Human protein lysine aminoacylation (K-AA) analysis using LC-MS/MS method
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo Sapiens
SUBMITTER: 
Tianxia Jiang
PROVIDER: PXD069870 | iProX | Fri Oct 24 00:00:00 BST 2025
REPOSITORIES: iProX
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Project description:Measurement of thiopurine metabolites is helpful to monitor adverse effects and assess compliance in patients on thiopurine treatment. The purpose of this study was to develop and validate an analytical method for measurement of thiopurine metabolites, thioguanine nucleotides (6-TGN) and 6-methylmercaptopurine nucleotide (6-MMPN), in RBCs. We developed and validated a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the quantification of 6-TGN and 6-MMPN and evaluated the stability of the thiopurine metabolites in RBC and whole blood states without any preprocessing at various storage conditions. The linear range was 0.1-10 μmol/L and 0.5-100 μmol/L for 6-TGN and 6-MMPN, respectively. The mean extraction recovery at the two concentrations was 71.0% and 75.0% for 6-TGN, and 102.2% and 96.4% for 6-MMPN. Thiopurine metabolites in preprocessed RBC samples were stable at 25°C and 4°C after storage for 4 hours and at -70°C for up to 6 months. However, 6-TGN decreased by 30% compared with the initial concentration when stored at -20°C for 180 days. In whole blood states, 6-TGN decreased by about 20% at four days after storage at 4°C. We validated a reliable LC-MS/MS method and recommend that the patient's whole blood sample be preprocessed as soon as possible.
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Project description:A highly sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for the determination of tadalafil (TAD) in human plasma. TAD and its deuterated internal standard (IS), tadalafil-d3, were extracted from 200 µL plasma using Phenomenex Strata-X-C 33 µ extraction cartridges. Chromatographic analysis was carried out on Synergi™ Hydro-RP C18 (100 mm × 4.6 mm, 4 µm) column with a mobile phase consisting of methanol and 10 mM ammonium formate, pH 4.0 (90:10, v/v), delivered at a flow rate of 0.9 mL/min. Quantitation of the protonated analyte was done on a triple quadrupole mass spectrometer using multiple reaction monitoring via electrospray ionization. The precursor to product ions transitions monitored for TAD and TAD-d3 were m/z 390.3 → 268.2 and m/z 393.1 → 271.2, respectively. The calibration curve was linear over the concentration range of 0.50-500 ng/mL with correlation coefficient, r2 ≥ 0.9994. Acceptable intra-batch and inter-batch precision (≤ 3.7%) and accuracy (97.8% to 104.1%) were obtained at five concentration levels. The recovery of TAD from spiked plasma was highly precise and quantitative (98.95% to 100.61%). Further, the effect of endogenous matrix components was minimal. TAD was found to be stable under different storage conditions in human plasma and also in whole blood samples. The validated method was successfully used to determine TAD plasma concentration in a bioequivalence study with 20 mg TAD tablets in 24 healthy volunteers. Method performance was evaluated by reanalyzing 115 study samples.
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Project description:BackgroundThe determination of antiretroviral drugs in hair is receiving considerable research interest to assess long-term adherence to antiretroviral therapy (ART). Currently in China, lamivudine, zidovudine, nevirapine, efavirenz, ritonavir, and lopinavir are combined as first-line and second-line free therapy regimens and are recommended for people living with HIV (PLWH). Simultaneous determination of the 6 antiretroviral drugs in human hair is important for accurately and widely assessing long-term adherence in Chinese PLWH receiving different ART regimens.MethodsSix drugs were extracted from 10-mg hair samples incubated in methanol for 16 hours at 37°C and then analyzed by liquid chromatography with tandem mass spectrometry using a mobile phase of 95% methanol, with an electrospray ionization source in multiple reaction monitoring and positive mode.ResultsThe LC-ESI+-MS/MS method exhibited a linear range (R2 > 0.99) within 6-5000, 10-5000, 6-50,000, 12-50,000, 8-5000, and 8-12,500 pg/mg for lamivudine, zidovudine, nevirapine, efavirenz, ritonavir, and lopinavir. For all 6 drugs, the limits of quantification ranged between 6 and 12 pg/mg. The intraday and interday coefficients of variation were within 15%, and the recoveries ranged from 91.1% to 113.7%. Furthermore, the other validation parameters (ie, selectivity, matrix effect, stability, and carryover) met the acceptance criteria stipulated by guidelines of the US Food and Drug Administration and European Medicines Agency. Significant intergroup differences were observed between high-adherence and low-adherence groups, with high intercorrelations in the hair content of the 6 drugs.ConclusionsThe developed method demonstrated good reliability, to comprehensively and accurately assess adherence in PLWH receiving different ART regimens.
