#METABOLOMICS WORKBENCH apulliam3_20240216_053758 DATATRACK_ID:4649 STUDY_ID:ST003093 ANALYSIS_ID:AN005063 PROJECT_ID:PR001922
VERSION             	1
CREATED_ON             	February 20, 2024, 11:47 am
#PROJECT
PR:PROJECT_TITLE                 	Integrative Analysis of Cytokine and Lipidomics Datasets Fol-lowing Mild
PR:PROJECT_TITLE                 	Traumatic Brain Injury in the Rat
PR:PROJECT_SUMMARY               	Traumatic brain injury (TBI) is a significant source of disability in the United
PR:PROJECT_SUMMARY               	States and around the world and may lead to long-lasting cognitive deficits and
PR:PROJECT_SUMMARY               	decreased quality of life for patients across injury severities. Following the
PR:PROJECT_SUMMARY               	primary injury phase, TBI is characterized by com-plex secondary cascades that
PR:PROJECT_SUMMARY               	involve altered homeostasis and metabolism, faulty signaling,
PR:PROJECT_SUMMARY               	neu-roinflammation, and lipid dysfunction. The objectives of the present study
PR:PROJECT_SUMMARY               	were to (1) assess po-tential correlations between lipidome and cytokine changes
PR:PROJECT_SUMMARY               	after closed-head mild TBI (mTBI), and (2) examine reproducibility of our acute
PR:PROJECT_SUMMARY               	lipidomic profiles following TBI. Cortices from 54 Sprague Dawley male and
PR:PROJECT_SUMMARY               	female rats were analyzed by ultra-high-performance liquid chromatography mass
PR:PROJECT_SUMMARY               	spectrometry (LC-MS) in both positive and negative ionization modes and
PR:PROJECT_SUMMARY               	multiplex cytokine analysis after single (smTBI) or repetitive (rmTBI)
PR:PROJECT_SUMMARY               	closed-head impacts, or sham conditions. Tissue age was a variable, given that
PR:PROJECT_SUMMARY               	two cohorts (n= 26 and n=28) were initially run a year-and-a-half apart,
PR:PROJECT_SUMMARY               	creating inter-batch variations. We annotated the lipidome datasets using an
PR:PROJECT_SUMMARY               	in-house data dictionary based on exact masses of precursor and fragment ions
PR:PROJECT_SUMMARY               	and removed features with statis-tically significant differences between sham
PR:PROJECT_SUMMARY               	control batches. Our results indicate that lipids with high fold change between
PR:PROJECT_SUMMARY               	injury groups moderately correlate with the cytokines eotaxin, IP-10, and TNF-a.
PR:PROJECT_SUMMARY               	Additionally, we show a significant decrease of the pro-inflammatory markers,
PR:PROJECT_SUMMARY               	IL-1b and IP-10, TNF-a, and RANTES in the rmTBI samples relative to sham
PR:PROJECT_SUMMARY               	control. We discuss the major challenges in correlating high dimensional
PR:PROJECT_SUMMARY               	lipidomic data with functional cytokine profiles and the implications for
PR:PROJECT_SUMMARY               	understanding the biological significance of two related but disparate analysis
PR:PROJECT_SUMMARY               	modes in the study of TBI, an inherently heterogeneous neurological disorder.
