A MALDI-MS-based quantitative analytical method for endogenous estrone in human breast cancer cells

Abstract

The level of endogenous estrone, one of the three major naturally occurring estrogens, has a significant correlation with the incidence of post-menopausal breast cancer. However, it is challenging to quantitatively monitor it owing to its low abundance. Here, we develop a robust and highly sensitive mass-assisted laser desorption/ionization mass spectrometry (MALDI-MS)-based quantitative platform to identify the absolute quantities of endogenous estrones in a variety of clinical specimens. The one-step modification of endogenous estrone provided good linearity (R2 > 0.99) and significantly increased the sensitivity of the platform (limit of quantitation: 11 fmol). In addition, we could identify the absolute amount of endogenous estrones in cells of the breast cancer cell line MCF-7 (34 fmol/106 cells) by using a deuterated estrone as an internal standard. Finally, by applying the MALDI-MS-based quantitative method to endogenous estrones, we successfully monitored changes in the metabolic expression level of estrones (17.7 fmol/106 letrozole-treated cells) in MCF-7 cells resulting from treatment with an aromatase inhibitor. Taken together, these results suggest that this MALDI-MS-based quantitative approach may be a general method for the targeted metabolomics of ketone-containing metabolites, which can reflect clinical conditions and pathogenic mechanisms.

Breast Cancer Metabolomics: From Analytical Platforms to Multivariate Data Analysis. A Review

“…The differentiation is based on variance between groups by rejection of the null hypothesis or acceptation the alternate hypothesis [101,107,108]. The most common methods used when the data is parametric are T-tests [31,47,48,50,53,57,59,62,68,70,71,73,74,76,77,78,79,84] and ANOVA [33,44,46,63,64,66,68,69,72,83] T-tests, such as Student and Welch’s tests, are recommended to analyze differences between two groups, and ANOVA-based methods, such as one-way ANOVA, two-way ANOVA, factorial ANOVA and MANOVA are used to evaluate more than two groups. Alternative univariate methods are implemented when the assumption of the normal distribution is non-parametric, such as Mann-Whitney test (MW-test) [51,81,102] and Wilcoxon test (W-test) [58].…”

“…Unsupervised methods are the preferential option for exploratory studies, where the modeling process is based only on the explanatory variables, without external intervention of user (Yi et al, 2016). The most commons are principal component analysis (PCA) [56,57,60,62,70,75,76,80,92,93] and hierarchical cluster analysis (HCA) [33,43,51,56,59,76] PCA provides the projection of dataset into low dimensional based on orthogonal transformation, converting the variables variability from a set of observations into score vectors and loadings, called principal components [100,109]. HCA methods are used to form subsets of samples at ordered levels based on variables similarities/dissimilarities (such as distances or correlations) and can be performed in agglomerative mode (samples are aggregate into clusters) or divisive mode (complete dataset is divided into clusters).…”

Lipid biosignature of breast cancer tissues by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

“…Namely, glycerophosphocholines are overexpressed in BC compared to luminal and HER2 subtypes. In addition, Cho et al [5] have found differences in the content of triacylglycerols (TGs) in healthy and BC tissues, as well as certain changes in the PC profile of the BC lipid extracts using MALDI MS. Kim et al [17] developed an analytical approach for the determination of ketone-containing metabolites in human BC cell lines (e.g., MCF-7 cell line) as they can reflect the clinical condition and pathogenic mechanisms. Another report by Phillips et al [18] demonstrated that MALDI MS imaging (MALDI MSI) of peptides obtained from BC tissues of patients with triple-negative BC (TNBC) were useful to discover proteins with higher abundance in TNBC when compared with noncancerous tissue and use them in disease prognosis.…”

Matrix-assisted laser desorption ionisation (MALDI) mass spectrometry (MS): basics and clinical applications

“…However, with the identification of further matrix formulations, sample enrichment, improved sensitivity and mass range capabilities, a range of biological analytes can be accurately detected and analysed. The type of analytes routinely studied includes lipids [28], carbohydrates [29], nucleotides [30], metabolites [31] and hormones [32].…”