Phosphoproteomic Effects of Acute Depletion of PP2A Regulatory Subunit Cdc55
- Organism: Saccharomyces cerevisiae
- Instrument: Orbitrap Fusion
targeted MS; PRM; protein phosphatase; PP2A; degron
- Lab head:
Protein phosphatase regulatory subunits are increasingly recognized as promising drug targets. In the absence of an existing drug, inducible degradation provides a means of predicting candidate targets. We here employed auxin-inducible degradation of S. cerevisiae PP2A regulatory subunit Cdc55 in combination with quantitative phosphoproteomics. A prevalence of hyperphosphorylated phosphopeptides and enrichment of proteins containing the PP2A consensus sequence indicates that the approach successfully identified direct PP2A-Cdc55 targets. PRM follow up of DDA-results confirmed that vacuolar amino acid transporters are among the proteins most strongly affected by Cdc55 depletion.
Yeast pellets from 50 ml cultures were lysed with 500 µl 0.5 mm glass beads in 0.5 ml MS-compatible lysis buffer (8 M urea, 100 mM ammonium bicarbonate (ABC), 5 mM EDTA, pH = 8.0) by 5 rounds of bead beating (45 s each with 5 min pause after 3 rounds and 45 s pause after other round; 6500 rpm; 4°C). Holes were punched into the bottom of the tubes with a hot 20 G needle and debris spun into a new tube at 1000 rpm for 1 min. The protein concentration was determined on cleared lysate using a BCA-assay (Pierce).
Lysates were diluted to 3 mg/ml and 2 mg protein subjected to in-solution digestion. They were incubated with 5 mM Tris(2-carboxyethyl)phosphine (TCEP) and 5 mM iodoacetamide (IAA) simultaneously for 30 min at room temperature (RT). Samples were diluted to an urea concentration of 5.5 M with 50 mM ABC and digested with LysC protease (Wako) at a 1:100 w/w protein ratio for 3 hours at 37°C. They were further diluted to 1 M urea before incubation with trypsin at a 1:100 w/w protein ratio o/n at 37°C. The digestion reaction was quenched by adding 10% tri-fluoroacetic acid (TFA) to a final of 2% v/v and debris cleared by centrifugation.
Samples were desalted on SepPak Plus cartridges (Waters) using a vacuum manifold in aliquots corresponding to 1 mg protein: Briefly, cartridges were conditioned with 65% acetonitrile (MeCN), 0.1% TFA, equilibrated in 2% MeCN, 0.1% TFA, samples loaded into an additional aliquot of equilibration solution and pulled slowly into the cartridges. After washing with the same solution, peptides were eluted in 2x 600 µl 65% MeCN, 0.1% TFA and lyophilized to dryness.
PRM acquisition and data analysis
Phospho-peptides with a p-value of less than 0.01 in DDA were selected for targeted acquisition using parallel reaction monitoring (PRM):
Three scheduled isolation lists were created in Skyline , specifying the appropriate medium and heavy label. The appropriate charge state was selected manually as detected in the DDA experiment. Each peptide was acquired in a 6 min retention-time window.
Samples were acquired using the same instrument and chromatographic conditions as for DDA. MS scans were acquired at 30 000 resolution and AGC-target of 400 000 (scan range 250-1800). For targeted MS/MS-scans, precursors were isolated with a quadrupole-isolation window of 1.6 Th and HCD-fragmented with 30% normalized collision energy. Fragment spectra were acquired in the Orbitrap at 30 000 resolution with an AGC-target of 50 000 and maximum fill time of 54 ms.
Data were analysed separately for runs with each of the inclusion lists. A spectral library based on the .dat file of a Mascot search of the acquired data was created in Skyline. Fragment traces of y-ions from the heavy and medium labelled precursor of each detectable phosphopeptide in the inclusion lists were extracted. Integration boundaries were adjusted manually where necessary and peaks that did not fall completely within the retention time window were removed. In case of alternative phospho-isoforms, the one with the higher dotp was retained. Ratios were calculated and log2-transformed and two-sided Student t-tests against null-hypothesis of a mean equal to zero performed.
Yeast cultures, SILAC-labelling, treatment and harvest
For stable isotope labelling with amino acids in cell culture (SILAC) mass spectrometry (MS) experiments, four yeast starter cultures were inoculated in synthetic complete dextrose (SCD) medium and grow over night (o/n) at 30oC, shaking at 120 rpm and the next morning diluted to an optical density (OD) of 0.05. In the evening, starter cultures were diluted into 70 ml SCD medium in which lysine and arginine had either been replaced with 220.127.116.11-D4-L-Lysine and L-Arginine 13C6 (‘medium’) or L-Lysine 13C6 15N2 and L-Arginine 13C6 15N4 (‘heavy’) (Silantes) to reach an OD of 0.6-0.8 the next morning. At this OD two heavy and two medium cultures were treated with 100 µM indol-acetic-acid (auxin; in ethanol), while the corresponding medium or heavy cultures were treated with the corresponding volume of ethanol.
After 10 min, 10 ml and 50 ml aliquots of each culture was harvested into 100% w/v trichloro-acetic acid (TCA) to a final of 3.5% v/v and incubated on ice for at least 10 minutes. Cells were spun down and washed twice in cold acetone.
Created on 6/2/20, 8:17 PM