PR:INSTITUTE                     	Georgia Institute of Technology
PR:DEPARTMENT                    	The Wallace H. Coulter Department of Biomedical Engineering
PR:LABORATORY                    	Michelle LaPlaca
PR:LAST_NAME                     	Pulliam
PR:FIRST_NAME                    	Alexis
PR:ADDRESS                       	313 Ferst Dr. NW, Atlanta, GA, 30332
PR:EMAIL                         	apulliam3@gatech.edu
PR:PHONE                         	404.385.0629
#STUDY
ST:STUDY_TITLE                   	Integrative Analysis of Cytokine and Lipidomics Datasets Following Mild
ST:STUDY_TITLE                   	Traumatic Brain Injury in the Rat
ST:STUDY_SUMMARY                 	Traumatic brain injury (TBI) is a significant source of disability in the United
ST:STUDY_SUMMARY                 	States and around the world and may lead to long-lasting cognitive deficits and
ST:STUDY_SUMMARY                 	decreased quality of life for patients across injury severities. Following the
ST:STUDY_SUMMARY                 	primary injury phase, TBI is characterized by com-plex secondary cascades that
ST:STUDY_SUMMARY                 	involve altered homeostasis and metabolism, faulty signaling,
ST:STUDY_SUMMARY                 	neu-roinflammation, and lipid dysfunction. The objectives of the present study
ST:STUDY_SUMMARY                 	were to (1) assess po-tential correlations between lipidome and cytokine changes
ST:STUDY_SUMMARY                 	after closed-head mild TBI (mTBI), and (2) examine reproducibility of our acute
ST:STUDY_SUMMARY                 	lipidomic profiles following TBI. Cortices from 54 Sprague Dawley male and
ST:STUDY_SUMMARY                 	female rats were analyzed by ultra-high-performance liquid chromatography mass
ST:STUDY_SUMMARY                 	spectrometry (LC-MS) in both positive and negative ionization modes and
ST:STUDY_SUMMARY                 	multiplex cytokine analysis after single (smTBI) or repetitive (rmTBI)
ST:STUDY_SUMMARY                 	closed-head impacts, or sham conditions. Tissue age was a variable, given that
ST:STUDY_SUMMARY                 	two cohorts (n= 26 and n=28) were initially run a year-and-a-half apart,
ST:STUDY_SUMMARY                 	creating inter-batch variations. We annotated the lipidome datasets using an
ST:STUDY_SUMMARY                 	in-house data dictionary based on exact masses of precursor and fragment ions
ST:STUDY_SUMMARY                 	and removed features with statis-tically significant differences between sham
ST:STUDY_SUMMARY                 	control batches. Our results indicate that lipids with high fold change between
ST:STUDY_SUMMARY                 	injury groups moderately correlate with the cytokines eotaxin, IP-10, and TNF-a.
ST:STUDY_SUMMARY                 	Additionally, we show a significant decrease of the pro-inflammatory markers,
ST:STUDY_SUMMARY                 	IL-1b and IP-10, TNF-a, and RANTES in the rmTBI samples relative to sham
ST:STUDY_SUMMARY                 	control. We discuss the major challenges in correlating high dimensional
ST:STUDY_SUMMARY                 	lipidomic data with functional cytokine profiles and the implications for
ST:STUDY_SUMMARY                 	understanding the biological significance of two related but disparate analysis
ST:STUDY_SUMMARY                 	modes in the study of TBI, an inherently heterogeneous neurological disorder.
ST:INSTITUTE                     	Georgia Institute of Technology
