Proteomic Analysis of the Increased Stress Tolerance of Saccharomyces cerevisiae Encapsulated in Liquid Core Alginate-Chitosan Capsules

Saccharomyces cerevisiae CBS8066 encapsulated in semi-permeable alginate or alginate-chitosan liquid core capsules have been shown to have an enhanced tolerance towards complex dilute-acid lignocellulose hydrolysates and the lignocellulose-derived inhibitor furfural, as well as towards high temperatures. The underlying molecular reasons for these effects have however not been elucidated. In this study we have investigated the response of the encapsulation on the proteome level in the yeast cells, in comparison with cells grown freely in suspension under otherwise similar conditions. The proteomic analysis was performed on whole cell protein extracts using nLC-MS/MS with TMT® labelling and 2-D DIGE. 842 and 52 proteins were identified using each method, respectively. The abundances of 213 proteins were significantly different between encapsulated and suspended cells, with good correlation between the fold change ratios obtained by the two methods for proteins identified in both. Encapsulation of the yeast caused an up-regulation of glucose-repressed proteins and of both general and starvation-specific stress responses, such as the trehalose biosynthesis pathway, and down-regulation of proteins linked to growth and protein synthesis. The encapsulation leads to a lack of nutrients for cells close to the core of the capsule due to mass transfer limitations. The triggering of the stress response may be beneficial for the cells in certain conditions, for example leading to the increased tolerance towards high temperatures and certain inhibitors.


Introduction
In the search for a replacement for fossil fuels bioethanol comes out as one of the most promising alternatives. For sustainable production without interference with food production it is necessary to use lignocellulosic sources such as agricultural or forestry residues as raw materials [1]. However, the inherent recalcitrance of these materials makes extensive pretreatment and hydrolysis necessary for efficient release of fermentable sugars [2]. This often creates significant amounts of by-products that act as inhibitors of the subsequent fermentation, lowering the efficiency and feasibility of the process [3][4][5].
The most widely used microorganism for production of fuel ethanol, be it 1 st or 2 nd generation, is Saccharomyces cerevisiae. This yeast is capable of in situ detoxification of toxic hydrolysates, however, the inhibitor to cell ratio has to be low [6]. A low ratio can be achieved by increasing the local cell concentration, by cell immobilization or flocculation and cell recirculation [7,8]. With a high local cell concentration, the ratio of inhibitors to cells becomes smaller locally and thus the cells can better handle the toxicity of a hydrolysate [9].
Cell immobilization can be done in a number of ways, but the one giving the highest local cell density is undoubtedly encapsulation in a semi permeable membrane. Local cell densities of several hundred grams dry weight per litre of capsule volume have been achieved [10]. Encapsulation of yeast cells has been shown to improve the fermentative performance in toxic lignocellulosic hydrolysates [8], as long as the inhibitors can be converted at a high rate [11]. Increased thermotolerance has also been observed upon encapsulation [12]. It is obvious that encapsulating cells will affect their growth and metabolism due to the close contact with other cells and due to the increased diffusion resistances that may lead to nutrient-limited conditions in the core of the capsule. It has been shown, that encapsulation leads to significantly lower cellular contents of RNA and protein as well as a lower RNA/protein ratio, and to higher cellular contents of trehalose, glycogen and total carbohydrates [13]. However, it is not clear how encapsulation affects the cells on a more molecular level, and how the responses facilitate increased tolerance to inhibitors or elevated temperatures. Furthermore, genome-wide investigations, on e.g. transcriptome or proteome level, of the physiological changes in yeast encapsulated in liquid core capsules have not yet been performed. A better understanding of the biochemical background of the improved tolerance may be used to design superior yeast strains, able to ferment toxic hydrolysates at high rates even without the need of an enclosing membrane.
Quantitative proteomics is of utmost importance for the understanding of changes in cellular physiology arising from different treatments of the cells, as different proteins, including post translationally modified variants, are directly linked to metabolic fluxes and cellular structure, and therefore ultimately determine the physiology. There are a number of different quantitative proteomic methods available, with two major types of protein separation, namely two dimensional gel electrophoresis  and multidimensional liquid chromatography (multidimensional protein identification, MudPIT), often called nLC-MS/MS. For identification of proteins both methods depend on mass spectrometry in combination with database searches. For 2-DE one of the currently most popular methods is 2-D difference gel electrophoresis , where proteins from different samples are labelled with different fluorescent probes, enabling quantification of proteins from different samples in the same gel [14]. In MudPIT the most common in vitro labelling method is by isobaric mass tags, such as iTRAQH or TMTH, that are applied after enzyme digestion of the protein samples to covalently label the peptides of different samples [15,16]. The isobaric mass tags have different isotopic substitutions that, as the tags are cleaved off the peptides in the MS/MS mode, result in reporter ions of different weight, thus enabling quantification of proteins from different samples.
In this study, we compare the protein expression levels in yeast cells growing anaerobically either in liquid core capsules enclosed by alginate-chitosan membranes or in suspension, using both the 2-D DIGE approach and a MudPIT approach with TMTH. In addition to the overall elucidation of physiological changes in the cells due to encapsulation, the aim was to find possible reasons for the increased tolerance towards lignocellulosic derived inhibitors as well as for the enhanced yield of ethanol and lower glycerol yield of encapsulated yeast [13]. We show that encapsulation leads to general down-regulation of proteins involved in growth and protein synthesis, and up-regulation of proteins involved in both specific responses to nutrient limitation, as well as general stress responses. We conclude that the triggering of general stress responses is the underlying mechanism of the improved inhibitor tolerance of encapsulated yeast.