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Project description:MotivationIn liquid chromatography-mass spectrometry/tandem mass spectrometry (LC-MS/MS), it is necessary to link tandem MS-identified peptide peaks so that protein expression changes between the two runs can be tracked. However, only a small number of peptides can be identified and linked by tandem MS in two runs, and it becomes necessary to link peptide peaks with tandem identification in one run to their corresponding ones in another run without identification. In the past, peptide peaks are linked based on similarities in retention time (rt), mass or peak shape after rt alignment, which corrects mean rt shifts between runs. However, the accuracy in linking is still limited especially for complex samples collected from different conditions. Consequently, large-scale proteomics studies that require comparison of protein expression profiles of hundreds of patients can not be carried out effectively.MethodIn this article, we consider the problem of linking peptides from a pair of LC-MS/MS runs and propose a new method, PeakLink (PL), which uses information in both the time and frequency domain as inputs to a non-linear support vector machine (SVM) classifier. The PL algorithm first uses a threshold on an rt likelihood ratio score to remove candidate corresponding peaks with excessively large elution time shifts, then PL calculates the correlation between a pair of candidate peaks after reducing noise through wavelet transformation. After converting rt and peak shape correlation to statistical scores, an SVM classifier is trained and applied for differentiating corresponding and non-corresponding peptide peaks.ResultsPL is tested in multiple challenging cases, in which LC-MS/MS samples are collected from different disease states, different instruments and different laboratories. Testing results show significant improvement in linking accuracy compared with other algorithms.Availability and implementationM files for the PL alignment method are available at http://compgenomics.utsa.edu/zgroup/PeakLink.Supplementary informationSupplementary data are available at Bioinformatics online.
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Project description:Nicotine is a highly addictive alkaloid and a neurostimulator found in tobacco that causes addiction in humans and makes tobacco a high-demand commercial product. It is popularly used for recreational purposes and is a harmful substance (Oral LD50 value for rat is 50 mg/kg) and causes addiction. The metabolites of nicotine such as the Tobacco-specific Nitrosamines (TSNAs) are hazardous substances whose metabolites are highly electrophilic and form DNA adducts, which will initiate the process of carcinogenesis. TSNAs are formed during curing, storage and fermentation due to the nitrosation of nicotine and other tobacco alkaloids. TSNAs are used as biomarkers for cancer risk assessment in humans exposed to tobacco and its products. To determine the occasional formation of TSNAs in tobacco-feeding insects, 5th instar larvae of Spodoptera litura and their faeces were analyzed for the presence of N'-nitrosonornicotine (NNN), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) along with the stored tobacco leaves (PT-76) using an Agilent 6470B LC-MS/MS system following ISO/DIS 19290:2015 protocol. The larvae are extracted in a buffered acetonitrile-water extraction and the amount of TSNAs are quantified in multiple reaction monitoring (MRM) mode. 20 [Formula: see text]l of each extracted and cleaned up sample was injected into the LC-MS/MS system for quantification. The Limit of Detection (LOD) and Limit of Quantification (LOQ) were 0.001 mg/kg and 0.005 mg/kg for all the tested nitrosamines. NNN was found to be 0.361 mg/kg, 0.340 mg/kg, and 5.66 mg/kg in insect whole-body samples, faeces, and tobacco leaves, respectively. NNK was found to be 0.060 mg/kg, 0.035 mg/kg and 0.93 mg/kg in insect whole body samples, faeces and tobacco leaves, respectively. However, NNAL was not detected in both the insect's whole body and faeces. Recoveries ranged between 95 and 98% for all compounds when spiked at LOD and LOQ. The presence of TSNAs is a biomarker for cancer risk and their presence in insects would point to cancer risk assessment in tobacco feeding insects and any possible TSNA-detoxifying pathways in insects that might prevent mutagenesis caused these compounds.
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Project description:BackgroundClonorchiasis is an important foodborne parasitic disease. The omics-based-techniques could illuminate parasite biology and further make innovations in the research for parasitic diseases. However, knowledge about the serum metabolic profiles and related metabolic pathways in clonorchiasis is very limited.MethodsA untargeted ultra-high performance liquid tandem chromatography quadrupole time of flight mass spectrometry (UHPLC-QTOF-MS) was used to profile the serum metabolites of rats at both 4 and 8 weeks post infection (wpi) with Clonorchis sinensis (C. sinensis). Additionally, multivariate statistical analysis methods were employed to identify differential metabolites. Next, serum amino acids and phosphatidylcholines (PCs) levels were determined by targeted metabolomics analysis.ResultA total of 10530 and 6560 ions were identified in ESI+ and ESI- modes. The levels of phosphatidylcholines, glycerophosphocholine and choline were significantly changed, with the shift in lipid metabolism. Significant changes were also observed in amino acids (isoleucine, valine, leucine, threonine, glutamate and glutamine). Targeted analysis showed that BCAAs (isoleucine, valine, leucine) levels significantly increased at 4 wpi and decreased at 8 wpi; threonine was increased at 8 wpi, whereas glutamate and glutamine showed a decreasing trend at 8 wpi. Additionally, the level of 17 PCs were significantly changed in infected rats. Marked metabolic pathways were involved in clonorchiasis, including glycerophospholipid metabolism, alanine, aspartate and glutamate metabolism, histidine metabolism and pyrimidine metabolism.ConclusionThese results show that C. sinensis infection can cause significant changes in the rat serum metabolism, especially in amino acids and lipids. The metabolic signature together with perturbations in metabolic pathways could provide more in depth understanding of clonorchiasis and further make potential therapeutic interventions.
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