Sep pak c18 column

The packed cell pellets (1×10⁷ cells) were lysed in 1 μg Rapigest SF Surfactant (Waters), in 50 mM AmBic and homogenized by sonication. Cleared lysates were heated (95°C, 15 min), diluted in 50 mM AmBic to a final concentration of 0.2% Rapigest and sonicated again followed by reduction (10 mM DTT, 60°C, 45 min), alkylation (20 mM iodoacetamide, in the dark at RT for 30 min), reduction (10 mM DTT, RT, 20 min) and digested with 25U trypsin (Roche) in 50mM AmBic (37°C, 12 h, 650 rpm). Each tryptic digest was acidified with TFA and incubated at 37°C for 30 min before centrifuging (max speed, RT, 15 min). Sep-Pak C18 columns (Waters) where washed with 100% methanol, 50% methanol 0.1% FA and equilibrated with 0.1% trifluoroacetic acid (TFA) before loading the supernatants containing the tryptic digests. After reloading the flow through, the column was washed with 0.1% TFA, twice with 0.1% FA and eluted twice with 50% methanol 0.1% FA. 10 ug of Freeze-dried samples were resuspended in 50 mM AmBic with 8U PNGaseF (Roche) and incubated at 37°C for 16 h. The digest was applied to Sep-pak C18 column, as described above, and flow through containing the released N-glycans was collected for further labeling with Rapifluor-MS glycan kit (Waters) as described by manufacturers.

The samples were lysed in lysis buffer containing 4% SDS, 100 mM DTT and 100 mM Tris pH 7.5. Tissues were homogenized and sonicated in lysis buffer. The protein concentration of the cleared lysates was estimated using BCA assay and equal amounts of protein from each donor was pooled for further fractionation. Proteins from SDS lysates were separated on SDS-PAGE and in-gel digestion was carried out as described earlier^{37 (link)}. Briefly, the protein bands were destained, reduced and alkylated and subjected to in-gel digestion using trypsin. The peptides were extracted, vacuum dried and stored at −80°C until further analysis. The samples (~450 µg proteins) were reduced, alkylated and digested using trypsin overnight at 37°C. The peptide digests were desalted using Sep-Pak C₁₈ columns (Waters Corporation, Milford, MA) and lyophilized. The lyophilized samples were reconstituted in solvent A (10 mM tetraethylammonium bicarbonate, pH 8.5) and loaded onto XBridge C₁₈, 5 µm 250 × 4.6 mm column (Waters, Milford, MA, USA). The digests were resolved by bRPLC^{38 (link)} method using a gradient of 0 to 100% solvent B (10 mM tetraethylammonium bicarbonate in acetonitrile, pH 8.5) in 50 minutes. The total fractionation time was 60 minutes. A total of 96 fractions were collected which were then concatenated to 24 fractions, vacuum dried and stored at −80 until further LC-MS analysis.

HEK293T cells were plated on 10 cm tissue-culture dishes at 80% confluency and transfected with 10 μg of total DNA per plate (5 μg PM-Cib; 5 μg optoPKA^W196R/F327A or Cry2-mCh) using Lipofectamine 2000 transfection reagent (32 μL Lipofectamine reagent:1 mL Opti-MEM) in Opti-MEM with 10% FBS. 4 h post-transfection, cell media was changed to DMEM + 10% FBS, and the cells were stored overnight, protected from light, in a humidified tissue culture incubator (37 °C; 5% CO₂). The next day, cells were serum starved in PBS for 15 min in a humidified tissue culture incubator (37 °C; 5% CO₂), and exposed to light (4 min; 470 nm LED flood lamp; continuous illumination) or kept in the dark (4 min). Cells were then lysed using 2 mL of M-PER + 1x HALT protease and phosphatase inhibitor per plate. Lysates were cleared by centrifugation (5 min; 1000 x g; 4 °C), and concentrations determi ned via the Bradford assay. The lysates were acetone precipitated, reconstituted in 7 M urea, reduced and alkylated, and then digested with trypsin overnight. The peptide samples were desalted using SepPak C18 columns (Waters) and then enriched for phosphopeptides with TiO₂ tips (GL Sciences). TiO₂ eluates were dried via vacuum centrifugation and then stored at −80 °C until analysis.