#METABOLOMICS WORKBENCH andreabonicelli_20230315_093104 DATATRACK_ID:3786 STUDY_ID:ST002558 ANALYSIS_ID:AN004217 PROJECT_ID:PR001650
VERSION             	1
CREATED_ON             	April 13, 2023, 8:02 am
#PROJECT
PR:PROJECT_TITLE                 	Extraction and Untargeted Analysis of Metabolome from Undemineralised Cortical
PR:PROJECT_TITLE                 	Bone Matrix for Forensic Application
PR:PROJECT_SUMMARY               	Analysis of bone biomolecules for post mortem and age at death estimation in
PR:PROJECT_SUMMARY               	forensic contexts
PR:INSTITUTE                     	University of Central Lancashire
PR:LAST_NAME                     	Bonicelli
PR:FIRST_NAME                    	Andrea
PR:ADDRESS                       	1 North Cliff street
PR:EMAIL                         	abonicelli@uclan.ac.uk
PR:PHONE                         	07383974949
PR:FUNDING_SOURCE                	UKRI Future Leaders Fellowship under grant number MR/S032878/1
#STUDY
ST:STUDY_TITLE                   	Extraction and Untargeted Analysis of Metabolome from Undemineralised Cortical
ST:STUDY_TITLE                   	Bone Matrix for Forensic Application
ST:STUDY_SUMMARY                 	Untargeted metabolomics has become the gold standard for the profiling of
ST:STUDY_SUMMARY                 	low-molecular-weight compounds. Recently, metabolomics has shown great potential
ST:STUDY_SUMMARY                 	in forensic science in the field of toxicology and postmortem interval
ST:STUDY_SUMMARY                 	estimation. The current study aims to evaluate three extraction protocol and
ST:STUDY_SUMMARY                 	four liquid chromatography coupled with mass spectrometry assays that could
ST:STUDY_SUMMARY                 	offer a valuable tool to identify biomarkers for PMI estimation. One fragment
ST:STUDY_SUMMARY                 	for anterior human skeletal tibia from a 82 years old male individual belonging
ST:STUDY_SUMMARY                 	to the Forensic Anthropology Center - Texas State University collection was
ST:STUDY_SUMMARY                 	powdered and extracted in five replicates to be extracted according to a the
ST:STUDY_SUMMARY                 	biphasic chloroform/methanol/water protocol and two single phase protocols based
ST:STUDY_SUMMARY                 	on methanol/water and methanol/acetonitrile/water. Formal analysis was carried
ST:STUDY_SUMMARY                 	out ThermoFisher Ultimate 3000 HPLC in hydrophilic interaction (HILIC) and
