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Mitochondrial OXPHOS efficiency and capacity
Last updated date: Jun 12, 2022 Views: 546 Forks: 0
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Acetyl-CoA and Acetyl-Carnitine analysis with LC-MS/MS
- Sample treatment (extraction)
1)Add 200ul of extraction solution (methanol: Water=4:1 (v:v), with internal standards, MalCoA_d3, Acetyl_Carnitine_d3) into the cell pellets.
2)Put cells into liquid nitrogen for 1min.
3)Thaw the samples in sonicate for 5min.
4) Repeat step 2 and 3 twice (3 times in total).
5) Centrifuge the samples at 13,000 rpm for 10 min at 4˚C.
6) Transfer 140 µL of the supernatant into auto-sampler vials for injection - LC-MS/MS analysis
1) LC/MS Assay
ACQUITY UPLC Conditions
Column | ZIC-pHILIC (150×2.1mm id, 5 μm, cat: sigma:1.50460.0001 | ||||
Column Temp. | 35 °C | ||||
Auto sampler Temp. | 10 °C | ||||
Mobile Phase A | 10% ACN, 20mM Ammonium bicarbonate (pH9.2) with 5uM PBS | ||||
Mobile Phase B | ACN | ||||
Injection Volume | 5 µL (partial Loop) | ||||
Gradient | Time (min) | Flow Rate (mL/min) | % A | % B | Curve |
Initial | 0.3 | 5 | 95 | Initial | |
0.5 | 0.3 | 5 | 95 | 6 | |
11 | 0.3 | 50 | 50 | 6 | |
12.5 | 0.3 | 50 | 50 | 6 | |
13.5 | 0.3 | 75 | 25 | 6 | |
14.5 | 0.3 | 75 | 25 | 6 | |
15 | 0.3 | 5 | 95 | 6 | |
20 | 0.3 | 5 | 95 | 6 | |
Sciex 6500+ MS Settings
1) MS Parameters in the method
| Positive | Negative |
Curtain Gas (CUR) | 20 | 20 |
Collision Gas (CAD) | High | High |
IonSpray Voltage (IS) | 5500 V | -4500 V |
Temperature (TMP) | 550 | 550 |
Ion Source Gas 1 (IS1) | 40 | 40 |
Ion Source Gas 2 (IS2) | 40 | 40 |
Declustering Potential (DP) | 60 | -60 |
Entrance Potential (EP) | 10 | -10 |
Collision Cell Exit Potential (EP) | 10 | -10 |
2) MRM Transitions Parameters (ES+, and ES-)
| Q1 | Q3 | CE | ID |
| 810.2 | 303.2 | 40 | AcetylCOA |
| 857 | 350 | 42 | MalCOA_d3_IS |
| 207 | 85 | 26.52 | Carnitine_C2_d3_IS |
| 204 | 85 | 23 | Carnitine_C2 |
- Pessin, J E and Tang, Y(2022). Mitochondrial OXPHOS efficiency and capacity. Bio-protocol Preprint. bio-protocol.org/prep1715.
- Tang, Y., Zong, H., Kwon, H., Qiu, Y., Pessin, J. B., Wu, L., Buddo, K. A., Boykov, I., Schmidt, C. A., Lin, C., Neufer, P. D., Schwartz, G. J., Kurland, I. J. and Pessin, J.(2022). TIGAR deficiency enhances skeletal muscle thermogenesis by increasing neuromuscular junction cholinergic signaling. eLife. DOI: 10.7554/eLife.73360
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