ST:DEPARTMENT                    	The Wallace H. Coulter Department of Biomedical Engineering
ST:LABORATORY                    	Michelle LaPlaca
ST:LAST_NAME                     	Pulliam
ST:FIRST_NAME                    	Alexis
ST:ADDRESS                       	313 Ferst Dr. NW, Atlanta, GA, 30332
ST:EMAIL                         	apulliam3@gatech.edu
ST:PHONE                         	404.385.0629
ST:NUM_GROUPS                    	3
ST:TOTAL_SUBJECTS                	54
ST:NUM_MALES                     	27
ST:NUM_FEMALES                   	27
#SUBJECT
SU:SUBJECT_TYPE                  	Mammal
SU:SUBJECT_SPECIES               	Rattus norvegicus
SU:TAXONOMY_ID                   	10116
SU:WEIGHT_OR_WEIGHT_RANGE        	400g
SU:GENDER                        	Male and female
SU:ANIMAL_ANIMAL_SUPPLIER        	Charles River
SU:ANIMAL_HOUSING                	Double Housed
SU:ANIMAL_LIGHT_CYCLE            	Reverse Light Cycle
SU:SPECIES_GROUP                 	Sprague Dawley
#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           	-	01_013	Sample source:Norm. Area  01_13.raw (F32) | Injury severity:1X | Sex:F | Batches:Batch 1	RAW_FILE_NAME=01_13.raw
SUBJECT_SAMPLE_FACTORS           	-	01_017	Sample source:Norm. Area  01_17.raw (F50) | Injury severity:1X | Sex:F | Batches:Batch 1	RAW_FILE_NAME=01_17.raw
SUBJECT_SAMPLE_FACTORS           	-	01_022	Sample source:Norm. Area  01_22.raw (F46) | Injury severity:1X | Sex:F | Batches:Batch 1	RAW_FILE_NAME=01_22.raw
SUBJECT_SAMPLE_FACTORS           	-	01_024	Sample source:Norm. Area  01_24.raw (F29) | Injury severity:1X | Sex:F | Batches:Batch 1	RAW_FILE_NAME=01_24.raw
SUBJECT_SAMPLE_FACTORS           	-	01_027	Sample source:Norm. Area  01_27.raw (F59) | Injury severity:1X | Sex:F | Batches:Batch 1	RAW_FILE_NAME=01_27.raw
SUBJECT_SAMPLE_FACTORS           	-	01_010	Sample source:Norm. Area  01_10.raw (F77) | Injury severity:1X | Sex:M | Batches:Batch 1	RAW_FILE_NAME=01_10.raw
SUBJECT_SAMPLE_FACTORS           	-	01_006	Sample source:Norm. Area  01_6.raw (F79) | Injury severity:1X | Sex:M | Batches:Batch 1	RAW_FILE_NAME=01_6.raw
SUBJECT_SAMPLE_FACTORS           	-	01_008	Sample source:Norm. Area  01_8.raw (F76) | Injury severity:1X | Sex:M | Batches:Batch 1	RAW_FILE_NAME=01_8.raw
SUBJECT_SAMPLE_FACTORS           	-	01_020	Sample source:Norm. Area  01_20.raw (F63) | Injury severity:3X | Sex:F | Batches:Batch 1	RAW_FILE_NAME=01_20.raw
SUBJECT_SAMPLE_FACTORS           	-	01_023	Sample source:Norm. Area  01_23.raw (F82) | Injury severity:3X | Sex:F | Batches:Batch 1	RAW_FILE_NAME=01_23.raw
SUBJECT_SAMPLE_FACTORS           	-	01_028	Sample source:Norm. Area  01_28.raw (F55) | Injury severity:3X | Sex:F | Batches:Batch 1	RAW_FILE_NAME=01_28.raw
SUBJECT_SAMPLE_FACTORS           	-	01_030	Sample source:Norm. Area  01_30.raw (F74) | Injury severity:3X | Sex:F | Batches:Batch 1	RAW_FILE_NAME=01_30.raw
SUBJECT_SAMPLE_FACTORS           	-	01_032	Sample source:Norm. Area  01_32.raw (F73) | Injury severity:3X | Sex:F | Batches:Batch 1	RAW_FILE_NAME=01_32.raw
SUBJECT_SAMPLE_FACTORS           	-	01_011	Sample source:Norm. Area  01_11.raw (F78) | Injury severity:3X | Sex:M | Batches:Batch 1	RAW_FILE_NAME=01_11.raw
SUBJECT_SAMPLE_FACTORS           	-	01_012	Sample source:Norm. Area  01_12.raw (F54) | Injury severity:3X | Sex:M | Batches:Batch 1	RAW_FILE_NAME=01_12.raw
SUBJECT_SAMPLE_FACTORS           	-	01_029	Sample source:Norm. Area  01_29.raw (F34) | Injury severity:3X | Sex:M | Batches:Batch 1	RAW_FILE_NAME=01_29.raw