ST:STUDY_SUMMARY                 	reverse phase (RP) liquid chromatography coupled with SCIEX 6600 TripleTOF Q-TOF
ST:STUDY_SUMMARY                 	mass spectrometer.
ST:INSTITUTE                     	University of Central Lancashire
ST:DEPARTMENT                    	Natural Sciences
ST:LAST_NAME                     	Bonicelli
ST:FIRST_NAME                    	Andrea
ST:ADDRESS                       	1 North Cliff street
ST:EMAIL                         	abonicelli@uclan.ac.uk
ST:PHONE                         	07383974949
ST:NUM_GROUPS                    	3
ST:TOTAL_SUBJECTS                	1
ST:NUM_MALES                     	1
#SUBJECT
SU:SUBJECT_TYPE                  	Human
SU:SUBJECT_SPECIES               	Homo sapiens
SU:TAXONOMY_ID                   	9606
SU:AGE_OR_AGE_RANGE              	82
SU:GENDER                        	Female
#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           	-	A1_Hilic_pos	class:Chlor_Meth | type:Sample	RAW_FILE_NAME=A1_Hilic_pos.mzML
SUBJECT_SAMPLE_FACTORS           	-	A2_Hilic_pos	class:Chlor_Meth | type:Sample	RAW_FILE_NAME=A2_Hilic_pos.mzML
SUBJECT_SAMPLE_FACTORS           	-	A3_Hilic_pos	class:Chlor_Meth | type:Sample	RAW_FILE_NAME=A3_Hilic_pos.mzML
SUBJECT_SAMPLE_FACTORS           	-	A4_Hilic_pos	class:Chlor_Meth | type:Sample	RAW_FILE_NAME=A4_Hilic_pos.mzML
SUBJECT_SAMPLE_FACTORS           	-	A5_Hilic_pos	class:Chlor_Meth | type:Sample	RAW_FILE_NAME=A5_Hilic_pos.mzML
SUBJECT_SAMPLE_FACTORS           	-	B1_Hilic_pos	class:Meth_Water | type:Sample	RAW_FILE_NAME=B1_Hilic_pos.mzML
SUBJECT_SAMPLE_FACTORS           	-	B2_Hilic_pos	class:Meth_Water | type:Sample	RAW_FILE_NAME=B2_Hilic_pos.mzML
SUBJECT_SAMPLE_FACTORS           	-	B3_Hilic_pos	class:Meth_Water | type:Sample	RAW_FILE_NAME=B3_Hilic_pos.mzML
SUBJECT_SAMPLE_FACTORS           	-	B4_Hilic_pos	class:Meth_Water | type:Sample	RAW_FILE_NAME=B4_Hilic_pos.mzML
SUBJECT_SAMPLE_FACTORS           	-	B5_Hilic_pos	class:Meth_Water | type:Sample	RAW_FILE_NAME=B5_Hilic_pos.mzML
SUBJECT_SAMPLE_FACTORS           	-	BLK A_Hilic_pos	class:Blank | type:Blank	RAW_FILE_NAME=BLK A_Hilic_pos.mzML
SUBJECT_SAMPLE_FACTORS           	-	BLK B_Hilic_pos	class:Blank | type:Blank	RAW_FILE_NAME=BLK B_Hilic_pos.mzML
SUBJECT_SAMPLE_FACTORS           	-	BLK C_Hilic_pos	class:Blank | type:Blank	RAW_FILE_NAME=BLK C_Hilic_pos.mzML
SUBJECT_SAMPLE_FACTORS           	-	C1_Hilic_pos	class:Meth_ACN | type:Sample	RAW_FILE_NAME=C1_Hilic_pos.mzML
SUBJECT_SAMPLE_FACTORS           	-	C2_Hilic_pos	class:Meth_ACN | type:Sample	RAW_FILE_NAME=C2_Hilic_pos.mzML