Yeast strain and medium
The S. cerevisiae CBS8066 obtained from Centraalbureau voor Schimmelcultures (Delft, the Netherlands) was used in all experiments. The strain was maintained on YPD agar plates with 10 g l 21 yeast extract (Scharlau), 20 g l 21 soy peptone (Fluka) and 20 g l 21 D-glucose (Fisher Scientific) as an additional carbon source.
Aerobic cultures for cell propagation were grown in 250 ml cotton-plugged conical flasks. Anaerobic batch cultivations were performed in 250 ml conical flasks equipped with a rubber stopper fitted with a loop trap filled with sterile water to permit produced CO 2 to leave the flasks, and stainless steel capillaries for sample removal. The growth medium used for the batch cultivations was a defined glucose medium (DGM), as previously reported [17], with ergosterol (Sigma) added during anaerobic cultivations.

Encapsulation procedure
The capsules were prepared by the liquid-droplet-forming method [18]. Yeast cells were grown in 100 ml DGM (50 g l 21 glucose) for 24 h in a shaker bath (130 rpm) at 30uC. Yeast from 50 ml of medium was harvested at 3500 g for 4 minutes and resuspended in 50 ml 1.3% (w/v) sterile CaCl 2 (Scharlau) solution containing 1.3% (w/v) carboxymethylcellulose (CMC) (Aldrich) with average Mw of 250 kDa and degree of substitution 0.9. CMC increases the viscosity of the CaCl 2 solution, thereby enhancing the formation of spherical capsules. A sterile solution containing 0.6% (w/v) sodium alginate (Product number 71238, Sigma) and 0.1% (v/v) Tween 20 (Sigma-Aldrich) was used for capsule formation. The surfactant Tween 20 improves the permeability of the capsule membrane, thereby preventing the capsules from rupturing due to CO 2 formation during the cultivation [19].
Capsules were formed by dripping the CMC-yeast-CaCl 2 solution into the stirred sodium alginate solution through syringe needles. The capsules were gelled for 10 minutes, washed with ultra-pure water for 10 minutes and hardened in 1.3% (w/v) CaCl 2 solution for 20 minutes. The calcium-alginate capsules were thereafter treated in a 0.2% (w/v) low molecular weight chitosan (Product number 448869, Aldrich) solution with 300 mM CaCl 2 in 0.040 M acetate buffer, pH 4.5, at a ratio of 1:10 of capsules to solution for 24 hours. The treatment was performed in 0.5 l Erlenmeyer flasks in a water bath at 30uC at 130 rpm. Chitosan molecules are incorporated in the alginate matrix, thereby improving the capsules' strength by creating alginatechitosan membranes [20].

