Quantitation of Protein Adducts of Aristolochic Acid I by Liquid Chromatography–Tandem Mass Spectrometry: A Novel Method for Biomonitoring Aristolochic Acid Exposure

Abstract

Emerging evidence suggests that chronic exposure to aristolochic acids (AAs) is one of the etiological pathways leading to chronic kidney disease (CKD). Due to the traditional practice of herbal medicine and AA-containing plants being used extensively as medicinal herbs, over 100 million East Asians are estimated to be at risk of AA poisoning. Given that the chronic nephrotoxicity of AAs only manifests itself after decades of exposure, early diagnosis of AA exposure could allow for timely intervention and disease risk reduction. However, an early detection method is not yet available, and diagnosis can only be established at the end stage of CKD. The goal of this study was to develop a highly sensitive and selective method to quantitate protein adducts of aristolochic acid I (AAI) as a biomarker of AA exposure. The method entails the release of protein-bound aristolactam I (ALI) by heat-assisted alkaline hydrolysis, extraction of ALI, addition of internal standard, and quantitation by liquid chromatography–tandem mass spectrometric analysis. Accuracy and precision of the method were critically evaluated using a synthetic ALI-containing glutathione adduct. The validated method was subsequently used to detect dose-dependent formation of ALI–protein adducts in human serum albumin exposed to AAI and in proteins isolated from the tissues and sera of AAI-exposed rats. Our time-dependent study showed that ALI–protein adducts remained detectable in rats even at 28 days postdosing. It is anticipated that the developed method will fill the technical gap in diagnosing AA intoxication and facilitate the biomonitoring of human exposures to AAs.

Determination of Aristolochic Acids in Soil, Water, and Herbal Plants in Medicinal Plant Cultivation Areas: An Emerging Environmental Contaminant Worth Concerning

“…Deionized water was further purified with a PALL Cascada I water purification system (Port Washington, NY). Reference standards of aristolactam I (AL-I) and aristolactam II (AL-II) and N -methyl-AL-I internal standard were synthesized previously. − Extract-Clean C18-HF solid phase extraction (SPE) cartridges were obtained from Grace Davison (Deerfield, IL). Primary and secondary amine-functionalized silica (PSA) were procured from Agela Technologies (Tianjin, China).…”

Polyurethane-Based Face Mask as a Sampling Device for Environmental Tobacco Smoke

“…Note that with only half of the mask used for analysis, the MDL of our developed method using PUF masks as the sampling media is lower than that of the sorbent tube-based NIOSH standard method (13 ng/sampling tube; 0.2 μg/m 3 ) . The lower detection limit of the newly developed mask-based method was in part attributed to the higher sensitivity of the LC–MS/MS method used for the analysis of the mask extracts than that of the gas chromatography-based method used in the NIOSH 2551 standard method. , Furthermore, the air sampling rate was also higher for the mask-based method (7.7 L/min) than that of the sampling tube-based method (1.0 L/min); thus, a higher volume of air is sampled when face masks are used as the sampling media.…”

Dual roles of drug or its metabolite−protein conjugate: Cutting‐edge strategy of drug discovery using shotgun proteomics

“…Researchers have come to know that classical parameters are unsuitable for assessing covalent drugs. In recent years, quantification based on drugs or their metabolite−amino acid adducts released from DMPC has been developed to enhance sensitivity and resolution at the amino acid level 59,103,131 . This strategy provided a solution for DMPC quantification, but without the specific information of modification sites and targets, for which quantification approaches based on modified peptides are still essentially needed.…”

“…One was "multiple digestions," which use diverse proteases to digest parallel samples (Figure 4). For example, the use of multiple proteases, trypsin, LysC, ArgC, AspN, and GluC, resulted Lys-N 1.7-fold increase in identifiable human N-terminal peptides [95,96] Lys-C and Asp-N 72% increase in detected phosphopeptides [97] Lys-C, Arg-C, Asp-N, and Glu-C 3 -fold increase in sequence coverage [98] Mixed digestion Trypsin Glu-C 2.4-fold increase in coverage of identified phosphorylation sites [99] Lys-N 72% increase of modified peptides [95] Lys-C High recovery of fully tryptic peptides; more unique peptides detected [100,101] Lys-C Arg-C 12.3% and 11.9% increase of identified peptides and proteins; 34% decrease of missed cleaved peptides [102] Pronase Chymotrypsin Complete hydrolysis of proteins [49] Carboxypeptidase Y/Leucine aminopeptidase Complete hydrolysis of proteins [103] Chemical digestion NaOH Fast hydrolysis (≤2 h) [59] T A B L E 4 Strategies based on digestion systems for protein digestion efficiency improvement. Unsuitable for chemical digestion [88] In-solution digestion FASP Removal of contaminates; efficient digestions Sample loss of peptides that molecular weight lower than MWCO; required long spins (>2 h) of highly complex protein samples; incompatible with most nonpolar organic solvents; easy to destroy the membrane by repeated centrifugation [106] Ultrasonic-Based FASP Fast digestion (2.5 h) Sample loss of peptides that molecular weight lower than MWCO (10 K); risk of deterioration of the microplate horn assembly surface iST Suitable for low-volume samples Sample loss during desalting step; limited removal capacity for some detergents (e.g., SDS) [112] nanoPOTS Suitable for low-nanoliter samples (down to 10 cells) [113,114] iPAD-1…”