SUBJECT_SAMPLE_FACTORS           	-	01_014	Sample source:Norm. Area  01_14.raw (F38) | Injury severity:SHAM | Sex:F | Batches:Batch 1	RAW_FILE_NAME=01_14.raw
SUBJECT_SAMPLE_FACTORS           	-	01_018	Sample source:Norm. Area  01_18.raw (F51) | Injury severity:SHAM | Sex:F | Batches:Batch 1	RAW_FILE_NAME=01_18.raw
SUBJECT_SAMPLE_FACTORS           	-	01_019	Sample source:Norm. Area  01_19.raw (F53) | Injury severity:SHAM | Sex:F | Batches:Batch 1	RAW_FILE_NAME=01_19.raw
SUBJECT_SAMPLE_FACTORS           	-	01_021	Sample source:Norm. Area  01_21.raw (F28) | Injury severity:SHAM | Sex:F | Batches:Batch 1	RAW_FILE_NAME=01_21.raw
SUBJECT_SAMPLE_FACTORS           	-	01_031	Sample source:Norm. Area  01_31.raw (F56) | Injury severity:SHAM | Sex:F | Batches:Batch 1	RAW_FILE_NAME=01_31.raw
SUBJECT_SAMPLE_FACTORS           	-	01_001	Sample source:Norm. Area  01_1.raw (F81) | Injury severity:SHAM | Sex:M | Batches:Batch 1	RAW_FILE_NAME=01_1.raw
SUBJECT_SAMPLE_FACTORS           	-	01_002	Sample source:Norm. Area  01_2.raw (F44) | Injury severity:SHAM | Sex:M | Batches:Batch 1	RAW_FILE_NAME=01_2.raw
SUBJECT_SAMPLE_FACTORS           	-	01_025	Sample source:Norm. Area  01_25.raw (F41) | Injury severity:SHAM | Sex:M | Batches:Batch 1	RAW_FILE_NAME=01_25.raw
SUBJECT_SAMPLE_FACTORS           	-	01_026	Sample source:Norm. Area  01_26.raw (F80) | Injury severity:SHAM | Sex:M | Batches:Batch 1	RAW_FILE_NAME=01_26.raw
SUBJECT_SAMPLE_FACTORS           	-	01_007	Sample source:Norm. Area  01_7.raw (F57) | Injury severity:SHAM | Sex:M | Batches:Batch 1	RAW_FILE_NAME=01_7.raw
SUBJECT_SAMPLE_FACTORS           	-	02_010	Sample source:Norm. Area  02_10.raw (F43) | Injury severity:1X | Sex:F | Batches:Batch 2	RAW_FILE_NAME=02_10.raw
SUBJECT_SAMPLE_FACTORS           	-	02_014	Sample source:Norm. Area  02_14.raw (F37) | Injury severity:1X | Sex:F | Batches:Batch 2	RAW_FILE_NAME=02_14.raw
SUBJECT_SAMPLE_FACTORS           	-	02_026	Sample source:Norm. Area  02_26.raw (F45) | Injury severity:1X | Sex:F | Batches:Batch 2	RAW_FILE_NAME=02_26.raw
SUBJECT_SAMPLE_FACTORS           	-	02_007	Sample source:Norm. Area  02_7.raw (F71) | Injury severity:1X | Sex:F | Batches:Batch 2	RAW_FILE_NAME=02_7.raw
SUBJECT_SAMPLE_FACTORS           	-	02_011	Sample source:Norm. Area  02_11.raw (F27) | Injury severity:1X | Sex:M | Batches:Batch 2	RAW_FILE_NAME=02_11.raw
SUBJECT_SAMPLE_FACTORS           	-	02_020	Sample source:Norm. Area  02_20.raw (F35) | Injury severity:1X | Sex:M | Batches:Batch 2	RAW_FILE_NAME=02_20.raw
SUBJECT_SAMPLE_FACTORS           	-	02_021	Sample source:Norm. Area  02_21.raw (F67) | Injury severity:1X | Sex:M | Batches:Batch 2	RAW_FILE_NAME=02_21.raw
SUBJECT_SAMPLE_FACTORS           	-	02_004	Sample source:Norm. Area  02_4.raw (F42) | Injury severity:1X | Sex:M | Batches:Batch 2	RAW_FILE_NAME=02_4.raw
SUBJECT_SAMPLE_FACTORS           	-	02_008	Sample source:Norm. Area  02_8.raw (F52) | Injury severity:1X | Sex:M | Batches:Batch 2	RAW_FILE_NAME=02_8.raw
SUBJECT_SAMPLE_FACTORS           	-	02_027	Sample source:Norm. Area  02_27.raw (F64) | Injury severity:3X | Sex:F | Batches:Batch 2	RAW_FILE_NAME=02_27.raw
SUBJECT_SAMPLE_FACTORS           	-	02_005	Sample source:Norm. Area  02_5.raw (F58) | Injury severity:3X | Sex:F | Batches:Batch 2	RAW_FILE_NAME=02_5.raw
SUBJECT_SAMPLE_FACTORS           	-	02_015	Sample source:Norm. Area  02_15.raw (F33) | Injury severity:3X | Sex:M | Batches:Batch 2	RAW_FILE_NAME=02_15.raw
