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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

MS/MS profile of GP-labeled estrone: (a) m/z 325.4; (b) m/z 253.4; (c) m/z 297.4; (d) m/z 157.5.
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f3: MS/MS profile of GP-labeled estrone: (a) m/z 325.4; (b) m/z 253.4; (c) m/z 297.4; (d) m/z 157.5.

Mentions: To demonstrate the chemical derivatization method, synthetic estrone and testosterone, sex hormones possessing one ketone group, were used. First, the unlabeled estrone (E1) was identified at 460 m/z corresponding to the estrone-matrix adduct form (i.e., [M + CHCA + H]+), as previously reported (Fig. 2A). The possible mechanism of matrix (i.e., CHCA) adduct formation in MALDI spectra has been reported as π-π interactions or hydrogen bonds between the estrone and CHCA matrix3738. However, chemical derivatization with Girard’s reagent P only enables the production of the [M′]+ ion for GP-E1 at 404 m/z without analyte-matrix adduct formation, which dramatically simplifies the MALDI profile (Fig. 2B). Moreover, liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis was carried out to validate the chemical conjugation of Girard’s reagent P onto the C17-ketone group of estrone. Figure 3 shows the positive ion MS/MS spectrum of the parent 404 m/z ion as GP-derivatized E1 ([M′]+). Among the MS/MS fragments, the appearance of a prominent a ion at 325 m/z, which contains the intact estrone and a partial GP fragment, confirms the coupling of Girard’s reagent P with an estrone molecule. More interestingly, the c ion at 297 m/z is structurally informative to demonstrate the position (i.e., C17-ketone of estrone) of the chemical derivatization, although it is relatively weak. The results indicate that the C17-ketone of estrone is highly reactive to the hydrazide moiety of Girard’s reagent P without steric hindrance. Likewise, the MALDI spectrum of synthetic testosterone shows both the protonated form at 289 m/z and the sodium adduct form at 311 m/z, while the two peaks were replaced with just one peak at m/z 422 after chemical derivatization with GP (Fig. S1). The GP-labeled estrone and testosterone showed improved signal resolution and signal-to-noise ratio (SNR) (up to 17 times) in the mass spectra compared to their unlabeled counterparts. Thus, our straightforward method enables a remarkable improvement in the quality of MALDI spectra for ketone-containing hormones and facilitates a more rapid and high-throughput analysis using a simple MALDI-MS instrument.


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)

MS/MS profile of GP-labeled estrone: (a) m/z 325.4; (b) m/z 253.4; (c) m/z 297.4; (d) m/z 157.5.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f3: MS/MS profile of GP-labeled estrone: (a) m/z 325.4; (b) m/z 253.4; (c) m/z 297.4; (d) m/z 157.5.
Mentions: To demonstrate the chemical derivatization method, synthetic estrone and testosterone, sex hormones possessing one ketone group, were used. First, the unlabeled estrone (E1) was identified at 460 m/z corresponding to the estrone-matrix adduct form (i.e., [M + CHCA + H]+), as previously reported (Fig. 2A). The possible mechanism of matrix (i.e., CHCA) adduct formation in MALDI spectra has been reported as π-π interactions or hydrogen bonds between the estrone and CHCA matrix3738. However, chemical derivatization with Girard’s reagent P only enables the production of the [M′]+ ion for GP-E1 at 404 m/z without analyte-matrix adduct formation, which dramatically simplifies the MALDI profile (Fig. 2B). Moreover, liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis was carried out to validate the chemical conjugation of Girard’s reagent P onto the C17-ketone group of estrone. Figure 3 shows the positive ion MS/MS spectrum of the parent 404 m/z ion as GP-derivatized E1 ([M′]+). Among the MS/MS fragments, the appearance of a prominent a ion at 325 m/z, which contains the intact estrone and a partial GP fragment, confirms the coupling of Girard’s reagent P with an estrone molecule. More interestingly, the c ion at 297 m/z is structurally informative to demonstrate the position (i.e., C17-ketone of estrone) of the chemical derivatization, although it is relatively weak. The results indicate that the C17-ketone of estrone is highly reactive to the hydrazide moiety of Girard’s reagent P without steric hindrance. Likewise, the MALDI spectrum of synthetic testosterone shows both the protonated form at 289 m/z and the sodium adduct form at 311 m/z, while the two peaks were replaced with just one peak at m/z 422 after chemical derivatization with GP (Fig. S1). The GP-labeled estrone and testosterone showed improved signal resolution and signal-to-noise ratio (SNR) (up to 17 times) in the mass spectra compared to their unlabeled counterparts. Thus, our straightforward method enables a remarkable improvement in the quality of MALDI spectra for ketone-containing hormones and facilitates a more rapid and high-throughput analysis using a simple MALDI-MS instrument.

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