SILAC-based phosphoproteomics reveals new PP2A-Cdc55-regulated processes in budding yeast.
Protein phosphatase 2A (PP2A) is a family of conserved serine/threonine phosphatases involved in several essential aspects of cell growth and proliferation. PP2ACdc55 phosphatase has been extensively related to cell cycle events in budding yeast, however few PP2ACdc55 substrates have been identified. Here, we performed a quantitative mass spectrometry approach to reveal new substrates of PP2ACdc55 phosphatase and new PP2A related processes in mitotic arrested cells. We identified 626 potential PP2ACdc55 substrates involved in a broad range of mitotic processes. As expected, several hyperphosphorylated proteins corresponded to Cdk1-dependent substrates, although other kinases’ consensus motifs were also enriched in our dataset, suggesting that PP2ACdc55 counteracts and regulates other kinases different than Cdk1. Indeed, Pkc1 and Cla4 kinases emerged as novel nodes of PP2ACdc55 regulation, highlighting a major role of PP2ACdc55 in membrane trafficking and cytokinesis, gene ontology terms significantly enriched in the PP2ACdc55-dependent phosphoproteome. In addition, we validated two new PP2ACdc55 substrates involved in early and late anaphase pathways, Slk19 and Lte1; and we also validated Zeo1, and other potential substrates, through protein interaction experiments. Finally, we performed docking models of Cdc55 and its substrate Mob1. We found that the predominant interface on Cdc55 is mediated by a protruding loop consisting of residues 84-90, thus highlighting the relevance of these aminoacids for substrate interaction.
Sample Processing Protocol
SILAC labelling, protein extraction and digestion Cells were grown in minimum media containing either 100 mg/L arginine and 100 mg/L lysine or 100 mg/L 13C6-arginine and 100 mg/L 13C6-lysine. Y859 (control) and Y858 (cdc55Δ) cells were grown in free-methionine minimum media containing 13C6-lysine and -arginine (heavy) or unmodified arginine and lysine (light), respectively. Both strains were synchronized at the metaphase-to-anaphase transition by adding methionine to the media. Protein extracts were prepared by mechanical lysis using glass beads in presence of protein inhibitors and 2X phosphatase inhibitors PhosStop. Cell lysates were mixed 1:1 and digestion with trypsin was performed. 400 μg of the mixed heavy/light protein sample were processed for in-solution digestion. Proteins were reduced (5 mM DTT; 30 min; 37ºC) and alkylated (10 mM IAA; 30 min; 30ºC). Samples were diluted five times with 25 mM ammonium bicarbonate, trypsin (ratio enzyme:protein 1:10) was added and incubated overnight at 37ºC. Digestion was stopped by addition of FA. Phosphopeptide enrichment Three strategies were used. In the first approach (SIMAC) peptides samples were added to an immobilized metal affinity chromatography (1h, RT). The flow-through was collected. The resin was washed once with 50μl 50% ACN and 0.1% TFA. The wash fraction was pooled with the flow-through. Elution was carried out by adding 50μl 30% ACN and 1% TFA (5 min; RT). Then, alkaline elution was done (50μl 0.5% NH4OH pH 10.5), followed by 30 min incubation at RT. For further enrichment, the flow-through fraction and the acid eluate were incubated with TiO2 beads and incubated with shaking for 1h at 30 °C. The TiO2 beads were washed twice with 80% ACN and 1% TFA and once with water. Bound peptides were eluted from the beads with 0.5% NH4OH pH 10.5 for 30 min at 30 °C. Eluted peptides were dried via centrifugal evaporation, resuspended with 1μl FA and 15μl water and analyzed using nano-LC-MS/MS on an LTQ-Orbitrap Velos mass spectrometer. In the second strategy, phosphopeptide enrichment was done using TiO2 chromatography. 100 μg were separated to be further analyzed without phosphopeptide enrichment. Both enriched and non-enriched samples were dried via centrifugal evaporation and subjected to fractionation with a high pH reversed phase peptide fractionation kit. The peptides were eluted in 9 fractions (ACN from 5% to 75%). The 9 fractions were dried via centrifugal evaporation, resuspended in 1% FA and analyzed in a nanoAcquity liquid chromatographer coupled to an LTQ-Orbitrap Velos mass spectrometer. In the third approach, a combination of enrichment and fractionation methods was used (The “TiSH” method: TiO2-SIMAC-HILIC). Briefly, peptide digest was first pre-enriched in phosphopeptides using TiO2 chromatography (5 μm, GL Sciences Inc, Japan) followed by SIMAC purification. The mono-phosphorylated peptide fraction from the SIMAC enrichment was further subjected to a second TiO2 purification. The mono-phosphorylated fraction was then pre-fractionated by HILIC chromatography using a 40 min gradient from 90% B buffer (95% ACN, 0.1% TFA) to 60% B buffer. Twenty-five fractions were collected, which were pooled into a final five fractions that were then analyzed by reverse phase LC-MS/MS. The multi-phosphorylated fraction from SIMAC was directly analyzed by LC-MS/MS after desalting and concentration using a Poros Oligo R3 Reversed phase (RP) micro-column.
