Lys c

Isolated mitochondria were lysed in Buffer D (8 M urea in 40 mM Tris, 30 mM NaCl, 1 mM CaCl₂, 1 × cOmplete ULTRA mini EDTA-free protease inhibitor tablet; pH = 8.0), as described previously^{37 (link)}. The samples were subjected to three freeze–thaw cycles, and sonication with a probe sonicator in three 5 s bursts (Q Sonica #CL-188; amplitude of 30). Samples were then centrifuged at 10,000 × g for 10 min at 4 °C. Protein concentration was determined by BCA protein assay. Equal amounts of protein were reduced with 5 mM DTT at 37 °C for 30 min, and then alkylated with 15 mM iodoacetamide at room temperature for 30 min in the dark. Unreacted iodoacetamide was quenched with DTT up to 15 mM. Initial digestion was performed with Lys C (ThermoFisher Cat# 90,307; 1:100 w:w; 2 µg enzyme per 200 µg protein) for 4 h at 37 °C. Following dilution to 1.5 M urea with 40 mM Tris (pH = 8.0), 30 mM NaCl, 1 mM CaCl₂, samples were digested overnight with trypsin (Promega; Cat# V5113; 50:1 w/w, protein:enzyme) at 37 °C. Samples were acidified to 0.5% TFA and then centrifuged at 4000×g for 10 min at 4 °C. Supernatant containing soluble peptides was desalted, as described previously^{37 (link)} and then eluate was frozen and lyophilized.

Lysis of spheroids from 3D culture (30 per each individual ATGL-KO clone [n = 3] or control clone [n = 3] of A549 cells) as well as of spheroids-derived small tumors grown on CAM was carried out in lysis buffer (100 mM Tris pH = 8, 1% sodium dodecyl sulphate, 10 mM tris(2-carboxyethyl) phosphine, 40 mM chloroacetamide), followed by several sonication cycles. Protein content was estimated using bicinchoninic acid assay (Thermo Fisher Scientific) after which 100 μg of protein per each sample was acetone precipitated overnight. The following day, protein pellets were re-dissolved in 25% trifluoroethanol (in 100 mM Tris pH = 8.5), diluted to 10% trifluoroethanol with ammonium bicarbonate, and digested for 2 h with LysC (Thermo Fisher Scientific) then overnight with trypsin (Thermo Fisher Scientific). Finally, samples were diluted in running buffer A (0.1% formic acid, 5% acetonitrile; running buffer B: pure acetonitrile containing 0.1% formic acid), and 1 μg of sample was injected for LC-MS/MS analysis.

Thirty microliters of soluble and pellet fractions from four separate biological replicates was normalized to 50 μl with 8M urea buffer. Next, 10 mM Dithiothreitol (DTT) in 50 mM ammonium bicarbonate (ABC) was added to a final concentration of 1 mM and incubated for 30 min at room temperature (RT), then 50 mM iodoacetamide (IAA) in 50 mM ABC was added to a final concentration of 5 mM and incubated in the dark for 30 min at RT. Samples were then digested overnight with 1 μg of Lys-C (Wako 121-05063). Following Lys-C digestion, samples were diluted to 1M urea and digested overnight with 1 μg of Trypsin (Thermo Fisher 90057). The next day samples were incubated with acidifying buffer [10% Formic Acid (FA), 1% trifluoroacetic acid (TFA)] and centrifuged for 2 min. Sample pH was verified as less than 3 using pH strips. Samples were desalted on an Oasis PRIME HLB 10 mg plate (Oasis 186008053) and washes were flowed through columns by a 96-well Positive Pressure processor (Waters 186006961). Samples were washed first with methanol, then Buffer A (0.1% TFA). The digested samples were then loaded onto Oasis PRIME HLB 10 mg plates (Oasis 186008053), washed with Buffer A twice, and then peptides were eluted with Buffer C [50% acetonitrile (ACN), 0.1% FA]. The elutant was lyophilized using a SpeedVac (Labconco 731022).