To obtain a global overview of the in vivo Yap1p targets at the proteome level of S. cerevisiae, a comparative analysis was performed using a yeast transformant harboring a control plasmid and a transformant with a plasmid carrying the YAP1 gene. Considering the possibility to control pH and maintain anaerobic conditions, yeast transformants were cultivated in a multi-bioreactor and the fermentation was discontinued when the cells were still in the exponential growth phase. Before 2-DE analysis, overexpression of Yap1p was validated by western-blot analysis. As expected, the Yap1 protein was present at elevated levels in the Yap1p-overexpressing transformant (Figure 1). The level was estimated to be approx. four-fold higher than in the control transformant.

Yeast cells from both cultures were harvested and proteins were extracted using the extraction protocol developed by Kolkman et al. 21, which we further optimized for our yeast samples. In the protocol developed by Kolkman et al., the cells are lyophilized and subsequently vortexed with glass beads prior to boiling with SDS. In order to improve cell disruption, we introduced an additional step. Before the SDS boiling, the yeast cells were disrupted in extraction buffer containing thiourea. Cell debris which was not dissolved in the extraction buffer was further exposed to boiling with SDS. More high-molecular-mass proteins (> 70 kDa) were observed on the 2-D gels when this optimized extraction protocol was adopted.

In Figure 2, the 2-D gel electrophoresis images of the control transformant (Figure 2A) and the Yap1p-overexpressing transformant (Figure 2B) are shown. By using the SYPRO Ruby staining method, more than 2,000 protein spots were detected on each 2-D gel. This number is higher than what has been achieved by silver staining, for which only a few hundred spots were detected 21. The 2-DE analyses were performed in triplicate to allow statistical analysis, and Student’s t-test was used to determine if the relative change in protein expression was statistically significant. Based on this analysis, protein spots that were significantly up-regulated upon Yap1p overexpression were identified on the 2-D gels. In total, 78 such spots were detected on the 2-D gels. Typical examples are shown in Figure 2C and D. These spots were further analyzed by MALDI-MS and LC-MS/MS, resulting in identification of 55 unique proteins (MALDI-MS was used for most of the protein spots (1-73), while LC-MS/MS was used for analysis of a few spots (74-78) for which MALDI-MS analysis did not give satisfactory results). Interestingly, some of the proteins were identified in more than one spot on the 2-D gels.

The 55 proteins that were identified are listed in Table 1 and the relative quantity is indicated. Of the averaged total spot volumes of the 55 identified proteins (Table 1), 16 changed significantly at 99% confidence level (P < 0.01), 33 changed significantly at the 95% confidence level (P < 0.05), and 6 changed significantly at 90% confidence level (P < 0.10). The identified proteins were divided into different categories, namely enzymes involved in carbon metabolism and proteins involved in pathways other than carbon metabolism, such as protein biosynthesis, cell cycle and growth regulation, etc. (Figure 3A). It is noteworthy that 16 proteins that play a role in carbon metabolism were up-regulated in the Yap1p-overexpressing yeast transformant. These proteins include ten glycolytic enzymes (Pgi1p, Hxk2p, Fba1p, Tdh1p, Tdh2p, Tdh3p, Pgk1p, Gpm1p, Eno2p, and Cdc19p), four enzymes involved in conversion of pyruvate to ethanol (Pdc1p, Adh1p, Ald6p, and Dld3p), and two enzymes (Tkl1p and Gnd1p) that are involved in the pentose phosphate pathway. Based on image analysis, we observed that the combined spot volumes of all identified enzymes involved in carbon metabolism enzymes increased about 1.5 fold in the Yap1p-overexpressing transformant (Figure 3B).

¹The normalized volume of spots from three replicate 2-D gels was averaged and the standard deviation was calculated for each yeast transformant. A Student’s t-test was performed to determine if the relative change was statistically significant. If proteins were identified and quantified in more than one spot on the 2-D gels, the total spot volume was calculated (indicated with “^#”). YBS, nucleotide sequence with (+) or without (−) predicted Yap1p-binding site; Avol, averaged value from three spot volumes; Y/C, the relative change (Yap1p-overexpression versus control).

