Mass spectrometry-based targeted proteomics method for the quantification of clinically relevant drug metabolizing enzymes in human specimens

Wenzel, Christoph; Droździk, Marek; Oswald, Stefan

doi:10.1016/j.jchromb.2021.122891

Cited by 12 publications

(8 citation statements)

References 54 publications

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“…Furthermore, the surrogate peptide we used for CYP2E1 was consistent with those reported in a previous study ( Ren et al, 2020 ), indicating the reproducibility of trypsin digestion across different laboratories. We also compared the surrogate peptides of the corresponding metabolic enzymes in rat and human liver microsomes, and found that the surrogate peptides of the reported enzyme isoforms were significantly different between species, except that the peptide of CYP2E1 was highly similar ( Groer et al, 2014 ; Li and Zhu, 2020 ; Wenzel et al, 2021 ).…”

Section: Resultsmentioning

confidence: 99%

“…The current LC-MS/MS-based CYP enzyme quantification methods mainly use the following three types of blank matrices to establish standard curves and QC samples: 1) Other tissues or serum of the same species or different species, such as 5% rat serum ( Ren et al, 2020 ), human serum albumin ( Groer et al, 2014 ), bovine serum albumin ( Wenzel et al, 2021 ); 2) the same matrix, standard curve correction by subtracting the substrate ( Sakamoto et al, 2011 ; Ohtsuki et al, 2012 ); 3) Standard solution without biological matrix ( Grangeon et al, 2019 ). Serum protein is significantly different from that in liver microsomes, which may lead to different matrix effects affecting the quantitative analysis.…”

Section: Resultsmentioning

confidence: 99%

“…Denaturation, reduction, alkylation and digestion of proteins can all affect the acquisition of surrogate peptides. In addition, the complex composition of biological samples may lead to the matrix effect that also affect peptide quantification ( An et al, 2019 ), so chromatography needs to be optimized for efficient separation, which has led many studies to take more than an hour for an analysis ( Grangeon et al, 2019 ; Wenzel et al, 2021 ). To correct for the matrix effect, an isotope internal standard is usually chosen because it has similar physicochemical properties to the target peptide.…”

Section: Introductionmentioning

confidence: 99%

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Simultaneous absolute protein quantification of seven cytochrome P450 isoforms in rat liver microsomes by LC-MS/MS-based isotope internal standard method

Zhong

Zhang

et al. 2022

Front. Pharmacol.

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The cytochrome P450 (CYP) enzymes play a pivotal role in drug metabolism. LC-MS/MS-based targeting technology has been applied to the analysis of CYP enzymes, promoting drug development and drug-drug interaction studies. Rat is one of the most commonly used models for drug metabolism assessment, but LC-MS/MS assay quantifying the abundance of CYP enzymes in rats is rarely reported. Herein, an accurate and stable LC-MS/MS based method was developed and validated for the simultaneous quantification of seven major rat CYP isoforms (CYP1A2, 2B1, 2C6, 2C11, 2D1, 2E1, and 3A1) in liver microsomes. The careful optimization of trypsin digestion and chromatography combined with isotope-labeled peptide as internal standard improved the efficiency and accuracy of the analysis. Highly specific surrogate peptides were obtained by a procedure including trypsin digestion for six hours and separated on a Hypersil Gold C18 column (100 × 2.1 mm, 3 μm) using gradient elution for 15 min with a mobile phase of water containing 0.1% formic acid and acetonitrile. In the method validation, linearity, matrix effect, recovery, stability, accuracy, and precision all meet the requirements. Subsequently, this method was applied to detect seven enzymes in rat liver microsomes from four different sources, and the correlation between the abundance and activity of CYP enzymes was further analyzed. The high-throughput detection method provided in this study will provide support for pertinent pharmaceutical research based on rat models.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Simultaneous absolute protein quantification of seven cytochrome P450 isoforms in rat liver microsomes by LC-MS/MS-based isotope internal standard method

Zhong

Zhang

et al. 2022

Front. Pharmacol.

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“…Protein abundance of ACE2, TMPRSS2, and the reference protein Na + /K + -ATPase in membrane protein fractions from kidney and colon samples were determined using a validated LC-MS/MS-based targeted proteomics assay [ 40 ]. The principle of this method is based on the quantification of proteospecific peptides, which are released during tryptic digestion, and allows determination of absolute protein abundance.…”

Section: Methodsmentioning

confidence: 99%

Importance of ACE2 for SARS-CoV-2 Infection of Kidney Cells

et al. 2023

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In late 2019, the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) as the causative agent of coronavirus disease 2019 (COVID-19) emerged in China and spread rapidly around the world, causing an ongoing pandemic of global concern. COVID-19 proceeds with moderate symptoms in most patients, whereas others experience serious respiratory illness that requires intensive care treatment and may end in death. The severity of COVID-19 is linked to several risk factors including male sex, comorbidities, and advanced age. Apart from respiratory complications, further impairments by COVID-19 affecting other tissues of the human body are observed. In this respect, the human kidney is one of the most frequently affected extrapulmonary organs and acute kidney injury (AKI) is known as a direct or indirect complication of SARS-CoV-2 infection. The aim of this work was to investigate the importance of the protein angiotensin-converting enzyme 2 (ACE2) for a possible cell entry of SARS-CoV-2 into human kidney cells. First, the expression of the cellular receptor ACE2 was demonstrated to be decisive for viral SARS-CoV-2 cell entry in human AB8 podocytes, whereas the presence of the transmembrane protease serine 2 (TMPRSS2) was dispensable. Moreover, the ACE2 protein amount was well detectable by mass spectrometry analysis in human kidneys, while TMPRSS2 could be detected only in a few samples. Additionally, a negative correlation of the ACE2 protein abundance to male sex and elderly aged females in human kidney tissues was demonstrated in this work. Last, the possibility of a direct infection of kidney tubular renal structures by SARS-CoV-2 was demonstrated.

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“…11 Additionally, immunoblotting, mass spectrometry, and proteomic techniques are commonly used; however, these methods have high costs, involve difficult sample preparation steps, and can give non-selective and inaccurate results. 12 To this end, development of more efficient imaging techniques is still needed. Fluorescent molecular probes, which provide non-invasive, non-destructive, and real-time monitoring with high spatial and temporal resolution, have attracted great attention in this direction as a highly competitive alternative to the conventional imaging modalities.…”

mentioning

confidence: 99%

Selective detection of carboxylesterase 2 activity in cancer cells using an activity-based chemiluminescent probe

et al. 2022

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Mass spectrometry-based targeted proteomics method for the quantification of clinically relevant drug metabolizing enzymes in human specimens

Cited by 12 publications

References 54 publications

Simultaneous absolute protein quantification of seven cytochrome P450 isoforms in rat liver microsomes by LC-MS/MS-based isotope internal standard method

Simultaneous absolute protein quantification of seven cytochrome P450 isoforms in rat liver microsomes by LC-MS/MS-based isotope internal standard method

Importance of ACE2 for SARS-CoV-2 Infection of Kidney Cells

Selective detection of carboxylesterase 2 activity in cancer cells using an activity-based chemiluminescent probe

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