SUBJECT_SAMPLE_FACTORS           	-	C3_Hilic_pos	class:Meth_ACN | type:Sample	RAW_FILE_NAME=C3_Hilic_pos.mzML
SUBJECT_SAMPLE_FACTORS           	-	C4_Hilic_pos	class:Meth_ACN | type:Sample	RAW_FILE_NAME=C4_Hilic_pos.mzML
SUBJECT_SAMPLE_FACTORS           	-	C5_Hilic_pos	class:Meth_ACN | type:Sample	RAW_FILE_NAME=C5_Hilic_pos.mzML
SUBJECT_SAMPLE_FACTORS           	-	QC01_Hilic_pos	class:QC | type:QC	RAW_FILE_NAME=QC01_Hilic_pos.mzML
SUBJECT_SAMPLE_FACTORS           	-	QC02_Hilic_pos	class:QC | type:QC	RAW_FILE_NAME=QC02_Hilic_pos.mzML
SUBJECT_SAMPLE_FACTORS           	-	QC03_Hilic_pos	class:QC | type:QC	RAW_FILE_NAME=QC03_Hilic_pos.mzML
SUBJECT_SAMPLE_FACTORS           	-	QC04_Hilic_pos	class:QC | type:QC	RAW_FILE_NAME=QC04_Hilic_pos.mzML
SUBJECT_SAMPLE_FACTORS           	-	QC05_Hilic_pos	class:QC | type:QC	RAW_FILE_NAME=QC05_Hilic_pos.mzML
SUBJECT_SAMPLE_FACTORS           	-	QC06_Hilic_pos	class:QC | type:QC	RAW_FILE_NAME=QC06_Hilic_pos.mzML
SUBJECT_SAMPLE_FACTORS           	-	QC07_Hilic_pos	class:QC | type:QC	RAW_FILE_NAME=QC07_Hilic_pos.mzML
SUBJECT_SAMPLE_FACTORS           	-	QC08_Hilic_pos	class:QC | type:QC	RAW_FILE_NAME=QC08_Hilic_pos.mzML
SUBJECT_SAMPLE_FACTORS           	-	A1_Hilic_neg	class:Chlor_Meth | type:Sample	RAW_FILE_NAME=A1_Hilic_neg.mzML
SUBJECT_SAMPLE_FACTORS           	-	A2_Hilic_neg	class:Chlor_Meth | type:Sample	RAW_FILE_NAME=A2_Hilic_neg.mzML
SUBJECT_SAMPLE_FACTORS           	-	A3_Hilic_neg	class:Chlor_Meth | type:Sample	RAW_FILE_NAME=A3_Hilic_neg.mzML
SUBJECT_SAMPLE_FACTORS           	-	A4_Hilic_neg	class:Chlor_Meth | type:Sample	RAW_FILE_NAME=A4_Hilic_neg.mzML
SUBJECT_SAMPLE_FACTORS           	-	A5_Hilic_neg	class:Chlor_Meth | type:Sample	RAW_FILE_NAME=A5_Hilic_neg.mzML
SUBJECT_SAMPLE_FACTORS           	-	B1_Hilic_neg	class:Meth_Water | type:Sample	RAW_FILE_NAME=B1_Hilic_neg.mzML
SUBJECT_SAMPLE_FACTORS           	-	B2_Hilic_neg	class:Meth_Water | type:Sample	RAW_FILE_NAME=B2_Hilic_neg.mzML
SUBJECT_SAMPLE_FACTORS           	-	B3_Hilic_neg	class:Meth_Water | type:Sample	RAW_FILE_NAME=B3_Hilic_neg.mzML
SUBJECT_SAMPLE_FACTORS           	-	B4_Hilic_neg	class:Meth_Water | type:Sample	RAW_FILE_NAME=B4_Hilic_neg.mzML
SUBJECT_SAMPLE_FACTORS           	-	B5_Hilic_neg	class:Meth_Water | type:Sample	RAW_FILE_NAME=B5_Hilic_neg.mzML
SUBJECT_SAMPLE_FACTORS           	-	BLK A_Hilic_neg	class:Blank | type:Blank	RAW_FILE_NAME=BLK A_Hilic_neg.mzML
SUBJECT_SAMPLE_FACTORS           	-	BLK B_Hilic_neg	class:Blank | type:Blank	RAW_FILE_NAME=BLK B_Hilic_neg.mzML