Eight proteins involved in stress response were identified that were significantly more abundant in the Yap1p-overexpressing transformant. These proteins include seven heat-shock and chaperone proteins (Ssa1p, Ssa2p, Ssb1p, Ssb2p, Hsp82p, Hsc82p, and Sse1p) and one peroxiredoxin (Tsa1p). Compared to the control transformant, most of the heat-shock and chaperone proteins showed more than 2-fold increase in the Yap1p-overexpressing transformant (Table 1). Moreover, 13 proteins involved in protein biosynthesis and 10 proteins involved in cell cycle and growth regulation were identified on the 2D gels. The combined spot volumes of all the identified proteins increased about two-fold (Figure 3B).

While the results reported were reproducibly obtained on all 2-D gels analyzed, it should be noted that some spots might have contained co-migrating proteins that were not detected in the MALDI-MS analysis. These proteins would have affected the relative quantification of the up-regulated proteins. As MALDI-MS identifies the prevalent proteins that are present in a gel sample, these errors are, however, considered to be negligible.

All yeast transformants (a Yap1p-overexpressing transformant and a control transformant) that were used in this study were previously constructed and stored in our laboratory 13. Yeast transformants designated Y (with the YAP1 gene under the control of PGK1 promoter) and C (negative control carrying the same plasmid but without any YAP1 gene) were streaked on SC–Ura agar plates (20 g/l glucose, 6.7 g/l yeast nitrogen base without amino acids, 10% amino-acid supplement solution × 10 excluding uracil, and 20 g/l agar) 43, which were then incubated at 30 °C for 72 h. Inoculum cultures of the two S. cerevisiae transformants were prepared in 500 ml shake flasks with 140 ml of SC-Ura medium (excluding the agar). The flasks were inoculated with cells from the agar plates and incubated for approximately 17 h at 30 °C with agitation (Infors Ecotron, Infors AG, Bottmingen, Switzerland). The cells were harvested in the exponential growth phase by centrifugation at 1,200 × g for 10 min at 4 °C (Allegra X-22R, Beckman Coulter, Brea, CA, USA). The cells were then resuspended in a suitable amount of sterile H₂O to yield an inoculum of 0.1 g/l (DW) in all bioreactor vessels.

The cultivation of the two transformants Y and C was carried out with a multi-bioreactor system (Sixfors from Infors AG, Bottmingen, Switzerland). Four 350-ml-bioreactor vessels (two vessels per transformant) equipped with condensers, FermProbe pH-electrodes (Broadley James Corporation, Irvine, CA) and OxyProbe polarographic dissolved-oxygen sensors (pO2-electrodes) (Broadley James Corporation) were sterilized through autoclavation and filled with 250 ml modified SC–Ura medium. The composition of the medium was: 40 g/l glucose, 13.4 g/l yeast nitrogen base without amino acids, 10% amino-acid supplement solution × 10 excluding uracil, 0.1% of an ergosterol/Tween 80 mixture (consisting of 10 g/l ergosterol and 420 g/l Tween 80), and 8 drops of antifoam.¹² The pH electrodes and the pO2 electrodes were calibrated prior to start-up. Two ml of inoculum were added to each bioreactor vessel to an initial biomass concentration of 0.1 g/l (DW). Throughout the fermentation, the temperature was kept at 30 °C, the stirring was kept at 300 rpm, and the pH was kept at 5.5 by automatic addition of 0.5 M NaOH. Nitrogen gas (15 l/h) was used to maintain anaerobic conditions. The fermentation was discontinued after seven hours when the cells were in the exponential growth phase and had reached a cell density of 1 g/l (DW). The yeast cells were harvested by centrifugation at 3,000 × g for 5 min at 4 °C, and stored at −80 °C before protein extraction.