SUBJECT_SAMPLE_FACTORS           	-	02_016	Sample source:Norm. Area  02_16.raw (F47) | Injury severity:3X | Sex:M | Batches:Batch 2	RAW_FILE_NAME=02_16.raw
SUBJECT_SAMPLE_FACTORS           	-	02_017	Sample source:Norm. Area  02_17.raw (F68) | Injury severity:3X | Sex:M | Batches:Batch 2	RAW_FILE_NAME=02_17.raw
SUBJECT_SAMPLE_FACTORS           	-	02_018	Sample source:Norm. Area  02_18.raw (F26) | Injury severity:3X | Sex:M | Batches:Batch 2	RAW_FILE_NAME=02_18.raw
SUBJECT_SAMPLE_FACTORS           	-	02_022	Sample source:Norm. Area  02_22.raw (F65) | Injury severity:3X | Sex:M | Batches:Batch 2	RAW_FILE_NAME=02_22.raw
SUBJECT_SAMPLE_FACTORS           	-	02_023	Sample source:Norm. Area  02_23.raw (F66) | Injury severity:3X | Sex:M | Batches:Batch 2	RAW_FILE_NAME=02_23.raw
SUBJECT_SAMPLE_FACTORS           	-	02_029	Sample source:Norm. Area  02_29.raw (F25) | Injury severity:3X | Sex:M | Batches:Batch 2	RAW_FILE_NAME=02_29.raw
SUBJECT_SAMPLE_FACTORS           	-	02_031	Sample source:Norm. Area  02_31.raw (F75) | Injury severity:3X | Sex:M | Batches:Batch 2	RAW_FILE_NAME=02_31.raw
SUBJECT_SAMPLE_FACTORS           	-	02_009	Sample source:Norm. Area  02_9.raw (F40) | Injury severity:3X | Sex:F | Batches:Batch 2	RAW_FILE_NAME=02_9.raw
SUBJECT_SAMPLE_FACTORS           	-	02_013	Sample source:Norm. Area  02_13.raw (F30) | Injury severity:SHAM | Sex:F | Batches:Batch 2	RAW_FILE_NAME=02_13.raw
SUBJECT_SAMPLE_FACTORS           	-	02_025	Sample source:Norm. Area  02_25.raw (F69) | Injury severity:SHAM | Sex:F | Batches:Batch 2	RAW_FILE_NAME=02_25.raw
SUBJECT_SAMPLE_FACTORS           	-	02_028	Sample source:Norm. Area  02_28.raw (F70) | Injury severity:SHAM | Sex:F | Batches:Batch 2	RAW_FILE_NAME=02_28.raw
SUBJECT_SAMPLE_FACTORS           	-	02_003	Sample source:Norm. Area  02_3.raw (F62) | Injury severity:SHAM | Sex:F | Batches:Batch 2	RAW_FILE_NAME=02_3.raw
SUBJECT_SAMPLE_FACTORS           	-	02_012	Sample source:Norm. Area  02_12.raw (F60) | Injury severity:SHAM | Sex:M | Batches:Batch 2	RAW_FILE_NAME=02_12.raw
SUBJECT_SAMPLE_FACTORS           	-	02_019	Sample source:Norm. Area  02_19.raw (F31) | Injury severity:SHAM | Sex:M | Batches:Batch 2	RAW_FILE_NAME=02_19.raw
SUBJECT_SAMPLE_FACTORS           	-	02_002	Sample source:Norm. Area  02_2.raw (F48) | Injury severity:SHAM | Sex:M | Batches:Batch 2	RAW_FILE_NAME=02_2.raw
SUBJECT_SAMPLE_FACTORS           	-	02_006	Sample source:Norm. Area  02_6.raw (F39) | Injury severity:SHAM | Sex:M | Batches:Batch 2	RAW_FILE_NAME=02_6.raw
#COLLECTION
CO:COLLECTION_SUMMARY            	Brain samples were harvested and collected following transcardial perfusion with
CO:COLLECTION_SUMMARY            	phosphate buffer (0.1 M, pH 7.4) 24 hr post-TBI. The perfused whole brains were
CO:COLLECTION_SUMMARY            	rapidly removed, and flash frozen in an isopentane-methanol ice slurry. Pieces
CO:COLLECTION_SUMMARY            	of parietal cortices (5 mm x 2 mm) were dissected from partially thawed brains
CO:COLLECTION_SUMMARY            	by removing the subcortical structures including the majority of white matter
CO:COLLECTION_SUMMARY            	and stored at -80° C in microcentrifuge tubes. The cortices were then
CO:COLLECTION_SUMMARY            	transferred to liquid nitrogen and manually pulverized with a pestle and mortar
CO:COLLECTION_SUMMARY            	submerged in liquid nitrogen and aliquoted in ~10-30 mg tissue samples.
