{
"METABOLOMICS WORKBENCH":{"STUDY_ID":"ST003072","ANALYSIS_ID":"AN005030","VERSION":"1","CREATED_ON":"February 9, 2024, 10:59 am"},

"PROJECT":{"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"},

"STUDY":{"STUDY_TITLE":"Investigation of polar metabolites 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 polar metabolite analysis, we employed targeted metabolomics profiling of a panel of 200 compounds. We interrogated relative changes between fresh and frozen adult (male or female) CSF compared to fresh and frozen embryonic CSF. This data presents results from polar HILIC chromatography","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":"617","NUM_GROUPS":"7","TOTAL_SUBJECTS":"19","NUM_MALES":"8","NUM_FEMALES":"6","STUDY_COMMENTS":"embryos also investigated. samples split for fresh and frozen conditions"},

"SUBJECT":{"SUBJECT_TYPE":"Mammal","SUBJECT_SPECIES":"Mus musculus","TAXONOMY_ID":"10090"},
"SUBJECT_SAMPLE_FACTORS":[
{
"Subject ID":"-",
"Sample ID":"RF377",
"Factors":{"Sample type":"Mock","Storage condition":"Frozen","Sex":"Male"},
"Additional sample data":{"Combined label":"Mock-Frozen Male","Genotype":"wild type","RAW_FILE_NAME":"20230303_QEF1_HILIC_T3T4Embryo_RF377"}
},
{
"Subject ID":"-",
"Sample ID":"RF378",
"Factors":{"Sample type":"Mock","Storage condition":"Frozen","Sex":"Female"},
"Additional sample data":{"Combined label":"Mock Frozen-Famale","Genotype":"wild type","RAW_FILE_NAME":"20230303_QEF1_HILIC_T3T4Embryo_RF378"}
},
{
"Subject ID":"-",
"Sample ID":"RF379",
"Factors":{"Sample type":"Mock","Storage condition":"Frozen","Sex":"Embryo"},
"Additional sample data":{"Combined label":"Mock-Frozen-Embryo","Genotype":"wild type","RAW_FILE_NAME":"20230303_QEF1_HILIC_T3T4Embryo_RF379"}
},
{
"Subject ID":"-",
"Sample ID":"RF380",
"Factors":{"Sample type":"Mock","Storage condition":"Fresh","Sex":"Male"},
"Additional sample data":{"Combined label":"Mock-Fresh-Male","Genotype":"wild type","RAW_FILE_NAME":"20230303_QEF1_HILIC_T3T4Embryo_RF380"}
},
{
"Subject ID":"-",
"Sample ID":"RF381",
"Factors":{"Sample type":"Mock","Storage condition":"Fresh","Sex":"Female"},
"Additional sample data":{"Combined label":"Mock-Fresh-Female","Genotype":"wild type","RAW_FILE_NAME":"20230303_QEF1_HILIC_T3T4Embryo_RF381"}
},
{
"Subject ID":"-",
"Sample ID":"RF382",
"Factors":{"Sample type":"Mock","Storage condition":"Fresh","Sex":"Embryo"},
"Additional sample data":{"Combined label":"Mock-Fresh-Embryo","Genotype":"wild type","RAW_FILE_NAME":"20230303_QEF1_HILIC_T3T4Embryo_RF382"}
},
{
"Subject ID":"-",
"Sample ID":"RF383",
"Factors":{"Sample type":"Adult CSF","Storage condition":"Frozen","Sex":"Male"},
"Additional sample data":{"Combined label":"Male-Frozen-1","Genotype":"wild type","RAW_FILE_NAME":"20230303_QEF1_HILIC_T3T4Embryo_RF383"}
},
{
"Subject ID":"-",
"Sample ID":"RF384",
"Factors":{"Sample type":"Adult CSF","Storage condition":"Frozen","Sex":"Male"},
"Additional sample data":{"Combined label":"Male Frozen 3","Genotype":"wild type","RAW_FILE_NAME":"20230303_QEF1_HILIC_T3T4Embryo_RF384"}
},
{
"Subject ID":"-",
"Sample ID":"RF385",
"Factors":{"Sample type":"Adult CSF","Storage condition":"Frozen","Sex":"Male"},
"Additional sample data":{"Combined label":"Male-Frozen-4","Genotype":"wild type","RAW_FILE_NAME":"20230303_QEF1_HILIC_T3T4Embryo_RF385"}
},
{
"Subject ID":"-",
"Sample ID":"RF386",
"Factors":{"Sample type":"Adult CSF","Storage condition":"Frozen","Sex":"Male"},
"Additional sample data":{"Combined label":"Male-Frozen-6 (small pellet)","Genotype":"wild type","RAW_FILE_NAME":"20230303_QEF1_HILIC_T3T4Embryo_RF386"}