Cultivation and cell sampling
Approximately 15 ml of cell seeded capsules were cultivated aerobically in 100 ml DGM containing 40 g l 21 glucose for 24 hours in a shaker bath (130 rpm) at 30uC. The capsules were thereafter rinsed with sterile 0.9% NaCl (Scharlau) and transferred to fresh medium for another 7 hours. Fifty capsules were subsequently transferred to 100 ml of DGM, 40 g l 21 glucose, for anaerobic batch cultivations, giving a starting cell concentration of 0.7760.04 g DW l 21 liquid volume. Samples for protein expression analysis were taken after 25.460.3 hours, when the glucose concentration had reached 12.660.7 g l 21 . Cells from 8 capsules were washed out with ice-cold sterile ultra-pure water, and harvested at 10,0006g for 1 minute at 4uC. The pellet was immediately frozen in N 2 (l) and subsequently kept at 280uC until analysis.
Propagation of suspended yeast was started from aerobic 24 hours cultivations in 100 ml DGM (50 g l 21 glucose), by a 1% dilution into fresh DGM. After 23.5 hours, cells were harvested (3500 g, 4 minutes) and resuspended in 100 ml fresh DGM (40 g l 21 glucose) for anaerobic cultivations, at an initial cell concentration of 0.7460.01 g DW l 21 . The cells were grown anaerobically and samples for protein expression analysis were taken at 8.360.1 hours, when 15.860.1 g l 21 glucose remained in the cultivations. Cells were harvested by centrifugation of 20 ml cell suspension for 1 min at 3,5006g, 4uC, followed by washing of the cell pellet with ice-cold ultrapure water and centrifugation for 1 min at 10,0006g, 4uC. The pellet was immediately frozen in N 2 (l) and subsequently kept at 280uC until preparation of protein extracts and analysis.
After a series of trial cultivations, the sampling times were chosen to obtain samples when the residual extracellular glucose concentration and total amount of cells in the cultivations were approximately equal, and glucose was being consumed at constant rates. Free cells were sampled at a slightly higher residual glucose concentration, due to a higher biomass yield on glucose.

Proteomic analysis by nLC-MS/MS and 2-D DIGE
Cells from five biological replicates each of free and encapsulated yeast were lysed and the proteins extracted and cleaned up prior to dividing the samples for 2-D DIGE and quantitative nLC-MS/MS.
For the nLC-MS/MS, precipitated protein pellets of 100 mg of each samples (three biological replicates each of free and encapsulated yeast) were digested with trypsin and labelled with a six-plex set of Tandem Mass Tag reagents following the manufacturer's instructions (Thermo Fisher Scientific). Nano LC-MS/MS analysis was performed on a LTQ Orbitrap Velos instrument (Thermo Fisher Scientific, Inc., Waltham, MA, USA) interfaced with an in-house constructed nano-LC column. MS data analysis was performed using Proteome Discoverer version 1.2 (Thermo Fisher Scientific).
2-D DIGE analyses [14] was performed across 5 gels. The samples were G-Dye labelled according to the manufacturers standard protocol (NH DyeAGNOSTICS), individual samples were labelled with G-Dye200 or G-Dye300 dyes using dye switching, while the internal standard was always G-Dye100 labelled. Isoelectric focusing was done in 24 cm pH 3-10 Nonlinear Imobiline DryStrips (GE Healthcare) on an Ettan IPGphore. The second dimension was run on an Ettan DALT II in in-house made polyacrylamide Bis-Tris gels. After 2D electrophoresis, the gels were scanned using the VersaDoc MP 4000 (BioRad) and the gel images were analysed using the Progenesis SameSpots software ver. 4.1 (Nonlinear Dynamics) for spot detection, spot quantification, inter-gel matching, and statistics. Spots with significant differences (Anova p,0.05) were selected for further identification, picked and digested with trypsin prior to identification using MALDI-TOF MS/MS.
Further details of the proteomic analyses are described in Methods S1. The nLC-MS/MS data associated with this manuscript may be downloaded from ProteomeCommons.org Tranche using the following hash: t7gxXL8oO/kgqNEGa2zWDwUVQKnEa90+l/ W++pR6JKuwNXrY1m7hHCrqnVsNn6M0pv3w0aebnXgnl8vXv6-N+YlyNFHcAAAAAAAACug = = .