Data Processing Protocol
LC-MS/MS Analysis For the first approach, peptides were separated on a BioBasic C-18 PicoFrit column (75 μm Øi, 10 cm, New Objective, Woburn, MA) at a flow rate of 200 nL/min. Water and ACN, both containing 0.1% FA, were used as solvents A and B, respectively. Peptides were trapped and desalted in the trap column for 5 min. The gradient was started and kept at 10% B for 5 min, ramped to 60% B over 60 min or 120 min, depending on the sample complexity, and kept at 90% B for another 5 min. Peptides (m/z 400-1400) were analyzed on the LTQ-Orbitrap velos in full Scan MS mode with a resolution of 60,000 FWHM at 400m/z; up to the 7 most abundant peptides were selected from each MS scan and then fragmented using collision induced dissociation in a linear ion trap using helium as collision gas at 7500 FWHM and 30 sec exclusion time. For the second approach, the peptides (enriched and non-enriched) were resuspended in 1% FA and were injected for chromatographic separation. Peptides were trapped on a Symmetry C18TM trap column, and were separated using a C18 reverse phase capillary column (75 μm Øi, 25 cm, nano Acquity, 1.7μm BEH column). The gradient used for the elution of the peptides was 1 to 35 % B in 90 min, followed by a gradient from 35% to 85% in 10 min (A: 0.1% FA; B: 100% ACN, 0.1%FA), with a 250 nL/min flow rate. Eluted peptides were subjected to electrospray ionization in an emitter needle (PicoTipTM) with an applied voltage of 2000V. Peptide masses (m/z 300-1700) were analyzed in data dependent mode where a full Scan MS was acquired in the Orbitrap with a resolution of 60,000 FWHM at 400m/z. Up to the 10 most abundant peptides (minimum intensity of 500 counts) were selected from each MS scan and then fragmented using CID (Collision induced Dissociation) in the linear ion trap using helium as collision gas. Multistage activation was enabled to favor the detection of phosphopeptides. The scan time settings were: Full MS: 250 ms and MSn: 120 ms. Generated .raw data files in the 1st and 2nd approach were collected with Thermo Xcalibur v.2.2. For the third strategy, the peptides were resuspended in 0.1 % TFA and analyzed using an Easy-nanoLC coupled to an LTQ-Orbitrap Fusion Tribride mass spectrometer. Peptides were loaded onto a pre-column of 2cm Reprosil –Pur C18 AQ 5 μm RP material using the EASY-LC system and eluted directly onto a 20 cm long fused silica capillary column (75 μm ID) packed with Reprosil- Pur C18 AQ 3 μm RP material. The peptides were separated using a gradient from 0-34% B (A buffer: 0.1 % FA (FA); B buffer: 90% ACN/0.1% FA) at a flow rate of 250 nL/min over 30-60min depending on the UV trace of the HILIC fractions. The peptides (m/z 400-1400) were analyzed in full MS mode using a resolution of 120.000 FWHM at 200 m/z and the peptides were selected and fragmented using helium as collision gas and the fragment ions were recorded in the LTQ with low resolution (rapid scan rate). A maximum of 3 sec were allowed between each MS and for MSMS the ion filling time was set to 40 ms and an AGC target value of 2E4 ions. Raw data was viewed in Xcalibur v2.0.7. Data Analysis for Peptide Identification and Quantitation Peptide identification was performed using Proteome Discoverer v220.127.116.11 and search against SwissProt /Uniprot Saccharomyces cerevisiae database (Jan. 2016) with SequestHT search engine. Both a target and a decoy database were searched to obtain a FDR. Percolator (semi-supervised learning machine algorithm) was used to discriminate correct from incorrect peptide spectrum matches. The PhosphoRS node was used to provide a confidence measure for the localization of phosphorylation in the peptide sequences identified with this modification. Database search parameters were: precursor mass tolerance 10 ppm, fragment mass tolerance 0.6 Da, cys carbamydomethylation as fixed modification and 2 missed cleavage for trypsin. Variable modifications were phosphorylation on S/T/Y and K/R label:13C6 and oxidation (M). Only peptides with FDR<1% were considered for further analyses. Peptide quantification was performed with Proteome Discoverer v1.4. The log2-ratio value associated with each peptide was calculated as a weighted average of the scans used to quantify the peptide. The data were normalized based on the median. Only quantified peptides detected as statistically significant (high confidence FDR< 0.01) were selected. The processing of the data was performed in R (v.3.3.1) with the help of the ‘rvest’, ‘Vennerable’ and ‘Venneuler’ packages. Briefly, the H/L ratios from the samples (TiSH, SIMAC and TiO2) were averaged for every phosphopeptide. The resulting list was filtered to keep only the phosphopeptides with a coefficient of variation (CV) between samples below 40%, peptides without CV (peptides only appearing in one sample) and peptides with a CV above 40% which show a H/L ratio in all the samples below 0.75. Statistical significance was assessed at 5% (p<0.05; two-tailed Student's t-test).
Joan Josep Bech-Serra, Posdoctoral Investigator -Proteomics Unit
Institut d'Investigació Biomèdica de Bellvitge (IDIBELL)
Hospital Duran i Reynals
Avinguda de la Granvia, 199 (3th Floor)
08908 L'Hospitalet de Llobregat
Barcelona - Spain
Ethel Queralt, Cell Cycle Group Cancer Epigenetics and Biology Program (PEBC) Bellvitge Isntitute of Biomedical Research (IDIBELL) Hospital Duran i Reynals Avinguda de la Gran Via de l'Hospitalet, 199-203 08908 - L'Hospitalet de Llobregat ( lab head )