SUBJECT_SAMPLE_FACTORS           	-	BLK C_Hilic_neg	class:Blank | type:Blank	RAW_FILE_NAME=BLK C_Hilic_neg.mzML
SUBJECT_SAMPLE_FACTORS           	-	C1_Hilic_neg	class:Meth_ACN | type:Sample	RAW_FILE_NAME=C1_Hilic_neg.mzML
SUBJECT_SAMPLE_FACTORS           	-	C2_Hilic_neg	class:Meth_ACN | type:Sample	RAW_FILE_NAME=C2_Hilic_neg.mzML
SUBJECT_SAMPLE_FACTORS           	-	C3_Hilic_neg	class:Meth_ACN | type:Sample	RAW_FILE_NAME=C3_Hilic_neg.mzML
SUBJECT_SAMPLE_FACTORS           	-	C4_Hilic_neg	class:Meth_ACN | type:Sample	RAW_FILE_NAME=C4_Hilic_neg.mzML
SUBJECT_SAMPLE_FACTORS           	-	C5_Hilic_neg	class:Meth_ACN | type:Sample	RAW_FILE_NAME=C5_Hilic_neg.mzML
SUBJECT_SAMPLE_FACTORS           	-	QC01_Hilic_neg	class:QC | type:QC	RAW_FILE_NAME=QC01_Hilic_neg.mzML
SUBJECT_SAMPLE_FACTORS           	-	QC02_Hilic_neg	class:QC | type:QC	RAW_FILE_NAME=QC02_Hilic_neg.mzML
SUBJECT_SAMPLE_FACTORS           	-	QC03_Hilic_neg	class:QC | type:QC	RAW_FILE_NAME=QC03_Hilic_neg.mzML
SUBJECT_SAMPLE_FACTORS           	-	QC04_Hilic_neg	class:QC | type:QC	RAW_FILE_NAME=QC04_Hilic_neg.mzML
SUBJECT_SAMPLE_FACTORS           	-	QC05_Hilic_neg	class:QC | type:QC	RAW_FILE_NAME=QC05_Hilic_neg.mzML
SUBJECT_SAMPLE_FACTORS           	-	QC06_Hilic_neg	class:QC | type:QC	RAW_FILE_NAME=QC06_Hilic_neg.mzML
SUBJECT_SAMPLE_FACTORS           	-	QC07_Hilic_neg	class:QC | type:QC	RAW_FILE_NAME=QC07_Hilic_neg.mzML
SUBJECT_SAMPLE_FACTORS           	-	QC08_Hilic_neg	class:QC | type:QC	RAW_FILE_NAME=QC08_Hilic_neg.mzML
SUBJECT_SAMPLE_FACTORS           	-	A1_RP_pos	class:Chlor_Meth | type:Sample	RAW_FILE_NAME=A1_RP_pos.mzML
SUBJECT_SAMPLE_FACTORS           	-	A2_RP_pos	class:Chlor_Meth | type:Sample	RAW_FILE_NAME=A2_RP_pos.mzML
SUBJECT_SAMPLE_FACTORS           	-	A3_RP_pos	class:Chlor_Meth | type:Sample	RAW_FILE_NAME=A3_RP_pos.mzML
SUBJECT_SAMPLE_FACTORS           	-	A4_RP_pos	class:Chlor_Meth | type:Sample	RAW_FILE_NAME=A4_RP_pos.mzML
SUBJECT_SAMPLE_FACTORS           	-	A5_RP_pos	class:Chlor_Meth | type:Sample	RAW_FILE_NAME=A5_RP_pos.mzML
SUBJECT_SAMPLE_FACTORS           	-	B1_RP_pos	class:Meth_Water | type:Sample	RAW_FILE_NAME=B1_RP_pos.mzML
SUBJECT_SAMPLE_FACTORS           	-	B2_RP_pos	class:Meth_Water | type:Sample	RAW_FILE_NAME=B2_RP_pos.mzML
SUBJECT_SAMPLE_FACTORS           	-	B3_RP_pos	class:Meth_Water | type:Sample	RAW_FILE_NAME=B3_RP_pos.mzML
SUBJECT_SAMPLE_FACTORS           	-	B4_RP_pos	class:Meth_Water | type:Sample	RAW_FILE_NAME=B4_RP_pos.mzML
SUBJECT_SAMPLE_FACTORS           	-	B5_RP_pos	class:Meth_Water | type:Sample	RAW_FILE_NAME=B5_RP_pos.mzML
