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MB Sample ID: SA218561
| Local Sample ID: | D2_TF_B_Hilic_Neg |
| Subject ID: | SU002369 |
| Subject Type: | Human |
| Subject Species: | Homo sapiens |
| Taxonomy ID: | 9606 |
| Age Or Age Range: | 61-91 |
| Gender: | Female |
Select appropriate tab below to view additional metadata details:
Subject:
| Subject ID: | SU002369 |
| Subject Type: | Human |
| Subject Species: | Homo sapiens |
| Taxonomy ID: | 9606 |
| Age Or Age Range: | 61-91 |
| Gender: | Female |
Factors:
| Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
|---|---|---|---|---|
| D2_TF_B_Hilic_Neg | SA218561 | FL026497 | 67 | Age |
| D2_TF_B_Hilic_Neg | SA218561 | FL026497 | 2-10 | PMI |
| D2_TF_B_Hilic_Neg | SA218561 | FL026497 | Pre deposition | Deposition |
| D2_TF_B_Hilic_Neg | SA218561 | FL026497 | Fresh | State |
Collection:
| Collection ID: | CO002362 |
| Collection Summary: | Bone samples (ca. 1 cm3) of the anterior midshaft tibia (left) were collected prior to placement of the body outdoors, and again upon retrieval of the completely skeletonized remains (right). Each body was in “fresh” stage of decomposition when pre-placement samples were taken, and in “skeletonization” stage when post-placement samples were collected, based on scoring of the gross morphological changes37. The duration of each placement and the deposition context are reported in Table 1. The soft tissue was incised with a disposable scalpel, and a 12 V Dremel cordless lithium-ion drill with a diamond wheel drill bit was used at max. 5000 revolutions to collect ~1 cm3 of bone. Sampling instruments were cleaned with bleach and deionised water between each individual sample collection. A total of eight samples were collected in Ziploc bags, transferred immediately to a -80 °C freezer, and subsequently shipped overnight on dry ice to the Forensic Science Unit at Northumbria University, U.K. The samples were then transferred to a lockable freezer at -20 °C as per UK Human Tissue Act regulations (licence number 12495). The bone samples were defrosted, and fine powder was obtained with a Dremel drill equipped with diamond-tipped drill bits operated at speed 5000 rpms, to avoid heat damage caused by the friction with the bone. The collected powder was homogenised and stored in 2 mL protein LoBind tubes (Eppendorf UK Limited, Stevenage, UK) at -80 °C until extraction and testing. The powder sample was later divided into 25 mg aliquots. The research and bone sample analyses were reviewed and approved by the Ethics committee at Northumbria University (ref. 11623). |
| Sample Type: | Bone |
| Storage Conditions: | -20℃ |
Treatment:
| Treatment ID: | TR002381 |
| Treatment Summary: | Anterior midshaft tibial bone was collected from four female body donors in a fresh stage of decomposition before placement of the bodies to decompose outdoors at the human taphonomy facility managed by the Forensic Anthropological Center at Texas State (FACTS). Bone samples were again collected at selected PMIs (219, 790, 834 and 872 days) |
Sample Preparation:
| Sampleprep ID: | SP002375 |
| Sampleprep Summary: | Chloroform (Chl), AnalaR NORMAPUR® ACS was purchased from VWR Chemicals (Lutterworth, UK). Water Optima™ LC/MS Grade, Methanol (MeOH) Optima™ LC/MS Grade, Pierce™ Acetonitrile (ACN), LC-MS Grade and Isopropanol (IPA), Optima™LC/MS Grade were purchased from Thermo Scientific (Hemel Hempstead, United Kingdom). In total two replicates for each of the six specimens were extracted according to a modified Folch et al. [17] as follow: 25 mg of bone powder was placed in tube A and 750μL of 2:1 (v/v) Chl:MeOH were added, vortexed for 30s and sonicated in ice for additional 20 min. 300μL of LC-MS grade water was added to induce phase separation and sonicate for another 15 mins. The sample were then centrifuged at 10°C for 5 mins at 2000 RPM. The