#METABOLOMICS WORKBENCH boryana_ptrva_20240111_060748 DATATRACK_ID:4567 STUDY_ID:ST003074 ANALYSIS_ID:AN005032 PROJECT_ID:PR001914 VERSION 1 CREATED_ON February 9, 2024, 9:46 pm #PROJECT PR:PROJECT_TITLE Optimized Mass Spectrometry Detection of Thyroid Hormones and Polar Metabolites PR:PROJECT_TITLE in Rodent Cerebrospinal Fluid PR:PROJECT_SUMMARY Thyroid hormones (TH) are required for brain development and function. PR:PROJECT_SUMMARY Cerebrospinal fluid (CSF), which bathes the brain and spinal cord, contains TH PR:PROJECT_SUMMARY as free hormone, or as bound to transthyretin (TTR). Tight TH level regulation PR:PROJECT_SUMMARY in the central nervous system is essential for de-velopmental gene expression PR:PROJECT_SUMMARY that governs neurogenesis, myelination, and synaptogenesis. This integrated PR:PROJECT_SUMMARY function of TH highlights the importance of developing precise and reliable PR:PROJECT_SUMMARY methods for assessing TH levels in CSF. We report an optimized liquid PR:PROJECT_SUMMARY chromatography-mass spectrome-try (LC-MS) based method to measure TH in rodent PR:PROJECT_SUMMARY CSF and serum, applicable to both fresh and frozen samples. Using this new PR:PROJECT_SUMMARY method, we find distinct differences in CSF TH in pregnant dams vs. non-pregnant PR:PROJECT_SUMMARY adults and in embryonic vs. adult CSF. Further, targeted LC-MS metabolic PR:PROJECT_SUMMARY pro-filing uncovers distinct central carbon metabolism in the CSF of these PR:PROJECT_SUMMARY populations. TH detection and metabolite profiling of related metabolic pathways PR:PROJECT_SUMMARY open new avenues of rigorous research into CSF TH and will inform future studies PR:PROJECT_SUMMARY on metabolic alterations in CSF during normal de-velopment. PR:INSTITUTE Boston Childrens Hospital PR:DEPARTMENT Pathology PR:LABORATORY Kanarek Lab PR:LAST_NAME Petrova PR:FIRST_NAME Boryana PR:ADDRESS 300 Longwood Av PR:EMAIL boryana.petrova@childrens.harvard.edu PR:PHONE 617 #STUDY ST:STUDY_TITLE Spectral information for 3,5,3’-L-triiodothyronine (T3) and ST:STUDY_TITLE 3,5,3’5’-L-tetraiodothyronine (T4) from synthetic standards ST:STUDY_SUMMARY We utilized synthetic standards for 3,5,3’-L-triiodothyronine (T3) and ST:STUDY_SUMMARY 3,5,3’5’-L-tetraiodothyronine (T4) to obtain high-quality MS2-level ST:STUDY_SUMMARY information that would aid compound identification from biological matrixes ST:INSTITUTE Boston Childrens Hospital ST:DEPARTMENT Patholody ST:LABORATORY Kanarek Lab ST:LAST_NAME Petrova ST:FIRST_NAME Boryana ST:ADDRESS 300 Longwood Av ST:EMAIL boryana.petrova@childrens.harvard.edu ST:PHONE 6173557433 #SUBJECT SU:SUBJECT_TYPE Synthetic sample #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Raw file names and additional sample data SUBJECT_SAMPLE_FACTORS - T3_6 Standard:2nM 3,5,3’-L-triiodothyronine HCD (NCE units)=20, 40, 60, 80; RAW_FILE_NAME=20220728_QEF2_C18_T3T4Dilutions_T3_6_PRM_pos SUBJECT_SAMPLE_FACTORS - T4_6 Standard:2nM 3,5,3’5’-L-tetraiodothyronine HCD (NCE units)=20, 40, 60, 80; RAW_FILE_NAME=20220728_QEF2_C18_T3T4Dilutions_T4_6_PRM_pos #COLLECTION CO:COLLECTION_SUMMARY The presented study is on chemical standards CO:SAMPLE_TYPE synthetic #TREATMENT TR:TREATMENT_SUMMARY no treatments were performed #SAMPLEPREP SP:SAMPLEPREP_SUMMARY standards were solubilized in 50/50 methanol/water with 10uM ammonium hydroxide #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Thermo Vanquish CH:COLUMN_NAME Merck Supelco Ascentis Express C18 (150 x 2.1mm,2.7um) CH:SOLVENT_A 100% water; 0.1% formic acid CH:SOLVENT_B 100% acetonitrile; 0.1% formic acid CH:FLOW_GRADIENT 0–5 min: gradient was held at 5% B; 2–12.1 min: linear gradient of 5% to CH:FLOW_GRADIENT 95% B; 12.1–17.0 min: 95% B; 17.1–21.0 min: gradient was returned to 5% CH:FLOW_GRADIENT B CH:FLOW_RATE 0.25 ml/min CH:COLUMN_TEMPERATURE 30 CH:METHODS_FILENAME C18_LC_Conditions.pdf #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Thermo Q Exactive Orbitrap MS:INSTRUMENT_TYPE Orbitrap MS:MS_TYPE ESI MS:ION_MODE POSITIVE MS:MS_COMMENTS MS data acquisition was performed using a Q Exactive Orbitrap benchtop orbitrap MS:MS_COMMENTS mass spectrometer equipped with an Ion Max source and a HESI II probe (Thermo MS:MS_COMMENTS Fisher Scientific, San Jose, CA, USA)in positive ionization mode and a PRM MS:MS_COMMENTS acquisition with the resolution set at 17,500, the AGC target at 2 × 10^5, and MS:MS_COMMENTS the maximum injection time (Max IT) at 40 msec. Collision energies were set at MS:MS_COMMENTS 20,40,60 and 80 NCE. Inclusion list was 651.7973 m/z for T3 and 777.6940 m/z for MS:MS_COMMENTS T4. HESI conditions were as follows: sheath gas flow rate: 40 units; Aug gas MS:MS_COMMENTS flow rate: 10 units; sweet gas flow rate: 0; spray voltage: 3.5 kV (pos), 2.8 kV MS:MS_COMMENTS (neg); capillary temperature: 380 °C; S-lens RF: 60; Aux gas heater MS:MS_COMMENTS temperature: 420 °C. MS spectra were consolidated using the mzVault software, MS:MS_COMMENTS automatically thresholded for noise and recalibrated for mass error. MS:MS_RESULTS_FILE ST003074_AN005032_Results.txt UNITS:NA Has m/z:No Has RT:No RT units:No RT data #END