**2. Proteomic methodology in micro and macroalgae**

Sample preparation, in particular the quality of protein extraction, is critical to the successful resolution of 2-DE patterns. In fact, when protein extraction protocols from higher plants are applied to algae, the 2-DE resolution is reduced (Contreras et al., 2008; Hippler et al., 2011). Due to the large variation in cellular biochemical composition among diverse organisms, which affects solubility and recovery of a complex mixture from the sample, there are no 2- DE sample preparation protocols accurate for all organisms. In macro and microalgae the protein extraction protocol must be optimized, due to the high concentration of photosynthetic pigments that are known to interfere with the resolution of the 2-DE gels (e.g. Contreras et al., 2008; Wang et al., 2003; Wong et al., 2006). Particularly, in macroalgae protein extraction is difficult due to a low concentration and the co-extraction of contaminants such as anionic polysaccharides, polyphenols and salts, which are highly concentrated in the tissue (Chinnasamy & Rampitsch, 2006; Cremer & Van de Walle, 1985; Flengsrub & Kobro, 1989; Mechin et al., 2003). These contaminants pose a significant difficulty for 2-DE, as they cause horizontal and vertical streaking, smearing and a reduction in the number of distinctly resolved protein spots. Thus, the selection of the most appropriate protein extraction method is necessary in order to obtain high quality extracts, and therefore, a high quality 2-DE pattern. For a better understanding and explanation of the current techniques and methodology in algae proteomic, this chapter has been divided in two sections: microalgae and macroalgae methodology.

### **2.1 Microalgae methodology**

24 Gel Electrophoresis – Advanced Techniques

biomolecular level, only a low production of oxidized proteins is recorded during

The results mentioned above, indicate that desiccation in *P. columbina* causes an overproduction of ROS, which is efficiently attenuated. Morphological and photosynthetic changes could be operating as tolerance mechanisms, due to the fact that these responses principally prevent biomolecular alterations, protein aggregation and cellular collapse. For example, it has been proposed that cell wall folding is a cellular strategy used to prevent tearing the plasmalemma from the cell wall during desiccation, ensuring cell integrity (Contreras-Porcia et al., 2011b). The activation of antioxidant enzymes and the photoinhibition of the photosynthetic apparatus help to explain the attenuation of ROS. Thus, ROS excess is buffered by the activation of several physiological and biochemical responses, which suggest a mechanism allowing this plant to tolerate desiccation (Contreras-Porcia et al., 2011b). The ecophysiological responses in this species help, in part, to account for its position and dominance at the highest level in the intertidal zone, and thereby, suggesting desiccation stress tolerance as a determinant trait for explaining that situation. In fact, our recent results demonstrate that the magnitude of the effects generated by desiccation in algae is related to the position of the species in the intertidal zone. Additionally, this work demonstrated the exceptional metabolism of *P. columbina* used to buffer this stress condition. Thus, the determinations of novel metabolic pathways are necessaries in order to fully understand the high desiccation tolerance in this species, for example at the proteomic level. In fact, in this time our forces are concentrated in resolving

the proteomic profile of this species under natural hydration and desiccation stress.

organisms in order to comprehend their ecophysiological behaviour.

**2. Proteomic methodology in micro and macroalgae** 

Finally, the need to unravel the mechanisms associated with tolerance to different environmental factors by algal species opens the electrophoretic and proteomic approximations as important tools in comprehending and explaining the observed tolerances. However, little information regarding electrophoretic and proteomic analysis is available in algal species. Compared with other group of organisms (e.g. vascular plant or animals) protein extraction in macroalgae has been extraordinary difficult, due principally to the limited knowledge at biochemical and molecular levels. In this context, the present chapter aims to understand the different proteomic approaches utilized in this group of

Sample preparation, in particular the quality of protein extraction, is critical to the successful resolution of 2-DE patterns. In fact, when protein extraction protocols from higher plants are applied to algae, the 2-DE resolution is reduced (Contreras et al., 2008; Hippler et al., 2011). Due to the large variation in cellular biochemical composition among diverse organisms, which affects solubility and recovery of a complex mixture from the sample, there are no 2- DE sample preparation protocols accurate for all organisms. In macro and microalgae the protein extraction protocol must be optimized, due to the high concentration of photosynthetic pigments that are known to interfere with the resolution of the 2-DE gels (e.g. Contreras et al., 2008; Wang et al., 2003; Wong et al., 2006). Particularly, in macroalgae protein extraction is difficult due to a low concentration and the co-extraction of contaminants such as anionic polysaccharides, polyphenols and salts, which are highly concentrated in the tissue (Chinnasamy & Rampitsch, 2006; Cremer & Van de Walle, 1985;

desiccation, due to the efficient antioxidant system of this alga.