respective upper and lower fractions were collected and transferred to fresh Eppendorf tubes and the samples were re-extracted with a second time using 750μL of 2:1 (v/v) Chl:MeOH. The two respective fractions were combined and concentrated. The organic lipid fraction was preconcentrated using a vacuum concentrator at 55oC for 2.5 hours or until all organic solvents has been removed. The aqueous metabolite fractions were flash frozen in liquid nitrogen and preconcentrated using a lyophilizer cold trap -65oC over night to remove all water content. The respective dry fractions were then stored at -80 until analysis. The metabolite fraction was resuspended in 100μL in 95:5 ACN/water (v/v) and sonicated for 15 mins and centrifuged for 15 min at 15K RPM at 4oC and supernatant was then transferred to 1.5mL autosampler vials with 200μL microinsert and caped. 20μL of each sample were collected and pooled to create the pooled QC. The lipid extracts were resuspended in 100μL of 1:1:2 (v/v) water:ACN:IPA and sonicated for sonicated for 15 min and centrifuged for 15 min at 15K RPM at 10oC and supernatant was then transferred to 1.5mL autosampler vials with 200μL microinsert and caped. 20μL of each sample were collected and pooled to create the pooled QC. The sample set was then submitted for analysis |
| Processing Storage Conditions: | On ice |
| Extraction Method: | Chloroform methanol biphasic extraction |
| Extract Storage: | -80℃ |
Combined analysis:
| Analysis ID | AN003728 | AN003729 | AN003730 | AN003731 |
|---|---|---|---|---|
| Analysis type | MS | MS | MS | MS |
| Chromatography type | HILIC | HILIC | Reversed phase | Reversed phase |
| Chromatography system | Thermo Vanquish | Thermo Vanquish | Thermo Vanquish | Thermo Vanquish |
| Column | Waters Acquity BEH Amide (150 x 2.1mm,1.7um) | Waters Acquity BEH Amide (150 x 2.1mm,1.7um) | Waters Acquity BEH Amide (150 x 2.1mm,1.7um) | Waters Acquity BEH Amide (150 x 2.1mm,1.7um) |
| MS Type | ESI | ESI | ESI | ESI |
| MS instrument type | Orbitrap | Orbitrap | Orbitrap | Orbitrap |
| MS instrument name | Obitrap ID-X Tribrid | Obitrap ID-X Tribrid | Obitrap ID-X Tribrid | Obitrap ID-X Tribrid |
| Ion Mode | POSITIVE | NEGATIVE | POSITIVE | NEGATIVE |
| Units | Area integration | Area integration | Area integration | Area integration |
Chromatography:
| Chromatography ID: | CH002761 |
| Chromatography Summary: | Hilic ESI+ |
| Methods Filename: | AB_Human_Bone_Chromatography.pdf |
| Instrument Name: | Thermo Vanquish |
| Column Name: | Waters Acquity BEH Amide (150 x 2.1mm,1.7um) |
| Column Temperature: | 45°C |
| Flow Gradient: | 95% B at T0 hold for 1.5 min, linear decrease to 50% B at 11 min, hold for 4 mins |
| Flow Rate: | 200 μL/min |
| Injection Temperature: | 300°C |
| Sample Injection: | 3μL |
| Solvent A: | 100% water; 0.1% formic acid; 10 mM ammonium formate |
| Solvent B: | 100% acetonitrile; 10 mM ammonium formate; 0.1% formic acid |
| Preconditioning: | 4.5 mins B |
| Chromatography Type: | HILIC |
| Chromatography ID: | CH002762 |
| Chromatography Summary: | Hilic ESI- |
| Methods Filename: | AB_Human_Bone_Chromatography.pdf |
| Instrument Name: | Thermo Vanquish |
| Column Name: | Waters Acquity BEH Amide (150 x 2.1mm,1.7um) |
| Column Temperature: | 45°C |
| Flow Gradient: | 95% B at T0 hold for 1.5 min, linear decrease to 50% B at 11 min, hold for 4 mins |
| Flow Rate: | 200 μL/min |
| Injection Temperature: | 300°C |
| Sample Injection: | 5μL |
| Solvent A: | 100% water; 0.1% formic acid; 10 mM ammonium formate |
| Solvent B: | 100% acetonitrile; 10 mM ammonium formate; 0.1% formic acid |
| Preconditioning: | 4.5 mins B |
| Chromatography Type: | HILIC |
| Chromatography ID: | CH002763 |
| Chromatography Summary: | RP ESI+ |
| Methods Filename: | AB_Human_Bone_Chromatography.pdf |
| Instrument Name: | Thermo Vanquish |
| Column Name: | Waters Acquity BEH Amide (150 x 2.1mm,1.7um) |
