 3-position carbonyl group of isatin moiety and therefore accelerate the ONOO--mediated nucleophilic attack, which eventually enabled prompt and efficient recognition reaction. For the decarbonylated product featured with a released primary aniline moiety, TCF acted as an acceptor for enabling an intramolecular charge transfer (ICT) process and the remarkable change in electronic feature upon reaction with ONOO-, which generated turn-on fluorescence with large contrast and therefore the basis for ONOO- sensing. The cell fluorescence imaging performed in this work definitely verified the capability of the probe for mapping intracellular ONOO-, despite the daunting elusive features of such physiological species.Biothiols, as basic biological reactive sulfur species, perform vital actions in many critical physiological processes. Simultaneous detection and direct visualization of intracellular biothiols has great significance to figure out their metabolic mechanisms and cellular functions in living organism. However, it is an enormous challenge to selectively sense biothiols, troubled by their similar chemical structures and properties. In this work, we reported a novel chlorinated coumarin based multi-signal fluorescent probe (CC) for discriminative detection of cysteine (Cys), homocysteine (Hcy) and glutathione (GSH) through three different emission channels. CC exhibited favorable sensing properties towards biothiols such as large fluorescence enhancement, low limits of detection (6, 3 and 200 nM for Cys, Hcy and GSH), fast response speed (15 min) and pretty good water solubility. Moreover, it was also utilized for discriminative visualization of endogenous and exogenous Cys, Hcy and GSH in A375 cells and Tcam-2 cells successfully.In this work, efficient, sensitive bifunctional-monomer chitosan magnetic molecularly imprinted polymers (BCMMIPs) were fabricated and successfully applied to concentrate the metabolites of Epimedium flavonoids in rat testis and bone that were later analyzed using UPLC-Q-TOF-MS. Using chitosan and methacrylic acid as co-functional monomers, BCMMIPs exhibited a large adsorption capacity (7.60 mg/g), fast kinetics (60 min), and good selectivity. Chitosan is bio-compatible and non-toxic, and methacrylic acid provides multiple hydrogen bond donors. The BCMMIPs were injected into rat testis to specifically enrich the total flavonoid metabolites in vivo and were used to extract metabolites from bone in vitro. The results showed that the BCMMIPs coupled with UPLC-Q-TOF-MS successfully identified 28 compounds from testis and 18 compounds from bone, including 19 new compounds. This study provided a reliable protocol for the concentration of metabolites from complex biological samples, and several new metabolites of Epimedium flavonoids were found in vivo and in vitro.Adenosine triphosphate (ATP) is the main energy currency of life that plays a vital role in supporting physiological activities in living organisms, including humans. Therefore, accurate and sensitive detection of ATP concentration is necessary in biochemical research and clinical diagnosis. Herein, a ratiometric fluorescence aptasensor was developed for ATP detection. A dual-function DNA strand comprising an ATP-binding aptamer (ABA) and berberine-binding aptamer (BBA) was designed and optimized, in which ABA can capture ATP and thioflavin T (ThT), whereas BBA can capture berberine. Interestingly, the fluorescence intensity of both berberine and ThT were enhanced as they were captured by this dual-function DNA strand. In the presence of ATP, the ABA on the 3'-end of the DNA bound specifically to its target, causing ThT release and a significant drop in ThT fluorescence. However, ATP had no significant effect on the interaction between berberine and DNA, remaining the enhanced fluorescence intensity of berberine stable. Based on this interesting phenomenon, a ratiometric fluorescence sensor was constructed that used the enhanced fluorescence intensity of berberine as reference to measure the fluorescence intensity of ThT for ATP detection. This ratiometric fluorescence strategy had excellent selectivity and high sensitivity towards ATP with a detection limit (3σ) as low as 24.8 nM. The feasibility of application of this method in biological samples was evaluated in human serum and urine samples, where it exhibited a good detection performance.In this work, electromagnetic heating was firstly explored as sample introduction approach in portable microplasma-atomic emission spectrometer to achieve the direct, rapid analysis of soil sample. The device primarily consists of an electromagnetic heating unit, a dielectric barrier discharge (DBD) excitation source and an optical signal acquisition unit. A W-boat was used as an electromagnetic heating medium and sample carrier, and copper coil spiraled around the tube was used as magnetic induction coil. With applying a voltage on copper coil, W-boat was electromagnetically heated to vaporize analyte-containing species for sample introduction into the microplasma. The portable battery-powered device is controlled by a miniature touch screen computer with the main advantages of small size (40.5 cm (l) × 30 cm (w) × 15 cm (h).), light weight (less than 7 kg), low-power consumption (the average power consumption is 118 W). By this method, Hg, Cd and Pb in soil were simultaneously analyzed within 4 min. Under the optimal conditions, the limits of detection for Hg, Cd and Pb in soils were 8.0 μg/kg, 17.8 μg/kg and 3.5 mg/kg, respectively, meeting the requirements for environmental quality standards for soils of China. Different types of CRM soils were analyzed, demonstrating good accuracy, stability and utility of this method. This technique could be a promising and powerful tool for on-site, rapid analysis of heavy metals in soil even other solid samples. Electromagnetic heating mode provides a good alternative for solid sampling to develop portable, miniaturized atomic spectrometers for solid sample analysis.N-glycan modification is reported to be important in regulating the structure and function of immunoglobulins in mammals. While, the study on teleost immunoglobulin glycosylation is still limitted. In this study, we constructed a TNP-antigen driven model, and detected the site-specific N-glycans of PBS-immunized and TNP-specific Oreochromis niloticus serum IgM through 18O-labeling and nanoLC-MS/MS. These methods are widely used for peptide enrichment and protein modification identification, but rarely used in detecting the level of N-glycosylation in teleost Igs that driven by specific antigen. The results revealed that there are four N-glycosylation sites in O.niloticus IgM heavy chain, namely, the Asn-315 site in the CH2 domain, the Asn-338 site in the CH3 domain, and the Asn-509 and Asn-551 sites in the CH4 domain, All of the four residues were efficiently N-glycosylated. After immunized with TNP-antigen, the signal strength of oligomannose in the TNP-specific IgM in primary mass spectrometry was significantly higher than that in the PBS-immunized IgM. Notably, the TNP-specific IgM had an Asn-509 site fully occupied with oligomannose, while only a small amount of oligomannose was found in the PBS-immunized IgM of this site. N-glycans in other sites were mainly complex-type with a low content of fucosylation and sialylated. The oligomannose in TNP-specific IgM was further verified to be essential for the binding of IgM and MBL. These results demonstrated that the TNP-antigen induced the site-specific oligomannose modification of O.niloticus IgM heavy chain, and played an important role in the interaction of IgM and MBL, which provided insights into the evolutionary understanding of the IgM oligomannose modification and function.