S trap micro spin column

EV isolation was performed from 1 mL plasma with one centrifugation at 20,000×g for 30 min at 4 °C using an Avanti J-30i centrifuge with a J A-30.50 fixed-angle rotor with a k-factor 280 (Beckman Coulter, Brea, CA, USA). The supernatant from the initial spin of the 20 K pellet was used to prepare the 100 K pellet (100,000×g for 1 h at 4 °C). Succeeding the initial centrifugation step for each pellet preparation, the resultant EVs were washed in 1 mL phosphate-buffered saline filtered by a 0.22 µm filter. The final enriched 20 K (microvesicles; large EVs) and 100 K (exosomes; small EVs) samples were resuspended in 20 µL filtered phosphate-buffered saline prior to MS analysis. The samples were lysed and solubilized in 5% sodium dodecyl sulfate containing 50 mM triethylammonium bicarbonate, pH 7.55. Alkylation and tryptic digestion were performed using S-TrapTM Micro Spin Columns (Protifi, NY, USA) essentially as previously described [16 ]. Proteins were cleaved using PierceTM Trypsin protease, MS Grade (Thermo Fisher Scientific, Waltham, MA, USA) and peptide concentrations were measured by fluorescence using an EnSpire microplate reader (Perkin Elmer, Waltham, MA, USA). Samples were resuspended in 0.1% formic acid and injected with an amount of 1 µg in case of 20 K sample and 0.75 µg in case of 100 K sample.

One hundred micrograms of protein per sample was diluted in RIPA buffer and SDS to reach a final concentration of 5% SDS and a volume of 25 μL. Proteins were alkylated in 20 mM Iodoacetamide for 30 min and were digested using S‐Trap™ micro spin columns (Protifi) according to the manufacturer's instructions. Shortly, 12% phosphoric acid was added to each sample (final concentration of phosphoric acid 1.2%) followed by the addition of S‐trap buffer (90% methanol, 100 mM TEAB [pH 7.1]) at a ratio of 6:1. Samples were mixed by vortexing and loaded onto S‐trap columns by centrifugation at 4000g for 1 min followed by three washes with S‐trap buffer. Digestion buffer (50 mM TEAB [pH 8.0]) containing sequencing‐grade modified trypsin (1/25, w/w; Promega) was added to the S‐Trap column and incubated for 1 h at 47°C. Peptides were eluted by the consecutive addition and collection by centrifugation at 4000g for 1 min of 40 μL digestion buffer, 40 μL of 0.2% formic acid, and finally 35 μL 50% acetonitrile containing 0.2% formic acid. Samples were dried under vacuum and stored at −20°C until further use.

At the indicated timepoints for each infection, cell lysates were collected from 2 mL wells or 6 cm cell culture dishes by rinsing and scraping cells into cold PBS, pelleting at 500x g for 3 minutes in a tabletop centrifuge, and freezing cell pellets at -80 °C until analysis. Immediately preceding analysis, cell pellets were lysed in prewarmed 5% SDS lysis buffer containing 100 mM Tris-HCL (pH 7.4), 0.5 mM EDTA, and 100 mM NaCl. Protein concentration was determined by BCA assay (Pierce), and 30 µg of protein was prepared for MS analysis. Samples were reduced and alkylated (25 mM TCEP and 50 mM chloroacetamide) at 70 °C for 20 min, then acidified to 1.2% phosphoric acid prior to protein extraction. S-Trap Micro Spin Columns (Protifi) were used for protein extraction, trypsin digest (using a 1:25 ratio of trypsin to sample protein for 1 h at 47 °C), and sample desalting according to the manufacturer’s protocol, apart from performing 5 total wash steps prior to trypsinization. Other peptide preparation methods tested, such as methanol-chloroform extraction with overnight trypsin digestion, often resulted in loss of hydrophobic peptides common to MCS proteins. Peptides were resuspended in 1% formic acid + 1% acetonitrile at a concentration of 0.75 µg/µl prior to loading on instrument.

Proteins from cellular lysates were reduced with a solution of 20 mM tris (2-carboxyethyl)phosphine (TCEP) in 50 mM NH₄HCO₃ as previously described (Kulak et al., 2014 (link)) supplemented with 55 mM iodoacetamide in 50 mM NH₄HCO₃, then incubated for 10 min at 56°C followed by 10 min incubation at room temperature in the dark. The proteins were acidified with a final concentration of 1.2% phosphoric acid and diluted with 6 volumes of S-Trap buffer [90% Methanol, 100 mM triethylammonium bicarbonate (TEAB), pH 7.1] to aggregate proteins in colloidal particles. The samples were then transferred into S-Trap Micro Spin columns (Protifi), trapped in filter by two centrifugations at 4,000 × g for 1 min, washed twice with 150 μL of S-Trap buffer, and centrifuged at 4,000 × g for 1 min. Proteolysis was initiated with the addition into the S-Trap cartridge of 20 μL of 50 mM NH₄HCO₃ supplemented with 2 μg of trypsin and 0.01% ProteaseMAX detergent, followed by a 15 min or 60 min incubation at 50°C. Peptide elution was conducted with: (i) 40 μL TEAB 50 mM, (ii) 0.2% formic acid (HCOOH) in H₂O, and (iii) 35 μL of 50% acetonitrile (CH₃CN) and 0.2% formic acid final concentration, with centrifugations at 4,000 × g for 1 min between each elution buffer. Eluates were pooled, lyophilized in a speed vacuum and re-suspended in 10 μL of 50 mM NH₄HCO₃ buffer containing 0.5% TFA.

Gray matter tissue (~20 mg) from DLPFC and NAc were collected from fresh-frozen coronal tissue blocks via cryostat to minimize contamination from white matter and other subregions [29 (link), 30 (link)]. Homogenate and synaptosome preparations were obtained using a variation of our enrichment protocol for postmortem human brain tissues [24 (link), 31 (link), 32 ] with SynPER reagent (ThermoFisher). From each sample, 10 µg total protein (as measured by Micro BCA) was reduced, alkylated, and trypsin digested on S-Trap™ micro spin columns (ProtiFi). Subject pairs were randomly assigned to TMT blocks and labeled with TMTPro channels 1–10, with brain regions and preparations assigned to separate blocks [33 ]. Additional aliquots from each sample were used for a pooled control, digested separately with S-Trap Midi™ columns, divided then labeled with TMTPro channels 1 and 12. TMT labeled preparations from the same block were pooled with 10 µg of the labeled pooled controls. The TMT labeled peptide pools were separated into eight fractions with the Pierce™ High pH Reversed-Phase Peptide Fractionation Kit (ThermoFisher Scientific), evaporated, and reconstituted in 20 µl 97% H₂O, 3% ACN, 0.1% formic acid.