Summary of Study ST003073
This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench, https://www.metabolomicsworkbench.org, where it has been assigned Project ID PR001914. The data can be accessed directly via it's Project DOI: 10.21228/M89T5V This work is supported by NIH grant, U2C- DK119886.
See: https://www.metabolomicsworkbench.org/about/howtocite.php
This study contains a large results data set and is not available in the mwTab file. It is only available for download via FTP as data file(s) here.
| Study ID | ST003073 |
| Study Title | Investigation of 3,5,3’-L-triiodothyronine (T3) and 3,5,3’5’-L-tetraiodothyronine (T4) by targeted LC-MS analysis from mouse adult or embryonic CSF and from adult serum. |
| Study Summary | To maximize discovery from samples with limited amounts, we optimized a method to detect the thyroid hormones ( 3,5,3’-L-triiodothyronine (T3) and 3,5,3’5’-L-tetraiodothyronine (T4)) as well as polar metabolites from central carbon metabolism from mouse adult or embryonic CSF and from adult mouse serum. Samples were ran on reverse phase and HILIC chromatography respectively. For T3 and T4 analysis we utilized targeted analysis using information from previously ran synthetic standards. We interrogated relative changes between fresh and frozen adult (male or female) CSF compared to fresh and frozen embryonic CSF. This data presents the levels of T3 and T4 as interrogated by reverse phase chromatography |
| Institute | Boston Childrens Hospital |
| Department | Pathology |
| Laboratory | Kanarek Lab |
| Last Name | Petrova |
| First Name | Boryana |
| Address | 300 Longwood Av |
| boryana.petrova@childrens.harvard.edu | |
| Phone | 6173557433 |
| Submit Date | 2024-01-10 |
| Num Groups | 7 |
| Total Subjects | 19 |
| Num Males | 8 |
| Num Females | 6 |
| Study Comments | embryos also investigated. samples split for fresh and frozen conditions |
| Raw Data Available | Yes |
| Raw Data File Type(s) | raw(Thermo) |
| Analysis Type Detail | LC-MS |
| Release Date | 2024-02-14 |
| Release Version | 1 |
Select appropriate tab below to view additional metadata details:
Project:
| Project ID: | PR001914 |
| Project DOI: | doi: 10.21228/M89T5V |
| Project Title: | Optimized Mass Spectrometry Detection of Thyroid Hormones and Polar Metabolites in Rodent Cerebrospinal Fluid |
| Project Summary: | Thyroid hormones (TH) are required for brain development and function. Cerebrospinal fluid (CSF), which bathes the brain and spinal cord, contains TH as free hormone, or as bound to transthyretin (TTR). Tight TH level regulation in the central nervous system is essential for de-velopmental gene expression that governs neurogenesis, myelination, and synaptogenesis. This integrated function of TH highlights the importance of developing precise and reliable methods for assessing TH levels in CSF. We report an optimized liquid chromatography-mass spectrome-try (LC-MS) based method to measure TH in rodent CSF and serum, applicable to both fresh and frozen samples. Using this new method, we find distinct differences in CSF TH in pregnant dams vs. non-pregnant adults and in embryonic vs. adult CSF. Further, targeted LC-MS metabolic pro-filing uncovers distinct central carbon metabolism in the CSF of these populations. TH detection and metabolite profiling of related metabolic pathways open new avenues of rigorous research into CSF TH and will inform future studies on metabolic alterations in CSF during normal de-velopment. |
| Institute: | Boston Childrens Hospital |
| Department: | Pathology |
| Laboratory: | Kanarek Lab |
| Last Name: | Petrova |
| First Name: | Boryana |
| Address: | 300 Longwood Av |
| Email: | boryana.petrova@childrens.harvard.edu |
| Phone: | 6173557433 |
Subject:
| Subject ID: | SU003188 |
| Subject Type: | Mammal |
| Subject Species: | Mus musculus |
| Taxonomy ID: | 10090 |
Factors:
Subject type: Mammal; Subject species: Mus musculus (Factor headings shown in green)
| mb_sample_id | local_sample_id | Sample source | Storage condition | Sex |
|---|---|---|---|---|
| SA332284 | RF401 | Adult CSF | Fresh | Female |
| SA332285 | RF402 | Adult CSF | Fresh | Female |
| SA332286 | RF400 | Adult CSF | Fresh | Female |
| SA332287 | RF403 | Adult CSF | Fresh | Female |
| SA332288 | RF398 | Adult CSF | Fresh | Male |
| SA332289 | RF397 | Adult CSF | Fresh | Male |
| SA332290 | RF396 | Adult CSF | Fresh | Male |
| SA332291 | RF399 | Adult CSF | Fresh | Male |
