Protein extraction, streptavidin enrichment, and on-bead trypsin digestion

Total protein was extracted by sonication in 400 µl lysis buffer (1% sodium dodecyl sulfate (SDS), 125 mM triethylammonium bicarbonate (TEAB), 75 mM NaCl, and Halt^TM protease and phosphatase inhibitors). Lysates were cleared by centrifugation at 12,000 g for 15 min at 4 °C. Supernatant was transferred to a new tube and used for subsequent procedure. Total protein (1 µl of samples were diluted to 100 µl with water) was estimated using microBCA assay according to the manufacturer instructions (Cat. No. 23235, Thermo Fisher).

For the experiment in Figs. 2–3 (the proteome across three APEX constructs), 300 µg of brain lysates (in 200 µl) were reduced with 20 µl 200 mM dithiothreitol (DTT) for 1 h and alkylated with 60 µl 200 mM iodoacetamide (IAA) in the dark for 45 min at 37 °C with shaking. Streptavidin magnetic beads (300 µl) (Cat. No. 88816, Thermo Fisher) were prewashed with 1 ml no-SDS lysis buffer and incubated with 240 µl of reduced and alkylated lysates for 60 min at RT with shaking. Enriched beads were washed twice with 1 ml no-SDS lysis buffer, 1 ml 1 M KCl, and five times with 1 ml 100 mM TEAB buffer. Washed beads were digested with trypsin/LysC solution (~6 µg in 100 µl 100 mM TEAB) overnight at 37 °C. Digested supernatant was collected. Beads were rinsed with 50 µl 100 mM TEAB. Supernatant was combined. Trace amounts of magnetic beads were removed twice by magnetization and tube changes. 10 µl from each sample was saved for Pierce fluorometric peptide quantification assay (Cat. No. 23290, Thermo Fisher), and 16 µl was taken from each sample and was combined to make an average reference sample for TMT batch effect correction. Peptides were frozen and dried in a vacuum concentrator before TMT labeling.

For experiments in Figs. 4–5 (Drd1^Cre vs A2a^Cre, and chemogenetic activation/DREADDs), 300 µg of brain lysates (in 250 µl) were reduced with 20 µl 200 mM DTT for 1 h and alkylated with 60 µl 200 mM IAA 100 mM TEAB in the dark for 45 min at 37 °C with shaking. Streptavidin magnetic beads (150 µl) were prewashed with 1 ml no-SDS lysis buffer and incubated with 250 µl of reduced and alkylated lysates for 60 min at RT with shaking. Enriched beads were washed twice with 1 ml no-SDS lysis buffer, 1 ml 1 M KCl, and five times with 1 ml 100 mM TEAB buffer. Washed beads were digested with trypsin solution (~4 µg in 150 µl 100 mM TEAB) overnight at 37 °C. Digested supernatant was collected. Beads were rinsed with 50 µl 100 mM TEAB. Supernatants were combined. Trace amounts of magnetic beads were removed twice by magnetization and tube changes. 10 µl from each sample was saved for Pierce fluorometric peptide quantification assay. Peptides were frozen and dried in a vacuum concentrator before TMT labeling. To make reference channel samples for TMT batch correction in Fig. 5, a separate set of H2B-hM3Dq, H2B-mCherry, and H2B-alone samples were prepared and pooled to make two 900 µg samples which were enriched with 400 µl of streptavidin beads, followed by the same bead washing and tryptic digestion (~6 µg trypsin in 200 µl + 50 µl rinse). Eluted peptides were pooled and dried in a vacuum concentrator.