#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