Tissue extraction of MitoA and MitoN

The protocol for extraction of MitoA and MitoN is based on that of Arndt et al. (2017) for mammalian tissue with some differences outlined below for P. mexicana. Frozen tissues were first weighed into chilled 2 ml Eppendorf tubes (in mg in part 1: gill 60±10, liver 21±3, muscle 93±10, brain 17±2.0; in part 2: gill 16±2.3, liver 12±1.7, muscle 35±6.1, brain 9.8±0.72). If available, more tissue (up to 100 mg) can be used for tissues that show lower uptake of MitoA to increase sensitivity. 210 µl 60% acetonitrile spiked with internal standards (100 pmol d₁₅-MitoA and 100 pmol of d₁₅-MitoN) was added to each sample. All chemicals for extraction and mass spectrometry analysis were of HPLC grade. P. mexicana tissue was homogenized using a Bullet Blender (Storm24; Next Advance) in 95% acetonitrile, which was used for mouse samples (Arndt et al., 2017), but yielded a viscous homogenate that was difficult to process (G.Y.L., personal observation). Instead, P. mexicana samples were homogenized using ∼50 mg beads (0.5 mm diameter zirconium oxide beads; Next Advance) with a Bullet Blender for 3 min at speed 8. Gill samples were homogenized for an extra minute at speed 9. The samples were then centrifuged at 16,000 g for 10 min after which the supernatant was transferred to a new tube. The tissue and beads were re-homogenized with another 200 µl 60% acetonitrile and re-centrifuged, the supernatants were combined and allowed to stand for 30 min at 4°C to precipitate proteins. The samples were then centrifuged at 16,000 g for 10 min to pellet the precipitates. The supernatants were loaded onto 96-well Millipore Multiscreen filter plates with low protein binding Durapore membranes (0.45 µm pore size) and centrifuged for 10 min at 1108 g. The filtrate was then transferred into fresh Eppendorf tubes and dried using a speed vacuum centrifuge (CentriVap benchtop vacuum concentrator, Labconco). Samples were stored dried at 20°C until analysis by LC-MS/MS. To prepare for LC-MS/MS analysis, the samples were resuspended in 20% acetonitrile with 0.1% formic acid, centrifuged for 10 min at 16,000 g and transferred to mass spectrometry vials (TrueView™ LCMS Certified, Waters).