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Analysis of testosterone and dihydrotestosterone in mouse tissues by liquid chromatography-electrospray ionization-tandem mass spectrometry.

ABSTRACT: A novel method was established for simultaneous quantitation of testosterone (T) and dihydrotestosterone (DHT) in murine tissue and serum samples. Endogenous T and DHT, together with the internal standards 17alpha-methyl-T and 17alpha-methyl-DHT, were extracted from tissues and then derivatized by reaction with 2-hydrazino-4-(trifluoromethyl)-pyrimidine (HTP). Analysis by liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) resulted in product ion spectra of HTP derivatives of both T and DHT that showed analyte-specific fragmentations; the latter fragmentations were characterized by the use of high-resolution Orbitrap MS/MS. These specific fragmentations enabled quantitation of T and DHT in the multiple-reaction monitoring (MRM) mode. The method was validated with charcoal-stripped serum as the matrix. The lower limit of quantitation (LLOQ) was 0.10ng/ml for T and 0.50ng/ml for DHT. The method was then used for determination of serum and tissue levels of T and DHT in transgenic mice carrying a hypomorphic NADPH-cytochrome P450 reductase gene (Cpr-low mice). Remarkably, ovarian T levels in Cpr-low mice were found to be 25-fold higher than those in wild-type mice, a finding that at least partly explains the female infertility seen in the Cpr-low mice. In conclusion, our method provides excellent sensitivity and selectivity for determination of endogenous levels of T and DHT in mouse tissues.

SUBMITTER: Weng Y

PROVIDER: S-EPMC2876209 | biostudies-literature | 2010 Jul

REPOSITORIES: biostudies-literature

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Analysis of testosterone and dihydrotestosterone in mouse tissues by liquid chromatography-electrospray ionization-tandem mass spectrometry.

Weng Yan Y Xie Fang F Xu Li L Zagorevski Dmitri D Spink David C DC Ding Xinxin X

Analytical biochemistry 20100331 2

A novel method was established for simultaneous quantitation of testosterone (T) and dihydrotestosterone (DHT) in murine tissue and serum samples. Endogenous T and DHT, together with the internal standards 17alpha-methyl-T and 17alpha-methyl-DHT, were extracted from tissues and then derivatized by reaction with 2-hydrazino-4-(trifluoromethyl)-pyrimidine (HTP). Analysis by liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) resulted in product ion spectra of HTP ...[more]

PMID: 20361922

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Project description:We present the first comprehensive capillary electrophoresis electrospray ionization mass spectrometry (CESI-MS) analysis of post-translational modifications derived from H1 and core histones. Using a capillary electrophoresis system equipped with a sheathless high-sensitivity porous sprayer and nano-liquid chromatography electrospray ionization mass spectrometry (nano-LC-ESI-MS) as two complementary techniques, we characterized H1 histones isolated from rat testis. Without any pre-separation of the perchloric acid extraction, a total of 70 different modified peptides, including 50 phosphopeptides, were identified in the rat linker histones H1.0, H1a-H1e, and H1t. Out of the 70 modified H1 histone peptides, 27 peptides could be identified with CESI-MS only, and 11 solely with LC-ESI-MS. Immobilized metal-affinity chromatography enrichment prior to MS analysis yielded a total of 55 phosphopeptides; 22 of these peptides could be identified only by CESI-MS, and 19 only by LC-ESI-MS, showing the complementarity of the two techniques. We mapped 42 H1 modification sites, including 31 phosphorylation sites, of which 8 were novel sites. For the analysis of core histones, we chose a different strategy. In a first step, the sulfuric-acid-extracted core histones were pre-separated using reverse-phase high-performance liquid chromatography. Individual rat testis core histone fractions obtained in this way were digested and analyzed via bottom-up CESI-MS. This approach yielded the identification of 42 different modification sites including acetylation (lysine and N(α)-terminal); mono-, di-, and trimethylation; and phosphorylation. When we applied CESI-MS for the analysis of intact core histone subtypes from butyrate-treated mouse tumor cells, we were able to rapidly detect their degree of modification, and we found this method very useful for the separation of isobaric trimethyl and acetyl modifications. Taken together, our results highlight the need for additional techniques for the comprehensive analysis of post-translational modifications. CESI-MS is a promising new proteomics tool as demonstrated by this, the first comprehensive analysis of histone modifications, using rat testis as an example.

Dataset Information

Analysis of testosterone and dihydrotestosterone in mouse tissues by liquid chromatography-electrospray ionization-tandem mass spectrometry.

Publications

Analysis of testosterone and dihydrotestosterone in mouse tissues by liquid chromatography-electrospray ionization-tandem mass spectrometry.

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