Mcx cartridges

As described in Searle et al.,^{13 (link)}S. cerevisiae strain BY4741 (Dharmacon) was cultured at 30 °C in YEPD and harvested at the mid-log phase. Cells were pelleted and lysed in a buffer of 8 M urea, 50 mM Tris (pH 8), 75 mM NaCl, 1 mM EDTA (pH 8) followed by seven cycles of 4 min bead beating with glass beads. After a 1 min rest on ice, lysate was collected by piercing the tube and centrifuging for 1 min at 3000 × g and 4 °C into an empty eppendorf. After further centrifugation at 21,000 × g and 4 °C for 15 min, the protein content of the supernatant was removed and estimated using BCA. Proteins were then reduced with 5 mM dithiothreitol for 30 min at 55 °C, alkylated with 10 mM iodoacetamide in the dark for 30 min at room temperature, and diluted to 1.8 M urea, before digestion with sequencing-grade trypsin (Pierce) at a 1:50 enzyme-to-substrate ratio for 16 h at 37 °C. In all, 5 N HCl was added to approximately pH 2 to quench the digestion, and the resulting peptides were desalted with 30 mg MCX cartridges (Waters). Peptides were dried with vacuum centrifugation and brought to 1 μg/3 μl in 0.1% formic acid (buffer A) prior to MS acquisition. All measurements of yeast were performed on the same biological replicate to assess technical variability in the method.

Yeast strain BY4741 (Dharmacon) was cultured at 30 °C in YEPD and harvested at mid-log phase. Cell pellets were lysed in a buffer of 8 M urea, 50 mM Tris (pH 8), 75 mM NaCl, 1 mM EDTA (pH 8) using 7 cycles of 4 min bead beating with glass beads followed by one minute rest on ice. Lysate was collected by piercing the tube, placing it into an empty eppendorf, and centrifuging for 1 min at 3000 × g and 4 C. Insoluble material was removed from the lysate by 15 min centrifugation at 21,000 × g and 4 C. The protein content of the supernatant was estimated using BCA. The proteins were reduced with 5 mM dithiothreitol for 30 min at 55 °C and alkylated with 10 mM iodoacetamide in the dark for 30 min at room temperature. The proteins were diluted to 1.8 M urea and then digested with sequencing grade trypsin (Pierce) at a 1:50 enzyme to substrate ratio for 16 h at 37 °C. The digestion was quenched using 5 N HCl to achieve approximately pH 2. Resulting peptides were desalted with 30 mg MCX cartridges (Waters) and dried with vacuum centrifugation. Peptides were brought to 1 μg / 3 μl in 0.1% formic acid (buffer A) prior to mass spectrometry acquisition.