Quantitative protein profiling of phenobarbital-induced drug metabolizing enzymes in rat liver by liquid chromatography mass spectrometry using formalin-fixed paraffin-embedded samples

Yamauchi, Hirofumi; Andou, Tomohiro; Watanabe, Takeshi; Gotou, Masamitsu; Anayama, Hisashi

doi:10.1016/j.vascn.2021.107107

Cited by 3 publications

(2 citation statements)

References 32 publications

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“…Metabolic enzyme expression was consistent with that in previous reports (18,19). This study showed that in addition to CYP2B, phenobarbital sodium significantly induced CYP2E1, which was consistent with the results of the study by Konstandi (20). Small doses of CCl4 have been repeatedly administered to induce CYP2E1 (21).…”

Section: Discussionsupporting

confidence: 92%

A New Method for Breath and Blood Alcohol Determination in Rats Using a Breath Alcohol Meter: An Experimental Study

Zeng¹,

Zou

Cao

et al. 2022

Iran J Pharm Res

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Background: The use of police breath alcohol detectors in rat breath alcohol detection experiments has always been a challenge because of the small lung capacity and inability of rats to actively inhale. However, the method of using gas chromatography to detect blood alcohol concentration is time-consuming, complex, relatively expensive, and cannot achieve on-site detection and multi-point unlimited non-invasive detection. Objectives: In this study, a laboratory method was validated for rat breath ethanol concentration (BrAC) measurement to estimate blood ethanol concentration (BAC) in rats. Methods: The rats were placed in a gas collection bottle, the breath sample was drawn out with a syringe, and injected into the mouthpiece of the breath alcohol detector through a rubber tube. The results were immediately detected and automatically converted to BAC. Male rats were randomly divided into three groups. The control group received an intraperitoneal injection of normal saline, the liver injury group received an intraperitoneal injection of 50% Carbon tetrachloride (CCL4 1 mL.kg-1), and the induction group received an intraperitoneal injection of phenobarbital sodium (75 mg.kg-1). Western blot analysis was used to detect the protein expression of CYP2E1. Similar grouping and experimental methods were used for female rats. Results: This method was reproducible. The metabolic activity of CYP2E1 was downregulated in the injury group and upregulated in the induction group, which was consistent with the results obtained for CYP2E1 protein expression. Conclusions: Our results confirmed that the rat gas cylinder breath alcohol assay can be used for multiple detections with immediate and non-invasive determination of alcohol metabolizing capacity. This is important for studies that require repeated assessment of blood alcohol levels.

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Section: Discussionsupporting

confidence: 92%

A New Method for Breath and Blood Alcohol Determination in Rats Using a Breath Alcohol Meter: An Experimental Study

Zeng¹,

Zou

Cao

et al. 2022

Iran J Pharm Res

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“…First, to achieve accurate quantification, efficient and quantitative recovery of signature peptides of the target proteins is critical but is much more challenging to achieve for FFPE tissues than fresh tissues, mostly owing to the extensive intra- and intermolecular cross-links generated by formalin fixation . To attain accurate and robust protein quantification, these cross-links must be efficiently reversed and the proteins must be quantitatively extracted with a high and reproducible yield, which is difficult. ,, Various methods were developed for proteomic analysis of FFPE tissues, such as a commercial Qproteome kit from Qiagen, , a popular approach using a high-pH buffer with sodium dodecyl sulfate (SDS) and its slightly modified versions published in more recent years, − and other approaches such as Zwittergent-containing buffer . While these methods were designed for the identification of a larger number of protein species in proteomic studies, optimization of methods with the aim of accurate and precise absolute protein quantification has not been conducted.…”

Section: Introductionmentioning

confidence: 99%

Highly Accurate and Robust Absolute Quantification of Target Proteins in Formalin-Fixed Paraffin-Embedded (FFPE) Tissues by LC–MS

Xue

Huo

et al. 2022

Anal. Chem.

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Accurate, absolute liquid chromatography–mass spectrometry (LC–MS)-based quantification of target proteins in formalin-fixed paraffin-embedded (FFPE) tissues would greatly expand sample availability for pharmaceutical/clinical investigations but remains challenging owing to the following issues: (i) efficient/quantitative recovery of target signature peptides from FFPE tissues is essential but an optimal procedure for targeted, absolute quantification is lacking; (ii) most FFPE samples are long-term-stored; severe immunohistochemistry (IHC) signal losses of target proteins during storage were widely reported, while the effect of storage on LC–MS-based methods was unknown; and (iii) the proper strategy to prepare calibration/quality-control samples to ensure accurate targeted protein analysis in FFPE tissues remained elusive. Using targeted quantification of monoclonal antibody (mAb), antigen, and 40 tissue markers in FFPE tissues as a model system, we extensively investigate those issues and develope an LC–MS-based strategy enabling accurate and precise targeted protein quantification in FFPE samples. First, we demonstrated a surfactant cocktail-based procedure (f-SEPOD), providing high/reproducible recovery of target signature peptides from FFPE tissues. Second, a heat-accelerated degradation study within a roughly estimated 5 year storage period recapitulated the loss of protein IHC signals while LC–MS signals of all targets remained constant. This indicates that the storage of FFPE tissues mainly causes decreased immunoreactivity but unlikely chemical degradation of proteins, which strongly suggests that the storage of FFPE tissues does not cause significant quantitative bias for LC–MS-based methods. Third, while a conventional spike-and-extract approach for calibration caused substantial negative biases, a novel approach, using FFPE-treated calibration standards, enabled accurate and precise quantification. With the pipeline, we conducted the first-ever pharmacokinetics measurement of mAb and its target in FFPE tissues, where time courses by FFPE vs fresh tissues showed excellent correlation.

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LC–MS Bioanalysis of Protein Biomarkers and Protein Therapeutics in Formalin-Fixed Paraffin-Embedded Tissue Specimens

G Jagadeeshaprasad,

Zeng,

Zheng

2024

Bioanalysis

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Formalin-fixed paraffin-embedded (FFPE) is a form of preservation and preparation for biopsy specimens. FFPE tissue specimens are readily available as part of oncology studies because they are often collected for disease diagnosis or confirmation. FFPE tissue specimens could be extremely useful for retrospective studies on protein biomarkers because the samples preserved in FFPE blocks could be stable for decades. However, LC–MS bioanalysis of FFPE tissues poses significant challenges. In this Perspective, we review the benefits and recent developments in LC–MS approach for targeted protein biomarker and protein therapeutic analysis using FFPE tissues and their clinical and translational applications. We believe that LC–MS bioanalysis of protein biomarkers in FFPE tissue specimens represents a great potential for its clinical applications.

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Quantitative protein profiling of phenobarbital-induced drug metabolizing enzymes in rat liver by liquid chromatography mass spectrometry using formalin-fixed paraffin-embedded samples

Cited by 3 publications

References 32 publications

A New Method for Breath and Blood Alcohol Determination in Rats Using a Breath Alcohol Meter: An Experimental Study

A New Method for Breath and Blood Alcohol Determination in Rats Using a Breath Alcohol Meter: An Experimental Study

Highly Accurate and Robust Absolute Quantification of Target Proteins in Formalin-Fixed Paraffin-Embedded (FFPE) Tissues by LC–MS

LC–MS Bioanalysis of Protein Biomarkers and Protein Therapeutics in Formalin-Fixed Paraffin-Embedded Tissue Specimens

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