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

Kim KJ, Kim HJ, Park HG, Hwang CH, Sung C, Jang KS, Park SH, Kim BG, Lee YK, Yang YH, Jeong JH, Kim YG - Sci Rep (2016)

Bottom Line: The one-step modification of endogenous estrone provided good linearity (R(2) > 0.99) and significantly increased the sensitivity of the platform (limit of quantitation: 11 fmol).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/10(6) 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemical Engineering, Soongsil University, Seoul 156-743, Korea.

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 (R(2) > 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/10(6) 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/10(6) 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.

No MeSH data available.


Related in: MedlinePlus

Relative quantitative analysis of estrone (E1) with different molar ratios of deuterated estrone (d4-E1) spiked in human normal sera.The molar ratios of E1 to d4-E1 were (A) 1:1, (B) 1:0.5, and (C) 1:0.2.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4836303&req=5

f4: Relative quantitative analysis of estrone (E1) with different molar ratios of deuterated estrone (d4-E1) spiked in human normal sera.The molar ratios of E1 to d4-E1 were (A) 1:1, (B) 1:0.5, and (C) 1:0.2.

Mentions: To verify that we can detect and quantify estrone and d4-estrone in complex media such as human serum and cell lysates, synthetic estrone and d4-estrone were spiked into human serum in various molar ratios (E1:d4-E1 = 1:1, 1:0.5, 1:0.2). After the MTBE extraction and subsequent GP labeling process, good quantitative recovery was observed without any purification process (Figs 4 and S2). Major peaks with a +4 Da difference in mass were clearly detected, as we intended. At the equivalent spiking (1:1) condition, the relative peak intensities were identical. In the more diluted ratios (1:0.5, 1:0.2), the peak intensities accurately reflected their theoretical molar ratios as well. In addition, we could determine the limit of quantitation (LOQ) of GP-estrone with the MALDI-MS-based quantitative method (Fig. S3). The GP derivatization significantly improved the LOQ of underivatized estrone (1.6 × 104-fold, from 185 pmol to 11 fmol on the MALDI plate). Moreover, the GP-estrone spiked in human serum could be assayed at the femtomole level by MALDI-MS. According to the sensitivity of the MALDI-MS-based estrone quantitation, the endogenous estrones are sufficiently detectable. Taken together, these results demonstrate that the GP derivatization method remarkably improves the sensitivity and reproducibility of the endogenous estrones and provides reliable results in MALDI-MS-based estrone quantitation.


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

Kim KJ, Kim HJ, Park HG, Hwang CH, Sung C, Jang KS, Park SH, Kim BG, Lee YK, Yang YH, Jeong JH, Kim YG - Sci Rep (2016)

Relative quantitative analysis of estrone (E1) with different molar ratios of deuterated estrone (d4-E1) spiked in human normal sera.The molar ratios of E1 to d4-E1 were (A) 1:1, (B) 1:0.5, and (C) 1:0.2.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4836303&req=5

f4: Relative quantitative analysis of estrone (E1) with different molar ratios of deuterated estrone (d4-E1) spiked in human normal sera.The molar ratios of E1 to d4-E1 were (A) 1:1, (B) 1:0.5, and (C) 1:0.2.
Mentions: To verify that we can detect and quantify estrone and d4-estrone in complex media such as human serum and cell lysates, synthetic estrone and d4-estrone were spiked into human serum in various molar ratios (E1:d4-E1 = 1:1, 1:0.5, 1:0.2). After the MTBE extraction and subsequent GP labeling process, good quantitative recovery was observed without any purification process (Figs 4 and S2). Major peaks with a +4 Da difference in mass were clearly detected, as we intended. At the equivalent spiking (1:1) condition, the relative peak intensities were identical. In the more diluted ratios (1:0.5, 1:0.2), the peak intensities accurately reflected their theoretical molar ratios as well. In addition, we could determine the limit of quantitation (LOQ) of GP-estrone with the MALDI-MS-based quantitative method (Fig. S3). The GP derivatization significantly improved the LOQ of underivatized estrone (1.6 × 104-fold, from 185 pmol to 11 fmol on the MALDI plate). Moreover, the GP-estrone spiked in human serum could be assayed at the femtomole level by MALDI-MS. According to the sensitivity of the MALDI-MS-based estrone quantitation, the endogenous estrones are sufficiently detectable. Taken together, these results demonstrate that the GP derivatization method remarkably improves the sensitivity and reproducibility of the endogenous estrones and provides reliable results in MALDI-MS-based estrone quantitation.

Bottom Line: The one-step modification of endogenous estrone provided good linearity (R(2) > 0.99) and significantly increased the sensitivity of the platform (limit of quantitation: 11 fmol).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/10(6) 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemical Engineering, Soongsil University, Seoul 156-743, Korea.

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 (R(2) > 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/10(6) 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/10(6) 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.

No MeSH data available.


Related in: MedlinePlus