},
{
"Subject ID":"-",
"Sample ID":"RF387",
"Factors":{"Sample type":"Adult CSF","Storage condition":"Frozen","Sex":"Female"},
"Additional sample data":{"Combined label":"Female-Frozen-1","Genotype":"wild type","RAW_FILE_NAME":"20230303_QEF1_HILIC_T3T4Embryo_RF387"}
},
{
"Subject ID":"-",
"Sample ID":"RF388",
"Factors":{"Sample type":"Adult CSF","Storage condition":"Frozen","Sex":"Female"},
"Additional sample data":{"Combined label":"Female-Frozen-2","Genotype":"wild type","RAW_FILE_NAME":"20230303_QEF1_HILIC_T3T4Embryo_RF388"}
},
{
"Subject ID":"-",
"Sample ID":"RF389",
"Factors":{"Sample type":"Adult CSF","Storage condition":"Frozen","Sex":"Female"},
"Additional sample data":{"Combined label":"Frmale-Frozen-3","Genotype":"wild type","RAW_FILE_NAME":"20230303_QEF1_HILIC_T3T4Embryo_RF389"}
},
{
"Subject ID":"-",
"Sample ID":"RF390",
"Factors":{"Sample type":"Adult CSF","Storage condition":"Frozen","Sex":"Female"},
"Additional sample data":{"Combined label":"Female-Frozen-4","Genotype":"wild type","RAW_FILE_NAME":"20230303_QEF1_HILIC_T3T4Embryo_RF390"}
},
{
"Subject ID":"-",
"Sample ID":"RF391",
"Factors":{"Sample type":"Embryonic CSF","Storage condition":"Frozen","Sex":"Embryo"},
"Additional sample data":{"Combined label":"Embryo-Frozen-1","Genotype":"wild type","RAW_FILE_NAME":"20230303_QEF1_HILIC_T3T4Embryo_RF391"}
},
{
"Subject ID":"-",
"Sample ID":"RF392",
"Factors":{"Sample type":"Embryonic CSF","Storage condition":"Frozen","Sex":"Embryo"},
"Additional sample data":{"Combined label":"Embryo-Frozen-2","Genotype":"wild type","RAW_FILE_NAME":"20230303_QEF1_HILIC_T3T4Embryo_RF392"}
},
{
"Subject ID":"-",
"Sample ID":"RF393",
"Factors":{"Sample type":"Embryonic CSF","Storage condition":"Frozen","Sex":"Embryo"},
"Additional sample data":{"Combined label":"Embryo-Frozen-3","Genotype":"wild type","RAW_FILE_NAME":"20230303_QEF1_HILIC_T3T4Embryo_RF393"}
},
{
"Subject ID":"-",
"Sample ID":"RF394",
"Factors":{"Sample type":"Embryonic CSF","Storage condition":"Frozen","Sex":"Embryo"},
"Additional sample data":{"Combined label":"Embryo-Frozen-4","Genotype":"wild type","RAW_FILE_NAME":"20230303_QEF1_HILIC_T3T4Embryo_RF394"}
},
{
"Subject ID":"-",
"Sample ID":"RF395",
"Factors":{"Sample type":"Embryonic CSF","Storage condition":"Frozen","Sex":"Embryo"},
"Additional sample data":{"Combined label":"Embryo-Frozen-5","Genotype":"wild type","RAW_FILE_NAME":"20230303_QEF1_HILIC_T3T4Embryo_RF395"}
},
{
"Subject ID":"-",
"Sample ID":"RF396",
"Factors":{"Sample type":"Adult CSF","Storage condition":"Fresh","Sex":"Male"},
"Additional sample data":{"Combined label":"Male-Fresh-1+4+6","Genotype":"wild type","RAW_FILE_NAME":"20230303_QEF1_HILIC_T3T4Embryo_RF396"}
},
{
"Subject ID":"-",
"Sample ID":"RF397",
"Factors":{"Sample type":"Adult CSF","Storage condition":"Fresh","Sex":"Male"},
"Additional sample data":{"Combined label":"Male-Fresh-2","Genotype":"wild type","RAW_FILE_NAME":"20230303_QEF1_HILIC_T3T4Embryo_RF397"}
},
{
"Subject ID":"-",
"Sample ID":"RF398",
"Factors":{"Sample type":"Adult CSF","Storage condition":"Fresh","Sex":"Male"},
"Additional sample data":{"Combined label":"Male-Fresh-5","Genotype":"wild type","RAW_FILE_NAME":"20230303_QEF1_HILIC_T3T4Embryo_RF398"}
},
{
"Subject ID":"-",
"Sample ID":"RF399",
"Factors":{"Sample type":"Adult CSF","Storage condition":"Fresh","Sex":"Male"},
"Additional sample data":{"Combined label":"Male-Fresh-8","Genotype":"wild type","RAW_FILE_NAME":"20230303_QEF1_HILIC_T3T4Embryo_RF399"}
},
{