Yeast protein extracts were prepared for analysis with 2-DE using a modified approach of Kolkman et al. 21. In brief, about 100 mg of lyophilized cell pellet were resuspended in 600 μl extraction buffer [7 M urea, 2 M thiourea, 4% (w/v) CHAPS (3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate), 40 mM dithiothreitol]. Protease inhibitor cocktail (Calbiochem, La Jolla, CA) and glass beads (acid washed, 425–600 μm, Sigma-Aldrich, St. Louis, MO) were added to the cell suspension. Cells were disrupted by vortexing six times 60 s (the samples were cooled on ice for 30 s in between the vortex steps). The cell extract was transferred to a fresh tube and centrifuged at 20,000 × g for 10 min at 4 °C. The supernatant was transferred completely to a fresh microcentrifuge tube and recovered as Fraction 1. The insoluble fractions were suspended in 400 μl SDS-buffer (2% SDS, 40 mM Tris, 60 mM DTT) by thorough vortexing and pipetting up and down with a 200 μl pipette tip for 10 times. The sample was boiled for 10 min and subsequently cooled on ice. After centrifugation for 10 min (20,000 × g, 4 °C), the supernatant (Fraction 2) was then transferred to a fresh microcentrifuge tube and mixed with Fraction 1. Subsequently, 75 μl of a DNase and RNase solution [1% (w/v) DNase I, 0.25% (w/v) RNase A, 50 mM MgCl₂, 0.5 M Tris–HCl, pH 7.0] were added and the combined fractions were incubated on ice. The mixed protein extract was then purified by using a 2-D Clean-Up Kit (GE Healthcare, Uppsala, Sweden), and the purified protein sample was dissolved in rehydration solution [7 M urea, 2 M thiourea, 4% (w/v) CHAPS, 0.002% (w/v) bromophenol blue] supplemented with 2% (v/v) 3–10 NL IPG buffer (GE Healthcare) and 5.4 mg/ml dithiothreitol. Total protein concentration was determined using the 2-D Quant Kit (GE Healthcare). Aliquots of extracellular protein samples were stored at −80 °C before proteomic assays.

The peaklist files of the processed mass spectra of spots 1 to 78 are provided in the appendix. Additional file 1: Spots 1 to 73 were analysed by MALDI-TOF-MS and the peaklist files are available in the supplementary zip files: Additional file 2: 1-5_MALDI_MS_data, Additional file 3: 6-10_MALDI_MS_data, Additional file 4: 11-15_MALDI_MS_data, Additional file 5: 16-20_MALDI_MS_data, Additional file 6: 21-25_MALDI_MS_data, Additional file 7: 26-30_MALDI_MS_data, Additional file 8: 31-35_MALDI_MS_data, Additional file 9: 36-40_MALDI_MS_data, Additional file 10: 41-45_MALDI_MS_data, Additional file 11: 46-50_MALDI_MS_data, Additional file 12: 51-55_MALDI_MS_data, Additional file 13: 56-60_MALDI_MS_data, Additional file 14: 61-65_MALDI_MS_data, Additional file 15: 66-69_MALDI_MS_data,_and Additional file 16: 70-73_MALDI_MS_data. As for spots 74 to 78, analysis was performed by LC-MS/MS and the corresponding mzML files are provided in the supplementary zip files Additional file 17: 74_LC_MS_MS_data, Additional file 18: 75_LC_MS_MS_data, Additional file 19: 76_LC_MS_MS_data, Additional file 20: 77_LC_MS_MS_data, and Additional file 21: 78_LC_MS_MS_data.

The crude protein extracts were separated by SDS-PAGE after adding 5× Laemmli sample buffer and boiling. The separated proteins were transferred onto a PVDF membrane by semi-dry blotting and probed (1:2000 dilution, incubated overnight at 4 °C) with a rabbit polyclonal antibody directed against amino-acid residues 351–650 at the C-terminus of S. cerevisiae Yap1p (Santa Cruz Biotechnology, Santa Cruz, CA). Goat anti-rabbit IgG-HRP (Santa Cruz Biotechnology) was used as secondary antibody. Bound antibodies were detected by the ECL Prime western blotting detection reagent (GE Healthcare) using a CCD-based imager (ImageQuant LAS 4000, GE Healthcare).