| SA332292 | RF390 | Adult CSF | Frozen | Female |
| SA332293 | RF389 | Adult CSF | Frozen | Female |
| SA332294 | RF387 | Adult CSF | Frozen | Female |
| SA332295 | RF388 | Adult CSF | Frozen | Female |
| SA332296 | RF384 | Adult CSF | Frozen | Male |
| SA332297 | RF383 | Adult CSF | Frozen | Male |
| SA332298 | RF386 | Adult CSF | Frozen | Male |
| SA332299 | RF385 | Adult CSF | Frozen | Male |
| SA332300 | RF381 | Adult Mock | Fresh | Female |
| SA332301 | RF380 | Adult Mock | Fresh | Male |
| SA332302 | RF378 | Adult Mock | Frozen | Female |
| SA332303 | RF377 | Adult Mock | Frozen | Male |
| SA332304 | RF411 | Adult Serum | Fresh | Male |
| SA332305 | RF412 | Adult Serum | Fresh | Male |
| SA332306 | RF410 | Adult Serum | Frozen | Male |
| SA332307 | RF409 | Adult Serum | Frozen | Male |
| SA332308 | RF406 | Embryonic CSF | Fresh | - |
| SA332309 | RF405 | Embryonic CSF | Fresh | - |
| SA332310 | RF407 | Embryonic CSF | Fresh | - |
| SA332311 | RF408 | Embryonic CSF | Fresh | - |
| SA332312 | RF404 | Embryonic CSF | Fresh | - |
| SA332313 | RF395 | Embryonic CSF | Frozen | - |
| SA332314 | RF391 | Embryonic CSF | Frozen | - |
| SA332315 | RF394 | Embryonic CSF | Frozen | - |
| SA332316 | RF392 | Embryonic CSF | Frozen | - |
| SA332317 | RF393 | Embryonic CSF | Frozen | - |
| SA332318 | RF382 | Embryonic Mock | Fresh | - |
| SA332319 | RF379 | Embryonic Mock | Frozen | - |
| Showing results 1 to 36 of 36 |
Collection:
| Collection ID: | CO003181 |
| Collection Summary: | Animals The Boston Children’s Hospital IACUC approved all experiments involving mice in this study. Adult CD-1 male and female mice and pregnant CD-1 dams were obtained from Charles River Laboratories. Sprague Dawley rats were purchased from Charles River Laboratories. Animals were housed in a temperature- and humidity-controlled room (70 ± 3 °F, 35–70% humidity) on a 12 h light/12 h dark cycle (7 a.m. on 7 p.m. off) and had free access to food and water. All animals younger than postnatal day 10 were al-located into groups based solely on the gestational age without respect to sex (both males and females were included). For studies involving rodents older than 10 days, sex was treated as a variable. Serum and CSF collection CSF was collected from the cisterna magna of adult (≥ 3 months old) or embryonic (E14.5) wild-type CD-1 mice. Independent samples (n) were defined as independent animals for adult samples, and as a CSF pool from all embryos in a single litter for embryonic CSF samples. Samples were maintained on ice, then spun 1000 × g for 10 min at 4 °C. The supernatant was collected and used for analysis. Blood was collected from cardiac puncture, allowed to clot for 10 minutes at room temperature, spun at 500 × g for 10 min at room temperature. The supernatant serum was collected and used for analysis. |
| Sample Type: | Cerebrospinal fluid |
| Storage Conditions: | -80℃ |
Treatment:
| Treatment ID: | TR003197 |
| Treatment Summary: | No treatments were performed. All animals were CD-1 wild type. We compared different groups based on samples storage condition (fresh, frozen, or 24h stored), animal sex (female or male), or type of biofluid (serum, adult CSF, and embryonic CSF) |
Sample Preparation:
| Sampleprep ID: | SP003194 |
| Sampleprep Summary: | Sample preparation for LC-MS analysis of thyroid hormone metabolites from CSF and serum in parallel to analysis for polar metabolites. Per condition, 5–10 μL of CSF or serum was extracted in 4:6:3 chloroform:methanol:water mixture supplemented with isotopically labeled T3 and T4 (at 100 nM, Cambridge Isotope Laboratories, CLM-7185-C and CLM-8931-PK) and isotopically labeled 17 amino acids (at 1/5000, Cambridge Isotope Laboratories, MSK-A2-1.2) and isotopically labeled reduced glutathione (at 1 µM, Cambridge Isotope Laboratories and CNLM-6245-10). After centrifugation for 10 min at maximum speed on a benchtop centrifuge (Eppendorf) the top, hydrophilic layer was transferred to a new tube, dried using a nitrogen dryer (ThermoFisher Scientific, TS-18826) and reconstituted in 20 µL 70% ac-etonitrile (supplemented with QReSS, Cambridge Isotope Laboratories, MSK-QRESS-KIT) by brief vortexing. Extracted metabolites were spun again and cleared supernatant was transferred to LC-MS micro vials. The protocol was used for both fresh and frozen CSF and serum samples. A small amount of each sample was also pooled and serially diluted 3- and 10-fold to be used as quality controls throughout the run of each batch. Serum and CSF sample pools were kept separate and serum and CSF sample sets were run consecutively on our chromatography to avoid interspersing the run of two different matrixes. |
