Protocol to extract and measure ubiquinone and rhodoquinone in murine tissues

TL;DR

This paper provides a detailed method to measure ubiquinone and rhodoquinone in mouse tissues using mitochondrial purification and LC-MS analysis.

Contribution

A novel protocol for extracting and quantifying ubiquinone and rhodoquinone in murine mitochondria using biphasic extraction and LC-MS.

Findings

Mitochondria can be effectively isolated from murine tissues using centrifugation.

Biphasic extraction enriches for ubiquinone and rhodoquinone, which are then quantified via LC-MS.

Protein normalization ensures accurate comparison of quinone levels across samples.

Abstract

Ubiquinone (UQ) and rhodoquinone (RQ) are electron carriers for the electron transport chain (ETC). Here, we present a protocol for measuring UQ and RQ in mitochondria purified from murine tissues. We describe steps for isolating mitochondria by centrifugation, isolating UQ and RQ by biphasic extraction, and normalizing samples to the protein content of the mitochondrial pellet. We then detail procedures for analyzing UQ and RQ by integrating peak areas for UQ-9 and RQ-9 (abundant in mice) or UQ-10 and RQ-10 (abundant in human). Thus, through enrichment of mitochondria, we establish a method to measure UQ and RQ in tissues.

For complete details on the use and execution of this protocol, please refer to Valeros et al.1

•A method to detect rhodoquinone in mammalian tissues by LC-MS•Details on mitochondrial purification from tissues for rhodoquinone detection•Biphasic metabolite extraction protocol to enrich for quinones•Protein quantification of extracted material to equalize LC-MS input

A method to detect rhodoquinone in mammalian tissues by LC-MS

Details on mitochondrial purification from tissues for rhodoquinone detection

Biphasic metabolite extraction protocol to enrich for quinones

Protein quantification of extracted material to equalize LC-MS input

Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Ubiquinone (UQ) and rhodoquinone (RQ) are electron carriers for the electron transport chain (ETC). Here, we present a protocol for measuring UQ and RQ in mitochondria purified from murine tissues. We describe steps for isolating mitochondria by centrifugation, isolating UQ and RQ by biphasic extraction, and normalizing samples to the protein content of the mitochondrial pellet. We then detail procedures for analyzing UQ and RQ by integrating peak areas for UQ-9 and RQ-9 (abundant in mice) or UQ-10 and RQ-10 (abundant in human). Thus, through enrichment of mitochondria, we establish a method to measure UQ and RQ in tissues.

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