SUBJECT_SAMPLE_FACTORS           	-	BLK A_RP_pos	class:Blank | type:Blank	RAW_FILE_NAME=BLK A_RP_pos.mzML
SUBJECT_SAMPLE_FACTORS           	-	BLK B_RP_pos	class:Blank | type:Blank	RAW_FILE_NAME=BLK B_RP_pos.mzML
SUBJECT_SAMPLE_FACTORS           	-	BLK C_RP_pos	class:Blank | type:Blank	RAW_FILE_NAME=BLK C_RP_pos.mzML
SUBJECT_SAMPLE_FACTORS           	-	C1_RP_pos	class:Meth_ACN | type:Sample	RAW_FILE_NAME=C1_RP_pos.mzML
SUBJECT_SAMPLE_FACTORS           	-	C2_RP_pos	class:Meth_ACN | type:Sample	RAW_FILE_NAME=C2_RP_pos.mzML
SUBJECT_SAMPLE_FACTORS           	-	C3_RP_pos	class:Meth_ACN | type:Sample	RAW_FILE_NAME=C3_RP_pos.mzML
SUBJECT_SAMPLE_FACTORS           	-	C4_RP_pos	class:Meth_ACN | type:Sample	RAW_FILE_NAME=C4_RP_pos.mzML
SUBJECT_SAMPLE_FACTORS           	-	C5_RP_pos	class:Meth_ACN | type:Sample	RAW_FILE_NAME=C5_RP_pos.mzML
SUBJECT_SAMPLE_FACTORS           	-	QC01_RP_pos	class:QC | type:QC	RAW_FILE_NAME=QC01_RP_pos.mzML
SUBJECT_SAMPLE_FACTORS           	-	QC02_RP_pos	class:QC | type:QC	RAW_FILE_NAME=QC02_RP_pos.mzML
SUBJECT_SAMPLE_FACTORS           	-	QC03_RP_pos	class:QC | type:QC	RAW_FILE_NAME=QC03_RP_pos.mzML
SUBJECT_SAMPLE_FACTORS           	-	QC04_RP_pos	class:QC | type:QC	RAW_FILE_NAME=QC04_RP_pos.mzML
SUBJECT_SAMPLE_FACTORS           	-	QC05_RP_pos	class:QC | type:QC	RAW_FILE_NAME=QC05_RP_pos.mzML
SUBJECT_SAMPLE_FACTORS           	-	QC06_RP_pos	class:QC | type:QC	RAW_FILE_NAME=QC06_RP_pos.mzML
SUBJECT_SAMPLE_FACTORS           	-	QC07_RP_pos	class:QC | type:QC	RAW_FILE_NAME=QC07_RP_pos.mzML
SUBJECT_SAMPLE_FACTORS           	-	QC08_RP_pos	class:QC | type:QC	RAW_FILE_NAME=QC08_RP_pos.mzML
SUBJECT_SAMPLE_FACTORS           	-	A1_RP_neg	class:Chlor_Meth | type:Sample	RAW_FILE_NAME=A1_RP_neg.mzML
SUBJECT_SAMPLE_FACTORS           	-	A2_RP_neg	class:Chlor_Meth | type:Sample	RAW_FILE_NAME=A2_RP_neg.mzML
SUBJECT_SAMPLE_FACTORS           	-	A3_RP_neg	class:Chlor_Meth | type:Sample	RAW_FILE_NAME=A3_RP_neg.mzML
SUBJECT_SAMPLE_FACTORS           	-	A4_RP_neg	class:Chlor_Meth | type:Sample	RAW_FILE_NAME=A4_RP_neg.mzML
SUBJECT_SAMPLE_FACTORS           	-	A5_RP_neg	class:Chlor_Meth | type:Sample	RAW_FILE_NAME=A5_RP_neg.mzML
SUBJECT_SAMPLE_FACTORS           	-	B1_RP_neg	class:Meth_Water | type:Sample	RAW_FILE_NAME=B1_RP_neg.mzML
SUBJECT_SAMPLE_FACTORS           	-	B2_RP_neg	class:Meth_Water | type:Sample	RAW_FILE_NAME=B2_RP_neg.mzML
SUBJECT_SAMPLE_FACTORS           	-	B3_RP_neg	class:Meth_Water | type:Sample	RAW_FILE_NAME=B3_RP_neg.mzML
