Mass Spectrometry Identification of <i>N</i>‑Chlorinated Dipeptides in Drinking Water

We report the identification of <i>N</i>-chlorinated dipeptides as chlorination products in drinking water using complementary high-resolution quadrupole time-of-flight (QTOF) and quadrupole ion-trap mass spectrometry techniques. First, three model dipeptides, tyrosylglycine (Tyr-Gly), tyrosylalanine (Tyr-Ala), and phenylalanylglycine (Phe-Gly), reacted with sodium hypochlorite, and these reaction solutions were analyzed by QTOF. <i>N</i>-Cl-Tyr-Gly, <i>N</i>,<i>N</i>-di-Cl-Tyr-Gly, <i>N</i>-Cl-Phe-Gly, <i>N</i>,<i>N</i>-di-Cl-Phe-Gly, <i>N</i>-Cl-Tyr-Ala, and <i>N</i>,<i>N</i>-di-Cl-Tyr-Ala were identified as the major products based on accurate masses, ³⁵Cl/³⁷Cl isotopic patterns, and MS/MS spectra. These identified <i>N</i>-chlorinated dipeptides were synthesized and found to be stable in water over 10 days except <i>N</i>,<i>N</i>-di-Cl-Phe-Gly. To enable sensitive detection of <i>N</i>-chlorinated dipeptides in authentic water, we developed a high-performance liquid chromatography–tandem mass spectrometry (HPLC–MS/MS) method with multiple reaction monitoring (MRM) mode. <i>N</i>-Cl-Tyr-Gly, <i>N</i>,<i>N</i>-di-Cl-Tyr-Gly, <i>N</i>-Cl-Phe-Gly, <i>N</i>-Cl-Tyr-Ala, and <i>N</i>,<i>N</i>-di-Cl-Tyr-Ala along with their corresponding dipeptides were detected in authentic tap water samples. The dipeptides were clearly detected in the raw water, but the <i>N</i>-chlorinated dipeptides were at background levels. These results suggest that the <i>N</i>-chlorinated dipeptides are produced by chlorination. This study has identified <i>N</i>-chlorinated dipeptides as new disinfection byproducts in drinking water. The strategy developed in this study can be used to identify chlorination products of other peptides in drinking water

Formation and Occurrence of Iodinated Tyrosyl Dipeptides in Disinfected Drinking Water

Iodinated disinfection byproducts (I-DBPs) are highly toxic, but few precursors of I-DBPs have been investigated. Tyrosine-containing biomolecules are ubiquitous in surface water. Here we investigated the formation of I-DBPs from the chloramination of seven tyrosyl dipeptides (tyrosylglycine, tyrosylalanine, tyrosylvaline, tyrosylhistidine, tyrosylglutamine, tyrosylglutamic acid, and tyrosylphenylalanine) in the presence of potassium iodide. High resolution mass spectrometry and tandem mass spectrometry (MS/MS) analyses of the benchtop reaction solutions found that all seven precursors formed both I- and Cl-substituted tyrosyl dipeptide products. Iodine substitutions occurred on the 3- and 3,5-positions of the tyrosyl-phenol ring while chlorine substituted on the free amino group. To reach the needed sensitivity to detect iodinated tyrosyl dipeptides in authentic waters, we developed a high performance liquid chromatography (HPLC)-MS/MS method with multiple reaction monitoring mode and solid phase extraction. HPLC-MS/MS analysis of tap and corresponding raw water samples, collected from three cities, identified four iodinated peptides, 3-I-/3,5-di-I-Tyr-Ala and 3-I-/3,5-di-I-Tyr-Gly, in the tap waters but not in the raw waters. The corresponding precursors, Tyr-Ala and Tyr-Gly, were also detected in the same tap and raw water samples. This study demonstrates that iodinated dipeptides exist as DBPs in drinking water

Tough Hydrophobic Hydrogels for Monitoring Human Moderate Motions in Both Air and Underwater Environments