Presentation of protein expression
The ratios between the abundances of proteins in suspended and encapsulated yeast are presented by one to three numbers in parentheses after the name of the protein. The first, and in most cases the only, number represents the ratio (fold change, FC) obtained by n-LC-MS/MS. When applicable, this is followed by the average ratio obtained from 2-D DIGE spots with unique significant protein hits, and lastly the average ratio obtained from spots with significant hits for more than one co-migrating proteins in 2-D DIGE. Thus, Tps1p (1.77, 1.59, 1.63) indicates a FC of 1.77 estimated by the nLC-MS/MS approach, an average FC of 1.59 estimated from spots with a unique significant hit for Tps1p, and an average FC of 1.63 estimated from spots with significant hits from both Tps1 and co-migrating proteins.

Analytical methods
The amounts of metabolites were quantified by HPLC using an Aminex HPX-87H column (Bio-Rad) at 60uC with 5 mM H 2 SO 4 as eluent at a flow rate of 0.6 ml min 21 . A refractive index detector was used for the detection and quantification of glucose, acetic acid, lactic acid, glycerol and ethanol.
The cell dry weight was measured in predried and preweighed glass tubes. Cells were separated by centrifugation and washed once with ultra-pure water before drying for approximately 24 h at 105uC. Cells from capsules were released by crushing the capsule followed by extensive washing of the capsule debris with ultra-pure water.

Statistics, yields and elemental balance calculations
The biomass and metabolite yields as well as the carbon balance were calculated from the determined concentrations at the end of the fermentations, i.e. the time of sampling for proteome analysis. Error intervals are given as 95% confidence intervals of the mean, unless otherwise stated.

Fermentation performance of free and encapsulated cells
Free and encapsulated cells were grown anaerobically in shake flasks starting at the same initial cell concentration, 0.7560.02 g DW l 21 medium volume. The cells were sampled at residual glucose concentrations of 14.261.2 g l 21 while consuming glucose and producing ethanol at constant rates ( Figure 1). The encapsulated cells consumed glucose markedly slower than the free cells, with a linear glucose consumption profile never reaching a true exponential growth phase. The sampling times were chosen as a compromise between residual extracellular glucose concentration and total cell amount in the cultivations in order to minimize the differences between the two modes of cultivation. The two systems are distinctly different, and the resulting differences in the proteome and other variables must be viewed as integrated responses to both direct and indirect effects of the encapsulation.
The yields of major metabolites and biomass were significantly different between cells grown in the two ways. The encapsulated cells had higher ethanol yield than the free cells, whereas free cells had higher glycerol, acetate and biomass yields (Table 1), similar to what has been observed in previous studies of encapsulated yeast [9]. Interestingly, low levels of lactate, the end-product of methylglyoxal degradation in anaerobic cultures, were also detected towards the end of the cultivations with encapsulated yeast (Table 1). Similar observations have been made in retentostat cultures with yeast growing extremely slowly [21,22].
The slower growth rate and lower biomass yields of encapsulated cells are likely an effect of mass transfer limitations to the cells in the middle of the capsules. The amount of biomass per capsule was 1.560.1 mg at the start of the cultivation, which increased to 3.960.1 mg per capsule at the time of sampling. This corresponds to approximately 300 g DW (l capsule volume) 21 . It has previously been shown, that for a flocculating yeast strain mass transfer limitations occur in flocs larger than 100 mm [23] and that mass transfer into capsules decrease as the capsules fill up with cells [13]. After chitosan treatment, the capsules used in this study had a mean diameter of 3.6360.03 mm. At the time of sampling, the capsules were full of cells that were likely not moving around freely ( Figure 2). This undoubtedly gives rise to mass transfer limitations into the interior of the capsule, considering the fact that glucose diffusivity in water alone is 600 mm 2 sec 21 [24]. Consumption of glucose by cells close to the membrane also leads to decreasing concentrations closer to the core of the capsule. For these reasons there are likely also physiological differences between cells along the radius towards the centre of the capsule. However, for the proteomic analysis in this study we consider only the mean values of all protein levels in the cell population, since cells were quantitatively washed out of the capsules at the time of sampling. The possible error introduced by this methodology would be an underestimate of the differences between cells in the core of the capsule and the suspended cells.