Combined analysis:
| Analysis ID | AN005031 |
|---|---|
| Analysis type | MS |
| Chromatography type | Reversed phase |
| Chromatography system | Thermo Vanquish |
| Column | Sigma Aldrich Ascentis Express C18 (150 × 2.1 mm, 2.7 um) |
| MS Type | ESI |
| MS instrument type | Orbitrap |
| MS instrument name | Thermo Q Exactive Orbitrap |
| Ion Mode | POSITIVE |
| Units | a.u. |
Chromatography:
| Chromatography ID: | CH003802 |
| Instrument Name: | Thermo Vanquish |
| Column Name: | Sigma Aldrich Ascentis Express C18 (150 × 2.1 mm, 2.7 um) |
| Column Temperature: | 30 |
| Flow Gradient: | The chromatographic gradient was run at a flow rate of 0.250 mL min−1 as follows: 0–2 min: held at 5% B; 2–12.1 min: linear gradient of 5% to 95% B; 12.1–17.0 min: 95% B; 17.1–21.0 min: gradient was returned to 5% B |
| Flow Rate: | 0.25 mL/min |
| Solvent A: | 100% water; 0.1% formic acid |
| Solvent B: | 100% acetonitrile; 0.1% formic acid. |
| Chromatography Type: | Reversed phase |
MS:
| MS ID: | MS004770 |
| Analysis ID: | AN005031 |
| Instrument Name: | Thermo Q Exactive Orbitrap |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | MS Data Acquisition Conditions for Targeted Analysis of Polar Metabolites and Thyroid Hormones MS data acquisition was performed using a Q Exactive Orbitrap benchtop orbitrap mass spectrometer equipped with an Ion Max source and a HESI II probe (Thermo Fisher Scientific, San Jose, CA, USA). A narrow scan in positive mode at m/z = 600–800 was used for more specific detection of TH. The resolution was set at 70,000, the AGC target was 5 × 105, and the max IT was 100 msec. HESI conditions were as follows: sheath gas flow rate: 40 units; Aug gas flow rate: 10 units; sweet gas flow rate: 0; spray voltage: 3.5 kV (pos), 2.8 kV (neg); capillary temperature: 380 °C; S-lens RF: 60; Aux gas heater temperature: 420 °C. Targeted Metabolomics Data Analysis Relative quantification of polar metabolites was performed with TraceFinder 5.1 (Thermo Fisher Scientific, Waltham, MA, USA) using a 5 ppm mass tolerance and referencing an in-house library of chemical standards (see associated Supplemental Dataset S1). We routinely queried 266 compounds (40 internal standards and 226 metabolites). Pooled samples and fractional dilutions were prepared as quality controls and injected at the beginning and end of each run. In addition, pooled samples were interspersed throughout the run to control for technical drift in signal quality as well as to serve to assess the coefficient of variability (CV) for each metabolite. Data from TraceFinder were further consolidated and normalized with an in-house R script, freely accessible at github (https://github.com/FrozenGas/KanarekLabTraceFinderRScripts/blob/main/MS_data_script_v2.4_20221018.R). Briefly, this script performs the following normalization and quality control steps: (1) extracts and combines the peak areas from TraceFinder output.csvs; (2) calculates and normalizes to an averaged factor from all mean-centered chromatographic peak areas of isotopically labeled amino acid and QReSS internal standards within each sample; (3) filters out low-quality metabolites based on user-inputted cut-offs calculated from pool reinjections and pool dilutions; (4) calculates and normalizes for biological material amounts based on the total integrated peak area values of high-confidence metabolites. In this study, the linear correlation between the dilution factor and the peak area cut-offs is set to RSQ > 0.95 and the coefficient of variation (CV) < 30%. Finally, data were log transformed and Pareto scaled within the MetaboAnalyst-based statistical analysis platform [42] to generate PCA, PLSDA, volcano plots, and heatmaps. Individual metabolite bar plots and statistics were calculated in Excel (v16.81) and GraphPad Prism (v.10). Data Analysis for TH Levels Data analysis for T3 and T4 levels was performed with TraceFinder 5.1 as described above, referencing empirically determined retention times using standards. Isotopically labeled T3 and T4 internal standards (Cambridge Isotope Laboratories, Tewksbury, MA, USA; CLM-7185-C and CLM-8931-PK) were used to normalize for technical signal variability and matrix effects. Equal volumes of samples were compared per condition. |
| Ion Mode: | POSITIVE |