| Column Temperature: | 45°C |
| Flow Gradient: | 60% B at T0 hold for 1.5 min, linear increase to 85% B at 7 min, increase to 95% B at 12.5 min and hold for 4.5 min |
| Flow Rate: | 200 μL/min |
| Injection Temperature: | 300°C |
| Sample Injection: | 3μL |
| Solvent A: | 40% water/60 % acetonitrile; 0.1% formic acid; 10 mM ammonium formate |
| Solvent B: | 90% isopropanol/10% acetonitrile; 0.1% formic acid; 10mM ammonium formate |
| Preconditioning: | 4.5 mins B |
| Chromatography Type: | Reversed phase |
| Chromatography ID: | CH002764 |
| Chromatography Summary: | RP ESI- |
| Methods Filename: | AB_Human_Bone_Chromatography.pdf |
| Instrument Name: | Thermo Vanquish |
| Column Name: | Waters Acquity BEH Amide (150 x 2.1mm,1.7um) |
| Column Temperature: | 45°C |
| Flow Gradient: | 60% B at T0 hold for 1.5 min, linear increase to 85% B at 7 min, increase to 95% B at 12.5 min and hold for 4.5 min |
| Flow Rate: | 200 μL/min |
| Injection Temperature: | 300°C |
| Sample Injection: | 5μL |
| Solvent A: | 40% water/60 % acetonitrile; 0.1% ammonia; 10 mM ammonium formate |
| Solvent B: | 90% isopropanol/10% acetonitrile; 0.1% ammonia solution; 10mM ammonium formate |
| Preconditioning: | 4.5 mins B |
| Chromatography Type: | Reversed phase |
MS:
| MS ID: | MS003476 |
| Analysis ID: | AN003728 |
| Instrument Name: | Obitrap ID-X Tribrid |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | Mass spectrometry (MS) data were acquired using the AcquieX acquisition workflow (data dependent analysis). The MS operating parameters were as follows: MS1 mass resolution 60K, for MS2 30K, stepped energy (HCD) 20, 25, 50, scan range 100-1000, RF len (%) 35, AGC gain, intensity threshold 2e4, 25% custom injection mode with an injection time of 54 ms. An extraction blank was used to create a background exclusion list and a pooled QC was used to create the inclusion list. |
| Ion Mode: | POSITIVE |
| MS ID: | MS003477 |
| Analysis ID: | AN003729 |
| Instrument Name: | Obitrap ID-X Tribrid |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | Mass spectrometry (MS) data were acquired using the AcquieX acquisition workflow (data dependent analysis). The MS operating parameters were as follows: MS1 mass resolution 60K, for MS2 30K, stepped energy (HCD) 20, 25, 50, scan range 100-1000, RF len (%) 35, AGC gain, intensity threshold 2e4, 25% custom injection mode with an injection time of 54 ms. An extraction blank was used to create a background exclusion list and a pooled QC was used to create the inclusion list. |
| Ion Mode: | NEGATIVE |
| MS ID: | MS003478 |
| Analysis ID: | AN003730 |
| Instrument Name: | Obitrap ID-X Tribrid |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | Mass spectrometry (MS) data were acquired using the AcquieX acquisition workflow (data dependent analysis). The MS operating parameters were as follows: MS1 mass resolution 60K, for MS2 30K, stepped energy (HCD) 20, 25, 50, scan range 100-1000, RF len (%) 35, AGC gain, intensity threshold 2e4, 25% custom injection mode with an injection time of 54 ms. An extraction blank was used to create a background exclusion list and a pooled QC was used to create the inclusion list. |
| Ion Mode: | POSITIVE |
| MS ID: | MS003479 |
| Analysis ID: | AN003731 |
| Instrument Name: | Obitrap ID-X Tribrid |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | Mass spectrometry (MS) data were acquired using the AcquieX acquisition workflow (data dependent analysis). The MS operating parameters were as follows: MS1 mass resolution 60K, for MS2 30K, stepped energy (HCD) 20, 25, 50, scan range 100-1000, RF len (%) 35, AGC gain, intensity threshold 2e4, 25% custom injection mode with an injection time of 54 ms. An extraction blank was used to create a background exclusion list and a pooled QC was used to create the inclusion list. |
| Ion Mode: | NEGATIVE |