Hydrophobic hydrogels with high strength and great stretchability hold immense potential in various fields, such as soft robots, 3D printing, and flexible sensors. However, the formation of large hydrophobic domains in a hydrophobic hydrogel can lead to a heterogeneous structure in the bulk hydrogel. This phenomenon will result in the hydrophobic hydrogel becoming opaque, having a large energy hysteresis during stretching, poor strain-sensitivity, and slow self-recovery. In this study, we successfully developed a series of transparent hydrophobic hydrogels that exhibit excellent mechanical properties (low hysteresis and high toughness of ∼1.8–2.5 MJ m–3) with a desirable strain-sensitivity. The key factor in achieving this was the ability to tune large, inhomogeneous hydrophobic structures into small, well-ordered domains at the scale of 16.50–52.08 nm by introducing a small number of electrostatic groups into the hydrophobic networks. The hydrophobic hydrogels were able to form strong dual physical interactions, including electrostatic interactions and hydrophobic associations, making them ideal materials for fabricating wearable sensors with both in air and underwater applications. This facile and effective approach provides a novel method to prepare hydrophobic hydrogels with good mechanical performance, low hysteresis, and good strain-sensitivity, opening up new potential for their applications in various fields

Cytotoxicity of Halogenated Tyrosyl Compounds, an Emerging Class of Disinfection Byproducts

Halogenated amino acids and peptides are an emerging class of disinfection byproducts (DBPs), having been detected in drinking water and in washed food products. However, the toxicological significance of these emerging DBPs remains unclear. In this study, the cytotoxicity of eight halogenated tyrosyl compounds was investigated in Chinese hamster ovary (CHO) cells using real-time cell analysis (RTCA). Dihalogenated tyrosyl compounds are more cytotoxic than their monohalogenated analogues. The cytotoxicity of the dihalogenated compounds is associated with their ability to induce intracellular reactive oxygen species (ROS), suggesting that oxidative stress is an important toxicity pathway of these compounds. Pearson correlation analysis of the cytotoxicity (IC50 values) of these compounds with eight physicochemical parameters showed strong associations with their lipophilicity (logP) and reactivity (polarizability, ELUMO). Finally, cytotoxicity testing of the concentrated extracts of a chloraminated mixture of eight dipeptides with bromide or iodide showed the cytotoxicity of these mixtures in the order: iodinated peptides > brominated peptides ≥ chlorinated peptides. These results demonstrate that halogenated peptide DBPs are toxicologically relevant, and further research is needed to understand the implications of long-term exposure for human health

High-Sensitivity N‑Glycoproteomic Analysis of Mouse Brain Tissue by Protein Extraction with a Mild Detergent of N‑Dodecyl β‑D-Maltoside

<i>N</i>-dodecyl β-D-maltoside (DDM), a mild detergent with the ability to maintain the enzyme activity and solubilize hydrophobic proteins without changing their structures, was applied for N-glycoproteomic analysis of minute protein sample from mouse brain tissue. After combining with the capillary-based glycoproteomic reactor, 281 N-glycosylation sites were successfully characterized from 50 μg of mouse brain tissue, which was 110% higher at least than those obtained by conventional strategies

Formation, Identification, and Occurrence of New Bromo- and Mixed Halo-Tyrosyl Dipeptides in Chloraminated Water

Dipeptides are widely present in surface water and serve as precursors to form disinfection byproducts (DBPs) during disinfection (e.g., chloramination). Bromide (Br–) and iodide (I–) are common in many source waters, enhancing Br- and I-DBP formation. Recently Cl-, I-, and Cl-I-dipeptides were identified after chloramination of tyrosyl dipeptides in the presence of I– and were detected in authentic disinfected drinking water samples. However, the formation and occurrence of Br- and mixed halogen (Cl, Br, and/or I)-dipeptides in disinfected water have not been studied. Here we investigated the formation of halogenated dipeptides from three aromatic dipeptides, phenylalanylglycine (Phe-Gly), tyrosylalanine (Tyr-Ala), and tyrosylglycine (Tyr-Gly), under chloramination in the presence of Br– and I– at environmentally relevant levels ([Br–] and [I–], 0 and 0 μg L–1, 6 and 30 μg L–1, 30 and 30 μg L–1, 150 and 30 μg L–1, 300 and 30 μg L–1, and 900 and 30 μg L–1, respectively). For the first time, N-Br- and N,N-di-Br- as well as N-Br-N-Cl- and N-Br-3-I-tyrosyl dipeptides were identified using infusion electrospray quadrupole time-of-flight mass spectrometry. Tyrosyl dipeptides produced N-Cl-, 3-I-/3,5-di-I-, and N-Cl-3-I-tyrosyl dipeptides, while Phe-Gly formed only N-Cl-/N,N-di-Cl-Phe-Gly. To determine halogenated dipeptides in authentic water samples, we developed a new method of solid phase extraction and high-performance liquid chromatography with quadrupole ion trap mass spectrometry using reaction monitoring. 3,5-Di-I-Tyr-Ala and N-Br-Tyr-Ala were detected in treated water but not in the corresponding raw water, warranting further investigation into the occurrence of halogenated peptides in other drinking water systems