It is important to highlight that due to the small size of microalgae, all of the protein extraction protocols for these organisms begin with a centrifugation step in order to pellet cells. This helps to concentrate cells, and consequently allows a correct extraction of the desired proteins.

#### **2.1.1 Early proteomic studies**

One of the first proteomics studies on microalgae dates from the year 1972, in which Mets & Bogorad showed alterations in the chloroplast ribosomes proteins of erythromycin-resistant mutants of *Chlamydomonas reinhardtii* (Plantae, Chlorophyta) compared to the wild-type. The ribosomal protein extraction performed on this work was the LiCl-urea method described by Leboy et al. (1971) that was developed for *Escherichia coli* (as cited in Mets & Bogorad, 1972). Thus, the Mets & Bogorad work was a precursor to microalgae proteomic studies. Here, ribosomes are disrupted and freed of RNA by adding LiCl. Then, the samples are centrifuged to precipitate total RNA and the supernatant, which contains the proteins, is retained.

Several studies in the same decade also focused their attention on characterizing ribosomal proteins (e.g. Götz & Arnold, 1980; Hanson et al., 1974). The Hanson et al. (1974) work based their protocols on the Mets & Bogorad (1972) research paper and also used *C. reinhardtii* as model species. Instead, in 1980 Götz & Arnold used a different ribosomal protein extraction after testing several protocols. The procedure chosen was the acetic-acid method in presence of MgCl2 according to Kaltschmidt & Wittmann (1972), method that was also first developed for *E. coli*. In this method, MgCl2 and glacial acetic acid are added to the ribosome suspension, and then the mixture is centrifuged to pellet RNA. For better mixture cleaning, the pellet can be extracted a second time in the same way.

Not all studies from this decade focused their attention on ribosomal proteins as was the case of the work of Piperno et al. (1977), in which the protein mixture came from *Chlamydomona* flagella and axonemes. It is important to highlight this research since the extraction method used was very rustic. After the flagella and axoneme separation, the proteins were dissolved only in SDS and kept for 2-DE analysis.

#### **2.1.2 Current proteomic studies**

Recent studies evaluate more complex protein mixtures, so the method chosen must be more accurate in extracting proteins with minimum contaminants and interferents. In fact, a work in *C. reinhardtii* that performed an analysis of all the thylakoid membranes proteins used a more complex protocol (Hippler et al., 2001) than the ones previously discussed in

Proteomics in Seaweeds: Ecological Interpretations 27

correct horizontal migration, however, the protocols varied according both to the biological model and the protein type extracted (i.e. soluble or membrane proteins). Finally, proteins separated in the IEF step are loaded in to the second dimension (SDS-PAGE). Thus, in this

In the work of Mets & Bogorad (1972), ribosomal proteins were only run in the IEF step at 1.5 mA for 4 h but it was enough to separate them due to the low quantity of proteins that were obtained in this extraction. The second dimension was run at 25 mA, enough time to allow the protein migration, since the 2-DE gel patterns are very clear and well resolved. Also, no vertical or horizontal streaking is present, thereby, permitting clear protein detection. It is not astonishing to observe similar 2-DE patterns in quality terms in the work by Hanson et al. (1974), since both the ribosomal protein extraction and the two-dimensional gel electrophoresis were performed essentially as described by Mets & Bogorad (1972). Therefore, no vertical or horizontal streaking was found, resulting in gels with high resolution. Both protein extraction and gel electrophoresis proved to be very efficient and adequate for protein separation. However, it should be emphasized that the patterns from both works are easier to obtain, since the protein mixture is very simple since it only came

