#METABOLOMICS WORKBENCH andreabonicelli_20230315_093104 DATATRACK_ID:3786 STUDY_ID:ST002558 ANALYSIS_ID:AN004216 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 POSITIVE 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_AN004216_Results.txt UNITS:area integration Has m/z:Yes Has RT:Yes RT units:Minutes #END