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MB Sample ID: SA288732
| Local Sample ID: | RJ_10QQ1 |
| Subject ID: | SU002843 |
| Subject Type: | Human |
| Subject Species: | Homo sapiens |
| Taxonomy ID: | 9606 |
| Gender: | Male and female |
Select appropriate tab below to view additional metadata details:
Subject:
| Subject ID: | SU002843 |
| Subject Type: | Human |
| Subject Species: | Homo sapiens |
| Taxonomy ID: | 9606 |
| Gender: | Male and female |
Factors:
| Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
|---|---|---|---|---|
| RJ_10QQ1 | SA288732 | FL035605 | Remote | Treatment |
Collection:
| Collection ID: | CO002836 |
| Collection Summary: | LV tissue from patients undergoing CABG was sampled from two or more pre-determined areas (as guided by CMR) on the beating heart during surgery: 1) a region of viable myocardium with inducible ischemia [‘ischemic’ biopsy]; 2) a region of remote myocardium with normal contractility, no qualitative evidence of inducible hypoperfusion on perfusion imaging, and without infarct pattern LGE [‘remote’ biopsy]. The a priori plan was to acquire paired biopsies, permitting high energy phosphate (HEP) quantification and LC-MS for each individual patient. Surgical discretion, however, resulted in some patients not undergoing paired tissue collection. Conversely, where deemed safe, extra tissue was acquired for single-nuclei RNA sequencing. The CABG biopsies were performed on the beating heart (prior to aortic cross-clamp, cardioplegia and hypothermia in the cases using cardiopulmonary bypass) with a Tru-Cut® needle or scalpel by the consultant cardiac surgeon. The majority of operations were performed off-pump without cardiopulmonary bypass. Samples were immediately clamp-frozen in theatre using a Wollenberg clamp (manufactured by Josh Firman, LMB Workshop, UK) which had been pre-cooled in liquid nitrogen until tissue acquisition. The clamps were then reopened, and the tissue rapidly transferred into Eppendorf tubes (pre-cooled in dry ice) before storage at -80ºC awaiting further analysis. |
| Collection Protocol Filename: | Protocol_AMBITION.pdf |
| Sample Type: | Heart |
Treatment:
| Treatment ID: | TR002852 |
| Treatment Summary: | 'Ischemic' or 'remote' biopsy as determined by preoperative stress perfusion cardiovascular magnetic resonance. |
Sample Preparation:
| Sampleprep ID: | SP002849 |
| Sampleprep Summary: | Frozen LV tissue samples (~1-5mg) were weighed into Precellys tubes (Stretton Scientific Ltd., Derbyshire, UK), and an exact volume of extraction solution (50% methanol, 30% acetonitrile and 20% water) was added to obtain 40 mg specimen per ml of extraction solution, permitting comparisons between experimental conditions for the same metabolite. The samples were subsequently lysed after the addition of 3 ceramic beads using a Precellys 24 tissue homogenizer (Bertin Corp, Rockville, MD 20850, USA. 5500 r.p.m for 15 seconds × 2) and finally centrifuged (16,162 × g for 10 min at 4 °C). The supernatant was transferred into glass vials (Microsolv Technology Corp., Leland, NC 28451, USA) and stored at −80 °C until LC–MS analysis. |
Combined analysis:
| Analysis ID | AN004438 |
|---|---|
| Analysis type | MS |
| Chromatography type | HILIC |
| Chromatography system | Thermo Dionex Ultimate 3000 |
| Column | SeQuant ZIC- pHILIC (150 x 2.1mm,5um) |
| MS Type | ESI |
| MS instrument type | Orbitrap |
| MS instrument name | Thermo Q Exactive Orbitrap |
| Ion Mode | UNSPECIFIED |
| Units | peak area |
Chromatography:
| Chromatography ID: | CH003334 |
| Chromatography Summary: | HILIC chromatographic separation of metabolites was achieved using a Millipore Sequant ZIC-pHILIC analytical column (5 µm, 2.1 × 150 mm) equipped with a 2.1 × 20 mm guard column (both 5 mm particle size) with a binary solvent system. Solvent A was 20 mM ammonium carbonate, 0.05% ammonium hydroxide; Solvent B was acetonitrile. The column oven and autosampler tray were kept at 40 °C and 4 °C, respectively. The chromatographic gradient was run at a flow rate of 0.200 mL/min as follows: 0–2 min: 80% B; 2-17 min: linear gradient from 80% B to 20% B; 17-17.1 min: linear gradient from 20% B to 80% B; 17.1-22.5 min: hold at 80% B. Samples were randomized and analysed with LC–MS in a blinded manner with an injection volume was 5 µl. Pooled samples were generated from an equal mixture of all individual samples and analysed interspersed, at regular intervals, within the sample sequence as a quality control. Each sample was analysed with three analytical replicates. |
| Instrument Name: | Thermo Dionex Ultimate 3000 |
| Column Name: | SeQuant ZIC- pHILIC (150 x 2.1mm,5um) |
| Column Temperature: | 40 |
| Flow Gradient: | 0–2 min: 80% B; 2-17 min: linear gradient from 80% B to 20% B; 17-17.1 min: linear gradient from 20% B to 80% B; 17.1-22.5 min: hold at 80% B |
| Flow Rate: | 0.200 mL/min |
| Solvent A: | 20 mM ammonium carbonate, 0.05% ammonium hydroxide |
| Solvent B: | acetonitrile |
| Chromatography Type: | HILIC |
MS:
| MS ID: | MS004185 |
| Analysis ID: | AN004438 |
| Instrument Name: | Thermo Q Exactive Orbitrap |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | Metabolites were measured using a Thermo Scientific Q Exactive Hybrid Quadrupole-Orbitrap Mass spectrometer (HRMS) coupled to a Dionex Ultimate 3000 UHPLC. The mass spectrometer was operated in full-scan, polarity-switching mode, with the spray voltage set to +4.5 kV/-3.5 kV, the heated capillary held at 320 °C, and the auxiliary gas heater kept at 280 °C. The sheath gas flow was programmed to 55 units, the auxiliary gas flow was programmed to 15 units, and the sweep gas flow was programmed to 0 unit. HRMS data acquisition was performed in a range of m/z = 70–900, with the resolution set at 70,000, the AGC target at 1 × 106, and the maximum injection time (Max IT) at 120 ms. Metabolite identities were confirmed using two parameters: (1) precursor ion m/z was matched within 5 ppm of theoretical mass predicted by the chemical formula; (2) the retention time of metabolites was within 5% of the retention time of a purified standard run with the same chromatographic method. Each sample underwent 3 analytical repeats with subsequent peak annotation and chromatogram review and peak area integration were performed using the Thermo Fisher software Tracefinder 5.0. The peak area for each detected metabolite was subjected to the “Filtering 80% Rule”, half minimum missing value imputation, and normalized against the total ion count (TIC) to correct any variations introduced from sample handling through instrument analysis. Samples were excluded after performing testing for outliers based on geometric distances of each point in the PCA score analysis as part of the muma package (v.1.4) |
| Ion Mode: | UNSPECIFIED |