Unlike the ribosomal protein mixture, others do not generate 2-DE patterns with the same resolution. One case may be flagella and axonemes of *C. reinhardtii* in which a larger number of proteins are founded. Piperno et al. (1977) compared proteins of this structure from both wild-type and paralyzed mutants strains of this species. The IEF step was performed at 300 V for 18-19 h and followed by 400 V for 1.5 h. The second dimension was first run at 25 mA (initial voltage: 60 V) for 1 h and then it was raised to 50 mA. The run continued until the dye in the molecular weight standard had reached the bottom according to Ames and Nikaido (1976) (as cited in Piperno et al., 1977). The 2-DE gels had minimum vertical streaking, but lot of horizontal streaking and big stains regardless of the sample. The horizontal streaking could be due to a more complex protein mixture; however, the protein extraction protocol of this work is very deficient since it only uses SDS. Regardless of this, some spots were easily detected in the gels allowing for comparison between wild-types and mutant strains. Finally, in the work of Götz & Arnold (1980) ribosomal proteins from eight species were evaluated with two gels showing clear and well-resolved 2-DE patterns. The protein extraction was well suited for all species. Therefore, the MgCl2-acetic acid method proved efficient in a large number of species, but again it was used to extract only ribosomal

In more recent papers, such as those described in the previous section, the rehydration buffer used to resuspend the proteins prior to gel loading is key for the proper migration of proteins. The most commonly used buffer contains the reagents thiourea, urea, CHAPS, DTT, ampholytes and bromophenol blue. However, the concentrations of the reagents vary among the different works, so choosing the most accurate one is no easy task. As an example, we chose the protocol described by Wang et al. (2003) in which several reagents were tested to determine which one that yielded the best 2-DE pattern (i.e. no streaking and more defined spots) (see Appendix A). The majority of researchers state in their works that

section rehydration buffers, IEF steps and second dimension gels will be analyzed.

**2.1.3.1 Early proteomic studies** 

from ribosome structures.

protein, so minimum contaminants are present.

**2.1.3.2 Current proteomic studies** 

this chapter. This method uses methanol in order to precipitate cell debris and retains proteins in the supernatant. Then, chloroform is added and the sample is vortexed and centrifuged. The upper phase containing DNA is discarded. Afterwards, methanol is added to the sample in order to pellet proteins and leave the RNA in the aqueous phase. Finally, the pellet is washed with methanol in order to remove contaminants.

In 2003, a study tested different protein extraction protocols in the microalga *Haematococcus pluvialis* (Plantae, Chlorophyta) in order to determine which ones yielded better results (Wang et al., 2003). After cell disruption, the samples were dialysed to remove any salt left in the samples, which are known to interfere in the IEF step. After the dyalisis, each sample was treated in three different ways: i) proteins were left to precipitate in a non-denaturing preparation, ii) a mixing of dialysate with acetone was kept at -20 °C o/n to allow complete precipitation and iii) a mixing of dialysate with TCA in acetone containing βmercaptoethanol also kept at -20°C o/n. Methods ii) and iii) were denaturing procedures but it was procedure iii) the one that yielded 2-DE gels with higher resolution (detailed protocol in Appendix A).

The work by Kim et al. (2005) is interesting since the protein extraction protocol used is relatively simple when compared to others (e.g. Wang et al., 2003; Contreras et al., 2008) (detailed protocol in Appendix A). Proteins of *Nannochloropsis oculata* (Chromista, Ochrophyta) are obtained in very short time compared to the other microalgae protocols, however, not with the same quality as the more complex protocols. In another *C. reinhardtii* work, but this time conducting a whole cell proteomic study (Förster et al., 2006), a protocol described by Mathesius et al. (2001) that is suited for root proteins was used (detailed protocol in Appendix A). This procedure is denaturing and relatively simple, but includes washing steps that help to improve the quality of the protein extracts compared to the one used on *N. oculata* (Kim et al., 2005). A work from 2009 in the microalga *Haematococcus lacustris* also had a denaturing protocol in which pelleted cells were grounded to a fine powder in liquid nitrogen (Tran et al., 2009). Then, they are disrupted with a lysis buffer containing urea, thiourea, DTT, CHAPS, Tris-base and a plant protease inhibitor cocktail tablet. Samples are centrifuged to separate cell debris, and then the pellet is resuspended in acetone to precipitate proteins and remove contaminants. Finally, the samples are again centrifuged, acetone is removed by air-drying and pellet is clean and ready for 2-DE gels.

*Chlamydomonas reinhardtii* is one of the most studied microalgae worldwide and as noted in this chapter, proteomics studies are no exception. Another protocol for this algae dates from 2011, in this one the cells are disrupted with a lysis buffer containing urea, CHAPS and thiourea (Mahong et al., 2012). The sample is centrifuged and the supernatant retained. To eliminate possible photosynthetic pigments and other hydrophobic compounds, the samples are washed with ice-cold acetone. Then, each sample is centrifuged, and the pellet is ready for electrophoretic processes.