Protein expression in response to encapsulation
Of the 842 proteins identified with nLC-MS/MS (Table S1), 116 were up-regulated and 95 down-regulated according to the set significance criteria (Figures 3 and 4). The large number of differentially expressed proteins indicates that the environmental conditions are quite different for yeasts growing in the capsules and in suspension. The localization and functional category of differentially expressed proteins were determined using the FunCatDB software ( Figure 4, Table S2). Notably, downregulated proteins were over-represented when compared to upregulated in cellular locations important for cell growth and division, such as the cell periphery, bud, cytoskeleton and Golgi. This was also the case for the functional categories transcription and protein synthesis ( Figure 4, Table 2). These results mirror the decreased growth inside the capsules, visualized by the decreased biomass yield (Table 1) and slower glucose consumption rate ( Figure 1). Up-regulated proteins were instead over-represented mainly in the cytoplasm and mitochondria ( Figure 4B), where many proteins involved in the two categories energy and metabolism are localized ( Figure 4A, Table 3, Figure S3). In these functional categories, up-regulated proteins were also overrepresented.

Nutrient limitation in capsules
Of utmost importance for the production of bioethanol are the metabolism and energy turnover of the yeast. The differences in metabolite yields between the two modes of cultivation indicate that the metabolism of the yeast changed significantly upon encapsulation (Table 1). Of the 112 quantified proteins classified as being involved in the functional category ''energy'', a total of 27 were up-regulated and only 3 were down-regulated. Included in this category are e.g. parts of the central carbon metabolism such as glycolysis and gluconeogenesis, the pentose phosphate pathway, the TCA cycle and the trehalose and glycogen metabolism ( Figure 5), all of which are localized to the cytoplasm and/or mitochondria.
The two most up-regulated proteins in encapsulated cells were the high affinity hexose transporters Hxt6p (10.63) and Hxt7p (13.04). The expression of these transporters is repressed by high glucose levels and they have high expression levels on nonfermentable carbon sources and at low concentrations of glucose [25,26]. Several enzymes in the glycolytic pathway were significantly up-regulated, in many cases seen also using the 2-D DIGE method. Only one, Pdc5p, was down-regulated ( Figure 5). The strong up-regulation of Hxk1p and Glk1p in the encapsulated yeast is notable, since their expressions, like the mentioned hexose transporters, are known to be repressed by glucose [27]. These are only some examples of the up-regulation of glucose-repressed proteins in the encapsulated cells and together they provide strong evidence for carbon limitation inside the capsules.
Another enzyme in the glycolytic pathway, Tdh1p catalysing the oxidative phosphorylation of glyceraldehyde-3-phosphate to 1,3-bisphosphoglycerate, is known to be expressed during stationary phase and other conditions of slow growth, while the Tdh2p and Tdh3p are detected in the exponential phase [28]. Tdh1p was detected at significantly higher levels in the encapsulated yeast than in the free cells, while there was no significant difference for Tdh2p and Tdh3p ( Figure 5).
The cytoplasmic alcohol dehydrogenases Adh1p and Adh5p, and the glucose-repressed mitochondrial Adh3p, that all reduce acetaldehyde to ethanol, were up-regulated in the encapsulated cells ( Figure 5). This is likely an effect of a higher maintenance energy requirement due to the low growth rate [29] and the increased anaerobicity. The glycerol-3-phosphatase Rhr2p was down-regulated, in line with the observed lower yield of glycerol for the encapsulated cells, which is to be expected since glycerol production is generally growth related when cells are not osmotically stressed.
The proteins involved in synthesis and utilization of the storage carbohydrates trehalose and glycogen were up-regulated ( Figure 5). This is in accordance with previous reports of increasing trehalose and glycogen levels in encapsulated yeast as the capsules filled up with cells (4 and 4.5-fold increase, respectively, in the end compared to the beginning of a series of 20 sequential batches) [13]. Trehalose and glycogen are important storage carbohydrates that accumulate in slowly growing or starved yeast, but trehalose is also an important protector of membranes and proteins against various stresses such as cold, heat, and starvation [30,31].
In addition to carbon starvation, it is possible that the cells in the middle of the capsules also experienced nitrogen and phosphate  limitation, since both glutamine and phosphate metabolism were significantly up-regulated in the encapsulated cells (  [32], and down-regulation of proteins repressed by anaerobic conditions, such as Sod1p (21.68) [33]. This shows that oxygen was absent in the capsules, meaning that the environment inside the capsule was more strictly anaerobic than in the suspension shake flasks. This could be a contributing factor to the observed increase in ethanol yield and decrease in biomass yield. Despite using air locks and rubber stoppers, some oxygen may diffuse into the shake flask culture.