CO:SAMPLE_TYPE                   	Brain
#TREATMENT
TR:TREATMENT_SUMMARY             	Experimental batch 1 contained female (n = 16) and male (n = 10) to either sham
TR:TREATMENT_SUMMARY             	procedure (n = 10), 1X, single mild traumatic brain injury (mTBI) (n = 8), and
TR:TREATMENT_SUMMARY             	3X, repetitive mTBI (n = 8) groups. Experimental batch 2 contained female (n =
TR:TREATMENT_SUMMARY             	11) and male (n = 17) to either sham procedure (n = 8), smTBI(n = 9), and rmTBI
TR:TREATMENT_SUMMARY             	(n = 11) groups.
#SAMPLEPREP
SP:SAMPLEPREP_SUMMARY            	Experimental batch 1 and 2 aliquoted tissue samples were thawed simultane-ously
SP:SAMPLEPREP_SUMMARY            	on ice prior to addition of solvent (IPA and Splash II Lipidomix in (1:3 v/v))
SP:SAMPLEPREP_SUMMARY            	to sep-arate lipids and small non-polar metabolites. LC-MS grade water was used
SP:SAMPLEPREP_SUMMARY            	to prepare sample blanks, and pooled quality control (QC) samples were prepared
SP:SAMPLEPREP_SUMMARY            	from 5 µL ali-quoted supernatant of all samples in the study. The brain and
SP:SAMPLEPREP_SUMMARY            	solvent (1:4 w/v), and beads were placed in a Tissuelyser II for 8 min and
SP:SAMPLEPREP_SUMMARY            	centrifuged at 16000 g for 7 min. The supernatant was collected for LC-MS.
SP:SAMPLEPREP_SUMMARY            	Pooled quality control samples were formed from combining 6 µL aliquots of all
SP:SAMPLEPREP_SUMMARY            	brain sample extracts. Sample blanks were prepared with the same procedure
SP:SAMPLEPREP_SUMMARY            	except instead of a brain sample, 50 µL of LC-MS grade water was used.
#CHROMATOGRAPHY
CH:CHROMATOGRAPHY_SUMMARY        	MS
CH:CHROMATOGRAPHY_TYPE           	Reversed phase
CH:INSTRUMENT_NAME               	Thermo Vanquish
CH:COLUMN_NAME                   	Thermo Accucore C30 (50 x 2.1mm,2.1um)
CH:SOLVENT_A                     	40% water/60% acetonitrile; 10 mM ammonium formate; 0.1% formic acid
CH:SOLVENT_B                     	10% acetonitrile/90% isopropanol; with 10 mM ammonium formate; 0.1% formic acid
CH:FLOW_GRADIENT                 	0 minutes 80% A; 1 minute 40% A; 5 minutes 30% A; 5.5 minutes 15% A; 8 minutes
CH:FLOW_GRADIENT                 	10% A; held 8.2 minutes to 10.5 minutes 0% A; 10.7 minutes 80% A; and held until
CH:FLOW_GRADIENT                 	12 minutes
CH:FLOW_RATE                     	0.40 mL/min
CH:COLUMN_TEMPERATURE            	50
#ANALYSIS
AN:ANALYSIS_TYPE                 	MS
#MS
MS:INSTRUMENT_NAME               	Thermo ID-X Orbitrap Tribrid
MS:INSTRUMENT_TYPE               	Orbitrap
MS:MS_TYPE                       	ESI
MS:ION_MODE                      	NEGATIVE
MS:MS_COMMENTS                   	LCMS Neg
MS:MS_RESULTS_FILE               	ST003093_AN005063_Results.txt	UNITS:m/z	Has m/z:Yes	Has RT:No	RT units:No RT data
#END