High-Throughput Determination of the Site-Specific N‑Sialoglycan Occupancy Rates by Differential Oxidation of Glycoproteins Followed with Quantitative Glycoproteomics Analysis

Sialylated glycoproteins, which play important roles in tumor progression, have been extensively analyzed for the discovery of potential biomarkers for cancer diagnosis and prognosis. The site-specific N-sialoglycan occupancy rates of glycoproteins reflect the activities of glycosyltransferases and glycosidases in vivo and could be novel disease biomarkers. However, a high-throughput method to determine the N-sialoglycan occupancy rates is not available. On the basis of the fact that dihydroxy of sialic acid of glycan chains in glycoproteins can be specifically oxidized to aldehyde in mild periodate concentration while all types of glycan chains can be oxidized in high periodate concentration, we developed a modified protein-level hydrazide chemistry method for the determination of the N-sialoglycan occupancy rates. This method was first applied to determine the N-sialoglycan occupancy rates of two glycosites on human transferrin. These two sites were found to be fully sialylated and the N-sialoglycan occupancy rates were found to under significant decrease after the neuraminidase treatment. This method was then applied to analyze N-sialoglycan occupancy rates in proteome samples. We determined 496 and 632 site-specific N-sialoglycan occupancy rates on 334 and 394 proteins from hepatocellular carcinoma (HCC) and normal human liver tissues, respectively. By comparing the N-sialoglycan occupancy rates between the above two samples, we determined 76 N-sialoglycosites with more than a 2-fold change. This method was demonstrated to be an effective and high-throughput method for the analysis of the N-sialoglycan occupancy rates

Aspartame-Sweetened Tap Water: Transformation Products and 2,6-Dichloro-1,4-Benzoquinone Formation

Aspartame (APM), a dipeptide of aspartic acid (ASP) and phenylalanine (PHE), is a widely used artificial sweetener in beverages. It is unclear whether residual chlorine in tap water can react with APM to form disinfection byproducts (DBPs). Therefore, we investigated the formation of DBPs from the reaction of APM with residual chlorine in authentic tap water. APM and a commercial sweetener (CS) packet containing APM were studied under authentic and simulated tap water conditions. Eight chlorinated products of APM were detected using solid-phase extraction (SPE) and high performance liquid chromatography quadrupole time-of-flight mass spectrometry (HPLC-QTOF-MS). These new chloro-products were tentatively identified based on accurate masses, isotopic patterns of 35,37Cl, and MS/MS spectra. Furthermore, we identified APM as a precursor to 2,6-dichloro-1,4-benzoquinone (DCBQ). DCBQ significantly increased to 2.3–12 ng/L with the addition of APM or CS in tap waters collected from different locations compared to 1.4–1.8 ng/L in the same tap water samples without sweetener. DCBQ and two of the chlorinated transformation products were identified in cold prepared tea containing APM. DCBQ formation was eliminated when the residual chlorine in tap water was reduced by ascorbic acid or boiling prior to the addition of APM or CS. This study found that eight new DBPs and DCBQ were produced by the reactions of residual chlorine with APM and CS. These findings show an unintended exposure source of emerging DBPs via APM sweetened beverages