Cell harvest, differential centrifugation, and isobaric labeling

Cell harvest and subcellular fractionation were performed based on the LOPIT-DC differential ultracentrifugation protocol as described in Geladaki et al. ¹⁷. Briefly, AC16 cells were treated, harvested with trypsinization, washed 3× with room temperature PBS, and resuspended in a detergent free gentle lysis buffer (0.25 M sucrose, 10 mM HEPES pH 7.5, 2 mM magnesium acetate). 1.5 mL of suspension at a time was lysed using an Isobiotec ball-bearing homogenizer with a 16 µM clearance size until ~80% of cell membranes were lysed, as verified with trypan blue (approximately 15 passes through the chamber). Lysates were spun 3 times each in a 4 °C swinging bucket centrifuge 200 × g, 5 min to remove unlysed cells. The supernatant was retained and used to generate the 9 ultracentrifugation pellets using spin parameters shown in Supplementary Table ².

The supernatant generated in the final spin was removed and all pellets and the final supernatant were stored at –80 °C until proceeding. Supernatant was thawed on ice and precipitated in 3× the volume of cold acetone overnight at –20 °C. This was used to generate pellet 10 by centrifuging at 13,000 × g for 10 min at 4 °C. Excess acetone was removed and the pellet was allowed to dry briefly before resuspension in a resolubilization buffer of 8 M urea, 50 mM HEPES pH 8.5, and 0.5% SDS with 1x Halt Protease and Phosphatase Inhibitor Cocktail (Thermo Scientific). The suspension was sonicated in a Biorupter with settings 20× 30 s on 30 s off at 4 °C.

Pellets from the ultracentrifugation fractions 1 to 9 were resuspended in RIPA buffer with Halt Protease and Phosphatase Inhibitor Cocktail (Thermo Scientific) with sonication in a Biorupter with settings 10x 30 s on 30 s off at 4 °C. Insoluble debris was removed from all samples (1-10) by centrifugation at 14,000 × g, 5 min. The protein concentration of all samples was measured with Rapid Gold BCA (Thermo Scientific). The samples were digested and isobarically tagged using the iFASP protocol⁵⁶. A total of 25 ug protein per sample in 250 uL 8 M urea was loaded onto Pierce Protein Concentrators PES, 10 K MWCO prewashed with 100 mM TEAB. The samples were again washed with 8 M urea to denature proteins and remove SDS. The samples were washed with 300 uL 100 mM TEAB twice. The samples were then reduced and alkylated with TCEP and CAA for 30 min at 37 °C in the dark. CAA and TCEP were removed with centrifugation and the samples were washed 3x with 100 mM TEAB. Samples were digested atop the filters overnight at 37 °C with mass spectrometry grade trypsin (Promega) at a ratio of 1:50 enzyme:protein. A total of 0.2 mg of TMT-10plex isobaric labels (Thermo Scientific) per differential centrifugation fraction were equilibrated to room temperature and reconstituted in 20 µL LC-MS grade anhydrous acetonitrile. In each experiment, labels were randomly assigned to each fraction (Supplementary Table ³, Supplementary Table ⁴) with a random number generator to mitigate the possible batch effect. Isobaric tags were added to peptides still atop the centrifugation filters and incubated at room temperature for 1 h with shaking. The reactions were quenched with 1 µL 5% hydroxylamine at room temperature for 30 min with shaking. Labeled peptides were eluted from the filters with centrifugation. To further elute labeled peptides 40 µL 50 mM TEAB was added and filters were again centrifuged. All 10 labeled fractions per experiment were combined and mixed well before dividing each experiment into two aliquots. Aliquots were dried with speed-vac and stored at –80 °C.