#### **2.1.3 Gel loading: From proteins to gels**

Another key step in obtaining 2-DE gels is gel loading and gel running. After protein extraction, the pellet must be resuspended in a rehydration buffer, which is generally the same in all works. Then, proteins are loaded in order to perform the IEF step for their correct horizontal migration, however, the protocols varied according both to the biological model and the protein type extracted (i.e. soluble or membrane proteins). Finally, proteins separated in the IEF step are loaded in to the second dimension (SDS-PAGE). Thus, in this section rehydration buffers, IEF steps and second dimension gels will be analyzed.

#### **2.1.3.1 Early proteomic studies**

26 Gel Electrophoresis – Advanced Techniques

this chapter. This method uses methanol in order to precipitate cell debris and retains proteins in the supernatant. Then, chloroform is added and the sample is vortexed and centrifuged. The upper phase containing DNA is discarded. Afterwards, methanol is added to the sample in order to pellet proteins and leave the RNA in the aqueous phase. Finally,

In 2003, a study tested different protein extraction protocols in the microalga *Haematococcus pluvialis* (Plantae, Chlorophyta) in order to determine which ones yielded better results (Wang et al., 2003). After cell disruption, the samples were dialysed to remove any salt left in the samples, which are known to interfere in the IEF step. After the dyalisis, each sample was treated in three different ways: i) proteins were left to precipitate in a non-denaturing preparation, ii) a mixing of dialysate with acetone was kept at -20 °C o/n to allow complete precipitation and iii) a mixing of dialysate with TCA in acetone containing βmercaptoethanol also kept at -20°C o/n. Methods ii) and iii) were denaturing procedures but it was procedure iii) the one that yielded 2-DE gels with higher resolution (detailed

The work by Kim et al. (2005) is interesting since the protein extraction protocol used is relatively simple when compared to others (e.g. Wang et al., 2003; Contreras et al., 2008) (detailed protocol in Appendix A). Proteins of *Nannochloropsis oculata* (Chromista, Ochrophyta) are obtained in very short time compared to the other microalgae protocols, however, not with the same quality as the more complex protocols. In another *C. reinhardtii* work, but this time conducting a whole cell proteomic study (Förster et al., 2006), a protocol described by Mathesius et al. (2001) that is suited for root proteins was used (detailed protocol in Appendix A). This procedure is denaturing and relatively simple, but includes washing steps that help to improve the quality of the protein extracts compared to the one used on *N. oculata* (Kim et al., 2005). A work from 2009 in the microalga *Haematococcus lacustris* also had a denaturing protocol in which pelleted cells were grounded to a fine powder in liquid nitrogen (Tran et al., 2009). Then, they are disrupted with a lysis buffer containing urea, thiourea, DTT, CHAPS, Tris-base and a plant protease inhibitor cocktail tablet. Samples are centrifuged to separate cell debris, and then the pellet is resuspended in acetone to precipitate proteins and remove contaminants. Finally, the samples are again centrifuged, acetone is removed by air-drying and pellet is clean and ready for 2-DE gels.

*Chlamydomonas reinhardtii* is one of the most studied microalgae worldwide and as noted in this chapter, proteomics studies are no exception. Another protocol for this algae dates from 2011, in this one the cells are disrupted with a lysis buffer containing urea, CHAPS and thiourea (Mahong et al., 2012). The sample is centrifuged and the supernatant retained. To eliminate possible photosynthetic pigments and other hydrophobic compounds, the samples are washed with ice-cold acetone. Then, each sample is centrifuged, and the pellet is ready

Another key step in obtaining 2-DE gels is gel loading and gel running. After protein extraction, the pellet must be resuspended in a rehydration buffer, which is generally the same in all works. Then, proteins are loaded in order to perform the IEF step for their

the pellet is washed with methanol in order to remove contaminants.

protocol in Appendix A).

for electrophoretic processes.

**2.1.3 Gel loading: From proteins to gels** 

In the work of Mets & Bogorad (1972), ribosomal proteins were only run in the IEF step at 1.5 mA for 4 h but it was enough to separate them due to the low quantity of proteins that were obtained in this extraction. The second dimension was run at 25 mA, enough time to allow the protein migration, since the 2-DE gel patterns are very clear and well resolved. Also, no vertical or horizontal streaking is present, thereby, permitting clear protein detection. It is not astonishing to observe similar 2-DE patterns in quality terms in the work by Hanson et al. (1974), since both the ribosomal protein extraction and the two-dimensional gel electrophoresis were performed essentially as described by Mets & Bogorad (1972). Therefore, no vertical or horizontal streaking was found, resulting in gels with high resolution. Both protein extraction and gel electrophoresis proved to be very efficient and adequate for protein separation. However, it should be emphasized that the patterns from both works are easier to obtain, since the protein mixture is very simple since it only came from ribosome structures.