"Subject ID":"-",
"Sample ID":"RF400",
"Factors":{"Sample type":"Adult CSF","Storage condition":"Fresh","Sex":"Female"},
"Additional sample data":{"Combined label":"Femal-Fresh-4","Genotype":"wild type","RAW_FILE_NAME":"20230303_QEF1_HILIC_T3T4Embryo_RF400"}
},
{
"Subject ID":"-",
"Sample ID":"RF401",
"Factors":{"Sample type":"Adult CSF","Storage condition":"Fresh","Sex":"Female"},
"Additional sample data":{"Combined label":"Female-Fresh-5","Genotype":"wild type","RAW_FILE_NAME":"20230303_QEF1_HILIC_T3T4Embryo_RF401"}
},
{
"Subject ID":"-",
"Sample ID":"RF402",
"Factors":{"Sample type":"Adult CSF","Storage condition":"Fresh","Sex":"Female"},
"Additional sample data":{"Combined label":"Female-Fresh-6","Genotype":"wild type","RAW_FILE_NAME":"20230303_QEF1_HILIC_T3T4Embryo_RF402"}
},
{
"Subject ID":"-",
"Sample ID":"RF403",
"Factors":{"Sample type":"Adult CSF","Storage condition":"Fresh","Sex":"Female"},
"Additional sample data":{"Combined label":"Female-Fresh-1 (small pellet)","Genotype":"wild type","RAW_FILE_NAME":"20230303_QEF1_HILIC_T3T4Embryo_RF403"}
},
{
"Subject ID":"-",
"Sample ID":"RF404",
"Factors":{"Sample type":"Embryonic CSF","Storage condition":"Fresh","Sex":"Embryo"},
"Additional sample data":{"Combined label":"Embryo-Fresh-1","Genotype":"wild type","RAW_FILE_NAME":"20230303_QEF1_HILIC_T3T4Embryo_RF404"}
},
{
"Subject ID":"-",
"Sample ID":"RF405",
"Factors":{"Sample type":"Embryonic CSF","Storage condition":"Fresh","Sex":"Embryo"},
"Additional sample data":{"Combined label":"Embryo-Fresh-2","Genotype":"wild type","RAW_FILE_NAME":"20230303_QEF1_HILIC_T3T4Embryo_RF405"}
},
{
"Subject ID":"-",
"Sample ID":"RF406",
"Factors":{"Sample type":"Embryonic CSF","Storage condition":"Fresh","Sex":"Embryo"},
"Additional sample data":{"Combined label":"Embryo-Fresh-3","Genotype":"wild type","RAW_FILE_NAME":"20230303_QEF1_HILIC_T3T4Embryo_RF406"}
},
{
"Subject ID":"-",
"Sample ID":"RF407",
"Factors":{"Sample type":"Embryonic CSF","Storage condition":"Fresh","Sex":"Embryo"},
"Additional sample data":{"Combined label":"Embryo-Fresh-4","Genotype":"wild type","RAW_FILE_NAME":"20230303_QEF1_HILIC_T3T4Embryo_RF407"}
},
{
"Subject ID":"-",
"Sample ID":"RF408",
"Factors":{"Sample type":"Embryonic CSF","Storage condition":"Fresh","Sex":"Embryo"},
"Additional sample data":{"Combined label":"Embryo-Fresh-5","Genotype":"wild type","RAW_FILE_NAME":"20230303_QEF1_HILIC_T3T4Embryo_RF408"}
},
{
"Subject ID":"-",
"Sample ID":"RF409",
"Factors":{"Sample type":"Serum","Storage condition":"Frozen","Sex":"Male"},
"Additional sample data":{"Combined label":"Male-1-Serum-Frozen","Genotype":"wild type","RAW_FILE_NAME":"20230303_QEF1_HILIC_T3T4Embryo_RF409"}
},
{
"Subject ID":"-",
"Sample ID":"RF410",
"Factors":{"Sample type":"Serum","Storage condition":"Frozen","Sex":"Male"},
"Additional sample data":{"Combined label":"Male-2-Serum-Frozen","Genotype":"wild type","RAW_FILE_NAME":"20230303_QEF1_HILIC_T3T4Embryo_RF410"}
},
{
"Subject ID":"-",
"Sample ID":"RF411",
"Factors":{"Sample type":"Serum","Storage condition":"Fresh","Sex":"Male"},
"Additional sample data":{"Combined label":"Male-1-Serum-Fresh","Genotype":"wild type","RAW_FILE_NAME":"20230303_QEF1_HILIC_T3T4Embryo_RF411"}
},
{
"Subject ID":"-",
"Sample ID":"RF412",
"Factors":{"Sample type":"Serum","Storage condition":"Fresh","Sex":"Male"},
"Additional sample data":{"Combined label":"Male-2-Serum-Fresh","Genotype":"wild type","RAW_FILE_NAME":"20230303_QEF1_HILIC_T3T4Embryo_RF412"}
}
],
"COLLECTION":{"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"},