SUBJECT_SAMPLE_FACTORS           	-	B4_RP_neg	class:Meth_Water | type:Sample	RAW_FILE_NAME=B4_RP_neg.mzML
SUBJECT_SAMPLE_FACTORS           	-	B5_RP_neg	class:Meth_Water | type:Sample	RAW_FILE_NAME=B5_RP_neg.mzML
SUBJECT_SAMPLE_FACTORS           	-	BLK A_RP_neg	class:Blank | type:Blank	RAW_FILE_NAME=BLK A_RP_neg.mzML
SUBJECT_SAMPLE_FACTORS           	-	BLK B_RP_neg	class:Blank | type:Blank	RAW_FILE_NAME=BLK B_RP_neg.mzML
SUBJECT_SAMPLE_FACTORS           	-	BLK C_RP_neg	class:Blank | type:Blank	RAW_FILE_NAME=BLK C_RP_neg.mzML
SUBJECT_SAMPLE_FACTORS           	-	C1_RP_neg	class:Meth_ACN | type:Sample	RAW_FILE_NAME=C1_RP_neg.mzML
SUBJECT_SAMPLE_FACTORS           	-	C2_RP_neg	class:Meth_ACN | type:Sample	RAW_FILE_NAME=C2_RP_neg.mzML
SUBJECT_SAMPLE_FACTORS           	-	C3_RP_neg	class:Meth_ACN | type:Sample	RAW_FILE_NAME=C3_RP_neg.mzML
SUBJECT_SAMPLE_FACTORS           	-	C4_RP_neg	class:Meth_ACN | type:Sample	RAW_FILE_NAME=C4_RP_neg.mzML
SUBJECT_SAMPLE_FACTORS           	-	C5_RP_neg	class:Meth_ACN | type:Sample	RAW_FILE_NAME=C5_RP_neg.mzML
SUBJECT_SAMPLE_FACTORS           	-	QC01_RP_neg	class:QC | type:QC	RAW_FILE_NAME=QC01_RP_neg.mzML
SUBJECT_SAMPLE_FACTORS           	-	QC02_RP_neg	class:QC | type:QC	RAW_FILE_NAME=QC02_RP_neg.mzML
SUBJECT_SAMPLE_FACTORS           	-	QC03_RP_neg	class:QC | type:QC	RAW_FILE_NAME=QC03_RP_neg.mzML
SUBJECT_SAMPLE_FACTORS           	-	QC04_RP_neg	class:QC | type:QC	RAW_FILE_NAME=QC04_RP_neg.mzML
SUBJECT_SAMPLE_FACTORS           	-	QC05_RP_neg	class:QC | type:QC	RAW_FILE_NAME=QC05_RP_neg.mzML
SUBJECT_SAMPLE_FACTORS           	-	QC06_RP_neg	class:QC | type:QC	RAW_FILE_NAME=QC06_RP_neg.mzML
SUBJECT_SAMPLE_FACTORS           	-	QC07_RP_neg	class:QC | type:QC	RAW_FILE_NAME=QC07_RP_neg.mzML
SUBJECT_SAMPLE_FACTORS           	-	QC08_RP_neg	class:QC | type:QC	RAW_FILE_NAME=QC08_RP_neg.mzML
#COLLECTION
CO:COLLECTION_SUMMARY            	Bone sample (∼1cm3) of the anterior midshaft tibia (left) were collected by
CO:COLLECTION_SUMMARY            	means of a 12V Dremel cordless lithium-ion drill with a diamond impregnated
CO:COLLECTION_SUMMARY            	wheel drill bit was used at maximum 5000 revolutions from an 82 years old male
CO:COLLECTION_SUMMARY            	individual belonging to the Forensic Anthropology Center - Texas State
CO:COLLECTION_SUMMARY            	University collection.
CO:SAMPLE_TYPE                   	Bone
CO:STORAGE_CONDITIONS            	-80℃
#TREATMENT
TR:TREATMENT_SUMMARY             	No treatment was applied to the material.