Down-regulated protein synthesis in encapsulated yeast
Another variable likely to change with a change in nutrient availability is the growth, and hence, also the protein synthesis should be affected. Previous reports have stated that both the total protein levels and the total RNA levels (with ribosomal RNA as the main contributor) decrease in prolonged growth of encapsulated yeast [13]. Of the identified proteins 163 were related to protein synthesis and 103 to ribosome biogenesis according to the FunCat analysis. Of these proteins, 26 and 15 were down-regulated, respectively (Table 2, Figure 4A), for example the most downregulated protein Drs1p (212.11), involved in ribosome assembly and function.
In the PRIMA analysis of the genes coding for regulated proteins, the usage of transcription factor Abf1p was enriched among the down-regulated proteins ( Figure S4). Among other things, Abf1p is involved in initiation of DNA replication as well as activation of transcription of many genes coding for ribosomal proteins [34]. In cells immobilized in alginate gel beads, the levels of ribosomal proteins have instead been seen to be up-regulated [35], showing that there are differences between the physiological responses to immobilization in porous gels and to liquid core encapsulation.
Certain proteins in the unfolded protein response (UPR) related to growth were also down-regulated, e.g. Ssz1p (21.49) and Zuo1p (21.34) that act together in a complex involved in ribosome biogenesis [36]. Two other down-regulated UPR proteins belong to the cytosolic chaperonin Cct ring complex, Cct2p (24.19) and Cct5p (21.82). This complex is required for assembly of actin and tubulins [37]. The slower growth of the encapsulated yeast would decrease the need of new actin assembly and of the Cct complex, hence its decreased expression. Other proteins involved in new actin assembly were also down-regulated, such as Bbc1p (21.57), involved in the reorganization of the actin cytoskeleton [38], and Tpm1p (21.80), involved in the assembly of actin cables [39], while actin itself, Act1p (1.13), showed invariant expression.

Stress response proteins in encapsulated yeast
Considering the seemingly starvation-stressed cells, the expression of stress response proteins are of particular interest to study. Of the 91 proteins in the data set identified as stress response proteins according to the FunCat analysis, 21 were up-regulated, while 16 were down-regulated. Notable is that proteins under the control of the transcription factors Msn2 and Msn4 were enriched in the encapsulated yeast ( Figure S4). These two transcription factors are activated in stress conditions and are an important part of the so called Environmental Stress Response (ESR) [40]. In accordance with the previously reported increased heat tolerance of similarly encapsulated cells [12], three proteins (Glc7p (1.58), Hsp12p (1.99) and Gre3p (1.46)) out of the five identified as involved in the ''heat shock response'' were up-regulated (Table 3), while the other two showed invariant (Get3p (1.17)) or statistically insignificantly increased (Bcy1 (1.39)) expression. This, together with the increase in intracellular trehalose, give the encapsulated cells an advantage compared to free cells in the response to an increased temperature. Among the proteins classified as involved in the response to osmotic and salt stress both up-regulated (Sip18p (7.77), Hsp12p (1.99) and Aip1p (1.78)) and down-  Proteins involved in the response to furfural One of the major benefits of using encapsulated yeast in 2 nd generation bioethanol production is its ability to tolerate otherwise too toxic dilute-acid hydrolysates. The tolerance of encapsulated yeast towards high levels of the pentose-derived furan aldehyde inhibitor furfural has been specifically studied [9]. Many of the proteomic responses to encapsulation observed in this study were similar to those in proteomic studies made on cells exposed to furfural [41,42]. The improved tolerance of the encapsulated cells can therefore plausibly be ascribed, at least partly, to the cells being in a state more prepared for the stress caused by the inhibitor. The reduction of furfural to furfuryl alcohol is an important part of S. cerevisiae's detoxification of lignocellulosic hydrolysates and this is believed to be performed mainly by the alcohol dehydrogenases [41,43]. It has been shown that the levels of Adh1p and Adh5p were increased in the presence of furfural [41], why an already increased level, as found in this study, would make the introduction of furfural seem less harsh to the cells and thus require less severe adaptations.
Lin et al. [41,42] suggested that yeast cells require an increased chaperone capacity when exposed to furfural, due to an increased amount of partially unfolded proteins. Of the up-regulated UPR proteins in our study, four (Ssa1p (1.32), Ssb1p (1.77), Hsp78p (1.39) and Ssb2p (1.71, -, 1.42)) belonged to the Hsp70 family, which has been shown to be up-regulated in the response to furfural [41]. In addition to these, the co-chaperone Mdj1p (1.31) and small heat shock protein Hsp26p (3.11) were also upregulated. Down-regulated UPR proteins were instead mainly related to the growth of the cells (see above). Taken together it is probable that the up-regulation of chaperones does indeed help encapsulated cells to cope with the stress caused by furfural.

