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 Children's Hospital
Department	Pathology
Laboratory	Kanarek Lab
Last Name	Petrova
First Name	Boryana
Address	300 Longwood Av
Email	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

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.

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