#SAMPLEPREP
SP:SAMPLEPREP_SUMMARY            	The bone piece was further powdered using a Spex SamplePrep 6775-115
SP:SAMPLEPREP_SUMMARY            	Freezer/Mill Small Cryogenic Grinder operated in liquid nitrogen at speed 10
SP:SAMPLEPREP_SUMMARY            	with 3min pre-cooling, 2min run and 2min cooling protocol. The powder was stored
SP:SAMPLEPREP_SUMMARY            	in a cryovial at -80◦C until further processing. Biphasic Extraction 50mg of
SP:SAMPLEPREP_SUMMARY            	bone powder was placed in a 2mL Pre-Filled Bead Mill Tubes (ceramic 1.4 mm in
SP:SAMPLEPREP_SUMMARY            	diameter) and 900µL of 2:1 (% v/v) Chlor:MeOH were added. Samples were vortexed
SP:SAMPLEPREP_SUMMARY            	for 30s and and homogenised (4x20s bursts at 7200RPMs, pause 2min between
SP:SAMPLEPREP_SUMMARY            	bursts) in a Precellys evolution. To induce phase separation, 400µL of LC‐MS
SP:SAMPLEPREP_SUMMARY            	grade water was added and homogenised (4x20s bursts at 7200RPMs, pause 2min
SP:SAMPLEPREP_SUMMARY            	between bursts). The samples were then centrifuged at 4°C for 10min at 2000RPMs
SP:SAMPLEPREP_SUMMARY            	and rest in ice for 5min. 600µL lower fraction (organic) was collected and
SP:SAMPLEPREP_SUMMARY            	transferred to fresh Eppendorf tubes and the samples were re‐extracted for a
SP:SAMPLEPREP_SUMMARY            	second time using 500µL of 2:1 (% v/v) Chlor:MeOH and the tube homogenised
SP:SAMPLEPREP_SUMMARY            	(4x20s bursts at 7200RPMs, pause 2min between bursts). The two respective
SP:SAMPLEPREP_SUMMARY            	fractions were combined and concentrated. 600µL lower fraction (organic) was
SP:SAMPLEPREP_SUMMARY            	collected and transferred the previous Eppendorf tube. This was centrifuged at
SP:SAMPLEPREP_SUMMARY            	13000rpm, 4°C for 10min and 1ml of the supernatant was collected and dried
SP:SAMPLEPREP_SUMMARY            	under nitrogen flow. 350µL of aqueous phase in a fresh Eppendorf tube and
SP:SAMPLEPREP_SUMMARY            	centrifuge at 13000rpm, 4°C for 10min and 300µL transferred to a fresh tube
SP:SAMPLEPREP_SUMMARY            	and dried under nitrogen flow. Dry extracts were store at -80°C until testing.
SP:SAMPLEPREP_SUMMARY            	Methanol-Water Extraction 50mg of bone powder was placed in a 2mL Pre-Filled
SP:SAMPLEPREP_SUMMARY            	Bead Mill Tubes (ceramic 1.4 mm in diameter) and 750µL of 8:2 (% v/v)
SP:SAMPLEPREP_SUMMARY            	MeOH:Water were added. Samples were vortexed for 30s and and homogenised (4x20s
SP:SAMPLEPREP_SUMMARY            	bursts at 7200RPMs, pause 2min between bursts) in a Precellys Evolution. The
SP:SAMPLEPREP_SUMMARY            	homogenisation tube was centrifuged at 13000RPMs, 4°C for 10min, 700µL were
SP:SAMPLEPREP_SUMMARY            	moved to a fresh tube. 750µL of 8:2 (% v/v) MeOH:Water were added and the
SP:SAMPLEPREP_SUMMARY            	homogenisation step was repeated. The homogenisation tube was centrifuged at
SP:SAMPLEPREP_SUMMARY            	13000RPMs, 4°C for 10min, 700µL were moved to the same collection tube. The
SP:SAMPLEPREP_SUMMARY            	tube with the two extracts was centrifuged at 13000RPMs, 4°C for 10min and
SP:SAMPLEPREP_SUMMARY            	1.2mL of supernatant were transferred in a fresh tube and dried under nitrogen
SP:SAMPLEPREP_SUMMARY            	flow. Dry extracts were store at -80°C until testing.
SP:SAMPLEPREP_SUMMARY            	Methanol-Acetonitrile-Water Extraction 50mg of bone powder was placed in a 2mL
SP:SAMPLEPREP_SUMMARY            	Pre-Filled Bead Mill Tubes (ceramic 1.4 mm in diameter) and 750µL of 2:2:1 (%
SP:SAMPLEPREP_SUMMARY            	v/v/v) MeOH:AC:Water were added. Samples were vortexed for 30s and and
SP:SAMPLEPREP_SUMMARY            	homogenised (4x20s bursts at 7200RPMs, pause 2min between bursts) in a Precellys
SP:SAMPLEPREP_SUMMARY            	Evolution. The homogenisation tube was centrifuged at 13000RPMs, 4°C for 10min,
SP:SAMPLEPREP_SUMMARY            	700µL were moved to a fresh tube. 750µL of 2:2:1 (% v/v/v) MeOH:ACN:Water were
SP:SAMPLEPREP_SUMMARY            	added and the homogenisation step was repeated. The homogenisation tube was
SP:SAMPLEPREP_SUMMARY            	centrifuged at 13000RPMs, 4°C for 10min, 700uL were moved to the same
SP:SAMPLEPREP_SUMMARY            	collection tube. The tube with the two extracts was centrifuged at 13000RPMs,
SP:SAMPLEPREP_SUMMARY            	4°C for 10min and 1.2mL of supernatant were transferred in a fresh tube and
SP:SAMPLEPREP_SUMMARY            	dried under nitrogen flow. Dry extracts were store at -80°C until testing.