2-D DIGE support nLC-MS/MS results
In addition to the nLC-MS/MS with TMTH labelling, relative quantification of proteins from cells encapsulated in alginatechitosan gel membranes compared to free cells was carried out also with 2-D DIGE. In this approach 103 spots with differential protein expression (Anova p,0.05) were chosen for subsequent MALDI TOF/TOF analysis for protein identification. Proteins were identified in 100 spots, of which 93 had highly significant hits, with a total of 52 different proteins detected (Table S3). After sorting out spots containing more than one identified protein, 59 spots with a total of 31 different proteins remained. Due to e.g. protein processing and posttranslational modifications, the same protein can be present in a number of spots [44]. For example, the abundant glycolytic proteins Hxk1p, Pdc1p and Pyk1p were found as the only protein in five spots each. The average FC values of these 31 proteins from the 59 spots were compared with the values from the nLC-MS/MS approach by dividing the ratios obtained for the same proteins by 2-D DIGE with that obtained by nLC-MS/MS ( Figure 6A and B). The comparison between 24 of the 31 proteins centred close to 1 (0.9160.07), the value expected for optimal correlation. The proteins with the highest FC values with the nLC-MS/MS approach (FC.2.5, triangles in Figure 6) were detected at lower FC in the 2-D DIGE approach. Three more outliers were found that showed different sign of the FC in the two approaches (squares in Figure 6). However, none of these showed significant changes in either of the two approaches, meaning that they centred close to 21. The average ratio determined by nLC-MS/MS followed a linear relationship with the average ratio obtained by 2-D DIGE, R MS/MS = 0.90 R DIGE , with an r 2 value of 0.96 ( Figure 6B).
In 34 of the 93 spots with significant protein hits, more than one protein was identified with significance. Such co-migration [44] is known to affect the accuracy of the quantification [45]. A total of 36 different proteins were found of which 15 were found also in the spots with only one significant hit, and 3 which were not identified in the nLC-MS/MS approach. By assigning the same FC to all significant protein hits in each spot and calculating the average ratio of the same protein from all spots with co-migrating proteins, a good correlation with the corresponding proteins quantified with the nLC-MS/MS method was obtained ( Figure 6C and D). After sorting the proteins as described in the previous section, 20 of the 33 proteins had a good correlation between the two proteomic methods, with an average ratio, R MSMS /R DIGE , of 1.0060.09 ( Figure 6C). A linear relationship R MS/MS = 0.99 R DIGE , with an r 2 value of 0.96, was obtained ( Figure 6D). It should be noted that there were 8 proteins showing different sign of the FC for the proteins quantified in spots with more than one significant protein hit, and of these, 5 proteins showed significant changes in one of the methods. Therefore it is important to keep track of whether the protein ratio is deduced from a spot with one or more significant protein hits when analysing data from 2-D DIGE. On average, the two different methods gave similar expression values and the combination of the methods is thus a good way of verifying proteomic results. A comparison of DIGE quantification with metabolic labelling quantification of spots picked from a gel showed correlations similar to what was observed in our study [46].
A FunCat localization analysis of the proteins detected as single proteins in the spots on the gels showed that all of them localized to the cytoplasm, nucleus, mitochondria or vacuole. This showed a drawback of the 2-D DIGE approach when it comes to proteomewide studies. Mainly abundant proteins are detected, such as the glycolytic ones that made up 30% of the proteins detected in the 2-D DIGE approach, and not those that are less abundant, such as those involved in e.g. transcription. Figure 5. The proteomic response on the central carbon metabolism upon encapsulation of yeast. The central carbon metabolism is presented with up-regulated proteins with fold changes (encapsulated cells compared to free cells) in green, down-regulated proteins with fold changes in red and unaffected proteins with the measured fold changes in grey. The first number represents the fold change obtained by n-LC-MS/ MS. Where applicable, this is followed by the average fold change obtained from 2-D DIGE spots with unique significant protein hits, and the average fold change obtained from spots with significant hits for co-migrating proteins in 2-D DIGE. doi:10.1371/journal.pone.0049335.g005