"TREATMENT":{"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)"},

"SAMPLEPREP":{"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 chloro-form: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% acetonitrile (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.","PROCESSING_STORAGE_CONDITIONS":"On ice","EXTRACT_STORAGE":"-80℃"},

"CHROMATOGRAPHY":{"CHROMATOGRAPHY_SUMMARY":"Analysis of polar metabolites by HILIC chromatography","CHROMATOGRAPHY_TYPE":"HILIC","INSTRUMENT_NAME":"Thermo Vanquish","COLUMN_NAME":"SeQuant ZIC- pHILIC (150 x 2.1mm,5um)","SOLVENT_A":"100% acetonitrile","SOLVENT_B":"100% water; 20 mM ammonium carbonate, 0.1% ammonium hydroxide","FLOW_GRADIENT":"0–20 min: 0–2 min: linear gradient from 20% to 80% B; 20–20.5 min: from 80% to 20% B; 20.5–28 min: hold at 20% B at 150 mL/min flow rate.","FLOW_RATE":"0.25 mL/min","COLUMN_TEMPERATURE":"25"},

"ANALYSIS":{"ANALYSIS_TYPE":"MS","LABORATORY_NAME":"Kanarek Lab","OPERATOR_NAME":"Boryana Petrova"},

"MS":{"INSTRUMENT_NAME":"Thermo Q Exactive Orbitrap","INSTRUMENT_TYPE":"Orbitrap","MS_TYPE":"ESI","ION_MODE":"UNSPECIFIED","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) in positive and negative ionization mode in a range of m/z = 70–1000, with the resolution set at 70,000, the AGC target at 1 × 106, and the maximum injection time (Max IT) at 20 msec. A narrower 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. For polar metabolites, HESI conditions were as follows: sheath gas flow rate: 35 units; Aug gas flow rate: 8 units; sweet gas flow rate: 1 unit; spray voltage: 3.5 kV (pos), 2.8 kV (neg); capillary temperature: 320 °C; S-lens RF: 50; Aux gas heater temperature: 350 °C. For T3/T4, 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)."},

"MS_METABOLITE_DATA":{
"Units":"a.u.",

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}

}