SP:PROCESSING_STORAGE_CONDITIONS 	Described in summary
SP:EXTRACT_STORAGE               	-80℃
#CHROMATOGRAPHY
CH:CHROMATOGRAPHY_SUMMARY        	RP ESI-
CH:CHROMATOGRAPHY_TYPE           	Reversed phase
CH:INSTRUMENT_NAME               	Thermo Dionex Ultimate 3000
CH:COLUMN_NAME                   	Thermo Accucore C18 (150 x 2.1mm,2.6um)
CH:SOLVENT_A                     	0.1 % formic acid in water
CH:SOLVENT_B                     	0.1 % formic acid in 98:2 acetonitrile / water
CH:FLOW_GRADIENT                 	0 min 5% B 1 min 5% B 8 min 100% B 10 min 100% B 10 min 5% B
CH:FLOW_RATE                     	0.3 ml/min
CH:COLUMN_TEMPERATURE            	40 °C
CH:SAMPLE_INJECTION              	5 μL
#ANALYSIS
AN:ANALYSIS_TYPE                 	MS
AN:LABORATORY_NAME               	Biological Mass Spectrometry Core Facility, The University of Manchester
AN:OPERATOR_NAME                 	George Taylor
AN:SOFTWARE_VERSION              	Chromeleon Xpress software
AN:DATA_FORMAT                   	.wiff
#MS
MS:INSTRUMENT_NAME               	ABI Sciex 6600 TripleTOF
MS:INSTRUMENT_TYPE               	QTOF
MS:MS_TYPE                       	ESI
MS:ION_MODE                      	NEGATIVE
MS:MS_COMMENTS                   	The mass spectrometer was ran under the following source conditions: curtain gas
MS:MS_COMMENTS                   	pressure, 50 psi; temperature, 400 °C; ESI nebulizer gas pressure, 50 psi;
MS:MS_COMMENTS                   	heater gas pressure, 70 psi; declustering potential, 80 V. Data was acquired in
MS:MS_COMMENTS                   	an information dependent manner across 10 high sensitivity product ion scans,
MS:MS_COMMENTS                   	each with an accumulation time of 100 ms and a TOF survey scan with accumulation
MS:MS_COMMENTS                   	time of 250 ms. Total cycle time was 1.3 s. Collision energy was determined
MS:MS_COMMENTS                   	using the formula CE (V) = 0.084 x m/z +12 up to a maximum of 55 V. Isotopes
MS:MS_COMMENTS                   	within 4 Da were excluded from the scan. Acquired data were checked in PeakView
MS:MS_COMMENTS                   	2.2 and imported into Progenesis Qi 2.4 for metabolomics, where they were
MS:MS_COMMENTS                   	aligned, peaks were picked, normalised to all compounds and deconvoluted
MS:MS_COMMENTS                   	according to standard Progenesis workflows. Annotations were made by searching
MS:MS_COMMENTS                   	the accurate mass, MS/MS spectrum and isotope distribution ratios of acquired
MS:MS_COMMENTS                   	data against the NIST MS/MS metabolite library. Metabolites were identified by
MS:MS_COMMENTS                   	searching retention times and accurate masses against an in-house chemical
MS:MS_COMMENTS                   	standard library.
MS:MS_RESULTS_FILE               	ST002558_AN004217_Results.txt	UNITS:area integration	Has m/z:Yes	Has RT:Yes	RT units:Minutes
#END