Sep pak tc18 cartridge

Proteins were reduced with dithiothreitol (DTT) (5 mM) for 1h at 37°C and subsequently alkylated with iodoacetamide IAA (10 mM) in the dark at room temperature for 30 min. Alkylation was stopped by adding DTT at a final concentration of 5 mM. Lysates were diluted with 50 mM ABC to a final urea concentration below 4 M. Proteins first were digested with Lys-C (Wako) at a 1:150 enzyme/protein ratio for 4 h at 37°C and then trypsin (1:80; Promega) was added followed by an overnight incubation at 37°C. Digestion was stopped by acidification with TFA to a final concentration of 1%. Peptide solution was desalted using Sep-Pak tC18 cartridges (Waters) according to the manufacturer’s protocol.
Dried peptides were dissolved in SCX Buffer A (7 mM KH₂PO₄, pH 2.65, 30% ACN) and separated by strong cation exchange (SCX) using an Agilent 1200 HPLC equipped with a PolySULFOETHYL A column (250 mm × 9.4 mm; 5 μm beads, pore size 200Å) (259-SE0502, PolyLC Inc.). Chromatography was performed by increasing SCX Buffer B concentration (7 mM KH₂PO₄, 350 mM KCl, pH 2.65, 30% ACN) from 1–30% over 40 min at a flow rate of 2 ml/min. Twelve 5-min fractions were collected over the full run, lyophilized and subsequently desalted using Sep-Pak tC18 cartridges (Waters). Fractions 1/2, 3/4, and 11/12 were pooled for phosphopeptide enrichment resulting in a total of 9 fractions.

Cells were lysed in ten volumes of modified SDT buffer (0.1 M Tris-HCL, pH 7.6, 0.1 M DTT, 1% SDS, 1% SDC) and incubated at 95 °C for 5 min. The lysate was sonicated to shear genomic DNA, and clarified by centrifugation at 20,000 × g for 15 min at 20 °C. The supernatant was transferred to ultrafiltration units (Millipore, Amicon Ultra 15 Ultracel 10 KD) and centrifuged at 4000 × g for 40 min. After centrifugation, the concentrates were mixed with 2 mL of 50 mM iodoacetamide in UA solution (8 M urea, 100 mM Tris-HCl pH 8.5) and incubated in darkness at room temperature (RT) for 30 min followed by centrifugation for 30 min. After alkylation, the filter units were washed four times with 10 ml UA buffer and two times 10 mL of 50 mM ammonium bicarbonate by centrifugation at 4000 × g. Proteins were then digested with Lys-C (1:100, w/w, Wako) for 6 h at 37 °C and trypsin (1:50, w/w, Promega) overnight at 37 °C. The resulting peptide mixture was acidified (pH 2.0) with formic acid, loaded onto Sep-Pak tC18 cartridges (Waters), desalted and eluted with 70% acetonitrile. The eluted peptides were lyophilized and stored at −80 °C before analysis.

Lysates were chloroform-methanol precipitated (26 (link)) and resuspended in 8 m urea, 100 mm Tris-HCl pH 8.0, 1 mm CaCl₂. After dilution to 1 m urea with 100 mm Tris-HCl, pH 8.0, 1 mm CaCl_2, the lysate was digested with trypsin at an enzyme:protein ratio of 1:50 (Promega). The peptides were then desalted 500 mg SepPak tC18 cartridges (Waters), dried in a centrifugal evaporator and resuspended in 50 mm borate, pH 9.3. NaOH was added to ensure pH was above pH 9.0 in samples prior to separation. Peptides were separated with an AS24 strong anion exchange column (Thermo Fisher Scientific) using an Ultimate 3000 UHPLC (Thermo Fisher Scientific) as described previously, with some modifications (27 (link)). Briefly, buffer A was 10 mm sodium borate-NaOH, pH 9.3, and buffer B was 10 mm sodium borate-NaOH, pH 9.3, 0.5 m sodium chloride. Peptides were eluted using an exponential gradient into 16 × 560 μl fractions. The peptide fractions were desalted using Sep-Pak 10 mg tC18 μ-Elution 96-well plates (Waters) and then resuspended in 5% formic acid for LC-MS/MS analysis.