For the first dimension (IEF), an amount of 200 μg of protein (300 μl) prepared as described in section Protein Extraction and Purification was loaded on a 13 cm Immobiline Dry-Strip pH 3–10 NL (GE Healthcare), and the IPG strips were rehydrated overnight at room temperature. Isoelectric focusing (IEF) was performed with a Multiphor II system (GE Healthcare) at 20 °C with a 3-phase gradient program: 500 V for 0.25 kVh, 3500 V for 5.25 kVh, and 3500 V for 45 kVh. Prior to the second dimension (SDS-PAGE), the IPG strips were incubated for 15 min in equilibration buffer [50 mM Tris–HCl, 6 M urea, 30% (v/v) glycerol, 2% (w/v) SDS, 0.002% (w/v) bromophenol blue] containing 1% (w/v) dithiothreitol, followed by 15 min incubation in equilibration buffer containing 2.5% (w/v) iodoacetamide. Second-dimension electrophoresis was performed on PROTEIN^TM II electrophoresis system (Bio-Rad, Hercules, CA). The IPG strips were placed on top of 12.5% polyacrylamide gels and sealed with a solution of 1% (w/v) agarose containing a trace of bromophenol blue. The vertical gels were run at 10 mA per gel for 30 min followed by 25 mA per gel until the bromophenol blue had migrated to the bottom of the gel. The temperature was maintained at 15 °C using MultiTemp III system (GE Healthcare). Proteins were visualized using SYPRO Ruby Protein Gel Stain (Invitrogen, Carlsbad, CA). The SYPRO Ruby-stained gels were scanned at 532 nm using a Typhoon 9400 scanner (GE Healthcare).

For each transformant, namely Yap1p-overexpressing transformant and control transformant, 2-D gels were run in triplicate. Additionally, a master 2-D gel was prepared, which contained a 1:1 mixture of the protein extract from the two yeast transformants. That gel, which should contain all protein spots present on the 2-D gels with samples from the Yap1p-overexpressing and the control transformant, was used during image analysis as a master gel. Image analysis was performed using the ImageMaster II software (GE Healthcare). The quantitative and statistical analyses were performed using suitable functions within the ImageMaster II software and Excel software (Microsoft, Redmond, WA). The normalized intensity of spots on three replicate 2-D gels was averaged and the standard deviation was calculated. The relative change in protein abundance for the Yap1p-overexpressing transformant (Y) versus the control transformant (C) (indicated with “fold change Y/C”) for each protein spot was calculated by dividing the averaged spot quantity from gels with samples from the Yap1p-overexpressing transformant by the averaged spot quantity from gels with samples from the control transformant. A two-tailed non-paired Student’s t-test was performed to determine if the relative change was statistically significant.

Protein spots of interest were picked from the 2-D gels using an Ettan Spotpicking Station (GE Healthcare) and destained three times using a fresh solution of 20 mM ammonium bicarbonate containing 35% (v/v) acetonitrile (ACN). Subsequently, the gel pieces were dried by two washes using 100% neat acetonitrile and re-hydrated on ice using a solution of sequencing grade modified trypsin (Promega, Madison, WI) in 20 mM ammonium bicarbonate. The trypsin concentration depended on the intensity of the spots and was 2 to 3 ng/µl. The re-hydrated gel samples were incubated in 37 °C for overnight digestion and either analyzed immediately or stored at −20 °C until further analysis.

MALDI-MS spectra for peptides were acquired using a Voyager DE-STR mass spectrometer (AB SCIEX, Framingham, MA, USA) as described by Yao et al. 44. LC-MS/MS combined with ESI-ion-trap MS was performed using an HCT-Ultra ETD II mass spectrometer from Bruker (Bremen, Germany) linked to an Easy-nLC system from Proxeon (Odense, Denmark). Spectra were acquired using the enhanced scanning mode covering a mass range from m/z 350 to m/z 1300. The LC separation of peptides was performed using a 5 µm C18 column (375 μm OD/75 μm ID × 10 cm) from NanoSeparations (Nieuwkoop, The Netherlands) and a 30 min gradient ranging from 0 to 60 percent of acetonitrile. The flow rate was 300 nl min^-1. Data processing was performed using the Data Analysis software (4.0 SP4) (Bruker, Bremen, Germany) using default setting for processing and AutoMSn detection of compounds.

Database searches using the peak list files (mgf-format) of the processed mass spectra were performed using an in-house license of Mascot (http://www.matrixscience.com), and searches were performed using the Swiss-Prot or NCBInr database. As for MALDI-MS spectra, a mass error of 50 ppm and one missed cleavage sites were permitted. In addition, variable modifications allowed included methionine oxidation and carbamidomethylation of cysteine residues. As for LC-MS/MS data a mass error of 0.3 Da was allowed for both the MS and MS/MS mode and variable modifications were set as for the database searches with the MALDI-MS data.