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Method for the simultaneous quantitation of apolipoprotein E isoforms using tandem mass spectrometry.

ABSTRACT: Using apolipoprotein E (ApoE) as a model protein, we developed a protein isoform analysis method utilizing stable isotope labeling tandem mass spectrometry (SILT MS). ApoE isoforms are quantitated using the intensities of the b and y ions of the (13)C-labeled tryptic isoform-specific peptides versus unlabeled tryptic isoform-specific peptides. The ApoE protein isoform analysis using SILT allows for the simultaneous detection and relative quantitation of different ApoE isoforms from the same sample. This method provides a less biased assessment of ApoE isoforms compared to antibody-dependent methods, and may lead to a better understanding of the biological differences between isoforms.

SUBMITTER: Wildsmith KR

PROVIDER: S-EPMC2749897 | biostudies-literature | 2009 Dec

REPOSITORIES: biostudies-literature

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Method for the simultaneous quantitation of apolipoprotein E isoforms using tandem mass spectrometry.

Wildsmith Kristin R KR Han Bomie B Bateman Randall J RJ

Analytical biochemistry 20090803 1

Using apolipoprotein E (ApoE) as a model protein, we developed a protein isoform analysis method utilizing stable isotope labeling tandem mass spectrometry (SILT MS). ApoE isoforms are quantitated using the intensities of the b and y ions of the (13)C-labeled tryptic isoform-specific peptides versus unlabeled tryptic isoform-specific peptides. The ApoE protein isoform analysis using SILT allows for the simultaneous detection and relative quantitation of different ApoE isoforms from the same samp ...[more]

PMID: 19653990

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Project description:UnlabelledBackgroundDespite considerable global investigation over several decades, the roles of vitamin D in health and disease development remains convoluted. One recognised issue is the difficulty of accurately measuring the active forms of vitamin D. Advances made include some new methods addressing the potential interference by excluding epimers and isobars. However, there is no evidence that epimers are without function. Therefore, the aim of this study was to develop and validate, for the first time, a new assay to simultaneously measure levels of six forms of vitamin D along with two epimers. The assay was applied to multilevel certified reference material (CRM) and 25 pooled human sera samples, obtained from the Vitamin D External Quality Assessment Scheme (DEQAS), to demonstrate its efficiency.ResultsThe assay is capable of simultaneously measuring eight vitamin D analogues over the calibration ranges and LODs (in nmol/L) of: 1?25(OH)2D2 [0.015-1; 0.01], 1?25(OH)2D3 [0.1-100; 0.01], 25OHD3 [0.5-100, 0.025], 3-epi-25OHD3 [0.1-100, 0.05], 25OHD2 [0.5-100, 0.025], 3-epi-25OHD2 [0.1-100, 0.05], vitamin D3 [0.5-100, 0.05] and vitamin D2 [0.5-100, 0.05], using stanozolol-d3 as internal standard. Certified reference material and external quality control samples (DEQAS) were analysed to meet the standards outlined by National Institute of Standards and Technology (NIST). Validation steps included recovery and both precision and accuracy under inter- and intra-day variation limit of detection, and analysis of each analyte over a linear range. All validation parameters were in line with acceptable Food and Drug Administration (FDA) guidelines. All eight analogues were quantified with the 25OHD levels being commensurate with DEQAS data.ConclusionsThis report details the application of a new LC-MS/MS based assay for the efficient analysis of eight analogues of vitamin D over a range of samples, which is a significant advance over the existing methods. Simultaneous measure of eight vitamin D analogues does not compromise the analytical capability of the assay to quantify the commonly used biomarker (25OHD) for vitamin D status. The results demonstrate the feasibility of applying the assay in research and clinical practice that i) excludes misleading measures owing to epimers and isobars and ii) is able to quantify the excluded component to facilitate further in vivo investigation into the roles of ubiquitous epimers.

Project description:Castration resistant prostate cancer (CRPC), the fatal form of prostate cancer, remains androgen dependent despite castrate levels of circulating testosterone (T) and 5?-dihydrotestosterone (DHT). To investigate mechanisms by which the tumor can synthesize its own androgens and develop resistance to abiraterone acetate and enzalutamide, methods to measure a complete androgen profile are imperative. Here, we report the development and validation of a stable isotope dilution liquid chromatography electrospray ionization tandem mass spectrometric (SID-LC-ESI-MS/MS) method to quantify nine human hydroxy-androgens as picolinates, simultaneously with requisite specificity and sensitivity. In the established method, the fragmentation patterns of all nine hydroxy-androgen picolinates were identified, and [13C3]-5?-androstane-3?, 17?-diol and [13C3]-5?-androstane-3?, 17?-diol used as internal standards were synthesized enzymatically. Intra-day and inter-day precision and accuracy corresponds to the U.S. Food and Drug Administration Criteria for Bioanalytical Method Validation. The lower limit of quantitation (LLOQ) of nine hydroxy-androgens is 1.0pg to 2.5pg on column. Diols which have been infrequently measured: 5-androstene-3?, 17?-diol and 5?-androstane-3?, 17?-diol can be determined in serum at values as low as 1.0pg on column. The method also permits the quantitation of conjugated hydroxy-androgens following enzymatic digestion. While direct detection of steroid conjugates by electrospray-ionization tandem mass spectrometry has advantages the detection of unconjugated and conjugated steroids would require separate methods for each set of analytes. Our method was applied to pooled serum from male and female donors to provide reference values for both unconjugated and conjugated hydroxy-androgens. This method will allow us to interrogate the involvement of the conversion of 5-androstene-3?, 17?-diol to T, the backdoor pathway involving the conversion of 5?-androstane-3?, 17?-diol to DHT and the inactivation of DHT to 5?-androstane-3?, 17?-diol in advanced prostate cancer.

Dataset Information

Method for the simultaneous quantitation of apolipoprotein E isoforms using tandem mass spectrometry.

Publications

Method for the simultaneous quantitation of apolipoprotein E isoforms using tandem mass spectrometry.

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