Concluding Remarks
Comparative proteomics of free and encapsulated S. cerevisiae CBS8066 revealed numerous changes in the cells arising from the encapsulation. Most changes could be attributed to stricter anaerobic conditions and nutrient starvation of cells arising from mass transfer limitations into the interior of the cell pellet inside the capsule. Notable were for example the up-regulation of proteins related to trehalose and glycogen synthesis and utilization, alcohol dehydrogenases and many stress-related proteins, and the downregulation of protein synthesis-related proteins. These changes mirrored the increased ethanol and decreased biomass yields compared to the free cells and also verified, on the protein level, previously reported findings of increased thermotolerance and increased levels of trehalose and glycogen inside similarly encapsulated cells. A number of changes in protein content that occurred due to the encapsulation can also be ascribed as contributing factors to the increased resistance of encapsulated cells towards furfural, one of the major lignocellulose-derived inhibitors. Figure S1 Heat map showing the proteomic differences between free and encapsulated S. cerevisiae. Heat map of the 211 significantly changed proteins between three biological replicates each of encapsulated (E1-3) and free (F1-3) yeast, with ratios normalized to the average value of the free yeast and converted to log 2 -space for centring to zero. Clusters were computed using the default settings of heatmap.2 in the gplots package of R [47]. The column clustering shows the biological homogeneity among the replicates as well as the large differences between free and encapsulated yeast, while the row clustering shows similarities in protein abundance change among the regulated proteins. Missing value is shown in white. (TIF) Figure S2 Proteome based pair-wise comparison of encapsulated and free S. cerevisiae. Volcano plot illustrating the distribution of all proteins identified with the nLC-MS/MS approach with protein names shown for statistically regulated proteins. Significantly up-and down-regulated proteins (|fold change| $1.3, x-axis; FDR adjusted p value#0.05, y-axis) are highlighted in green and red respectively. Statistically up-and down-regulated proteins with non-significant biological changes (|fold change| ,1.3) are shown in light green and orange, respectively, and proteins with non-significant differences between the free and encapsulated yeast are shown in grey. (TIF) Figure S3 GO functional enrichment analysis of differentially expressed proteins in free and encapsulated S. cerevisiae. GO categories identified by TANGO, showing enriched categories and the percentage of the up-or downregulated proteins belonging to the respective category above each bar. (TIF) Figure S4 Promoter enrichment analysis of differentially expressed proteins in free and encapsulated S. cerevisiae. A promoter enrichment analysis (p values are shown above the bars) of the genes coding for regulated proteins showed that the genes controlled by the transcription factor ABF1, for example controlling the expression of many ribosomal proteins, were over-represented among proteins down-regulated (light grey) in the encapsulated yeast. Enriched promoter usage among the upregulated proteins (dark grey) in the encapsulated yeast were instead seen among the stress sensitive promoters MSN2 and 4, as well as the stress response element, STRE, and ADR1, required for transcription of genes necessary for ethanol, glycerol and fatty acid utilization, mirroring the starvation response from lack of glucose in the inner of the capsule.