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Quantitation of steroid hormones in thin fresh frozen tissue sections.

ABSTRACT: As analytical technologies in proteomics and metabolomics continue to mature, there is an increasing need to apply these to clinically relevant biologic samples. In this study, a liquid chromatography-tandem mass spectrometry method that utilizes selected reaction monitoring was used to measure the absolute quantity of estrogens and estrogen metabolites and testosterone in 8-microm tissue sections obtained from a fresh frozen lymph node tumor infiltrated by metastatic breast carcinoma. Total (conjugated plus unconjugated) and unconjugated levels of these steroid hormones were measured using two cohorts, each containing five adjacent serial sections cut from this tumor. The results were highly reproducible across replicate samples, showing that typical histological tissue sections represent an important sample type for the measurement of these specific metabolites.

SUBMITTER: Blonder J

PROVIDER: S-EPMC7236062 | biostudies-literature | 2008 Nov

REPOSITORIES: biostudies-literature

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Quantitation of steroid hormones in thin fresh frozen tissue sections.

Blonder Josip J Johann Donald J DJ Veenstra Timothy D TD Xiao Zhen Z Emmert-Buck Michael R MR Ziegler Regina G RG Rodriguez-Canales Jaime J Hanson Jeffrey A JA Xu Xia X

Analytical chemistry 20081021 22

As analytical technologies in proteomics and metabolomics continue to mature, there is an increasing need to apply these to clinically relevant biologic samples. In this study, a liquid chromatography-tandem mass spectrometry method that utilizes selected reaction monitoring was used to measure the absolute quantity of estrogens and estrogen metabolites and testosterone in 8-microm tissue sections obtained from a fresh frozen lymph node tumor infiltrated by metastatic breast carcinoma. Total (co ...[more]

PMID: 18937426

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Project description:Determination of disease-relevant proteomic profiles from limited tissue specimens, such as pathological biopsies and tissues from small model organisms, remains an analytical challenge and a much needed clinical goal. In this study, a transgenic mouse disease model of cardiac-specific H-Ras-G12V induced hypertrophic cardiomyopathy provided a system to explore the potential of using mass spectrometry (MS)-based proteomics to obtain a disease-relevant molecular profile from amount-limited specimens that are routinely used in pathological diagnosis. Our method employs a two-stage methanol-assisted solubilization to digest lysates prepared from 8-?m-thick fresh-frozen histological tissue sections of diseased/experimental and normal/control hearts. Coupling this approach with a nanoflow reversed-phase liquid chromatography (LC) and a hybrid linear ion trap/Fourier transform-ion cyclotron resonance MS resulted in the identification of 704 and 752 proteins in hypertrophic and wild-type (control) myocardium, respectively. The disease driving H-Ras protein along with vimentin were unambiguously identified by LC-MS in hypertrophic myocardium and cross-validated by immunohistochemistry and western blotting. The pathway analysis involving proteins identified by MS showed strong association of proteomic data with cardiovascular disease. More importantly, the MS identification and subsequent cross-validation of Wnt3a and ?-catenin, in conjunction with IHC identification of phosphorylated GSK-3? and nuclear localization of ?-catenin, provided evidence of Wnt/?-catenin canonical pathway activation secondary to Ras activation in the course of pathogenic myocardial hypertrophic transformation. Our method yields results indicating that the described proteomic approach permits molecular discovery and assessment of differentially expressed proteins regulating H-Ras induced hypertrophic cardiomyopathy. Selected proteins and pathways can be further investigated using immunohistochemical techniques applied to serial tissue sections of similar or different origin.

Dataset Information

Quantitation of steroid hormones in thin fresh frozen tissue sections.

Publications

Quantitation of steroid hormones in thin fresh frozen tissue sections.

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