Unlike the ribosomal protein mixture, others do not generate 2-DE patterns with the same resolution. One case may be flagella and axonemes of *C. reinhardtii* in which a larger number of proteins are founded. Piperno et al. (1977) compared proteins of this structure from both wild-type and paralyzed mutants strains of this species. The IEF step was performed at 300 V for 18-19 h and followed by 400 V for 1.5 h. The second dimension was first run at 25 mA (initial voltage: 60 V) for 1 h and then it was raised to 50 mA. The run continued until the dye in the molecular weight standard had reached the bottom according to Ames and Nikaido (1976) (as cited in Piperno et al., 1977). The 2-DE gels had minimum vertical streaking, but lot of horizontal streaking and big stains regardless of the sample. The horizontal streaking could be due to a more complex protein mixture; however, the protein extraction protocol of this work is very deficient since it only uses SDS. Regardless of this, some spots were easily detected in the gels allowing for comparison between wild-types and mutant strains. Finally, in the work of Götz & Arnold (1980) ribosomal proteins from eight species were evaluated with two gels showing clear and well-resolved 2-DE patterns. The protein extraction was well suited for all species. Therefore, the MgCl2-acetic acid method proved efficient in a large number of species, but again it was used to extract only ribosomal protein, so minimum contaminants are present.

#### **2.1.3.2 Current proteomic studies**

In more recent papers, such as those described in the previous section, the rehydration buffer used to resuspend the proteins prior to gel loading is key for the proper migration of proteins. The most commonly used buffer contains the reagents thiourea, urea, CHAPS, DTT, ampholytes and bromophenol blue. However, the concentrations of the reagents vary among the different works, so choosing the most accurate one is no easy task. As an example, we chose the protocol described by Wang et al. (2003) in which several reagents were tested to determine which one that yielded the best 2-DE pattern (i.e. no streaking and more defined spots) (see Appendix A). The majority of researchers state in their works that

Proteomics in Seaweeds: Ecological Interpretations 29

Finally, the protocols that yielded the 2-DE images of higher quality were those developed by Wang et al. (2003) and Mahong et al. (2012). In both works, total proteins were extracted from two different microalgae, *H. pluvialis* and *C. reinhardtii* respectively. Highlight that both protocols are the most complex ones among all six analyzed. Gels from both works succeeded in having reduced streaking as well as defined, highly stained and high number of spots. Nevertheless, if one must choose between both, it is Mahong et al. (2012) protocol the one with the best results since gels in this work have minimum background allowing an

The difficulty in obtaining high quality 2-DE gels from macroalgae was first highlighted by Wong et al. (2006), who obtained algal proteins from *Gracilaria changii* (Plantae, Rhodophyta) using four different extraction methods: 1) direct precipitation by trichloroacetic acid/acetone, 2) direct lysis using urea buffer, 3) tris buffer and 4) phenol/chloroform. However, only methods 3) and 4) were compared for their suitability to generate *G. changii* proteins for two-dimensional gel electrophoresis. It was stated in this work that the phenol/chloroform method (detailed protocol in Appendix B) was Ideal for obtaining well resolved 2-DE patterns. Nevertheless, the quality of the 2-DE profiles was poor due to the presence of high amounts of interfering substances accompanied by low protein yield and horizontal and vertical streaking along gels regardless the pH gradient.

As part of an on-going work focused on unravelling the metabolic processes occurring in physiologically stressed brown macroalgae, a new method for protein extraction that minimizes the co-extraction of non-protein compounds using two structurally distinct brown algal species *Scytosiphon gracilis* (Chromista, Ochrophyta) (Contreras et al., 2007b) and *Ectocarpus siliculosu*s (Chromista, Ochrophyta) (Contreras et al., 2008) was developed. In order to do this, several protein extraction methods available in the literature were tested. However, neither of the previous protocols was ideal for obtaining a good quality algal protein extraction, due to high background noise, band distortion, and more importantly, very low protein dissolution. The protocol developed in this work allowed the use of a highly resolving 2-DE protein analyses, providing the opportunity to unravel potentially novel physiological processes unique to this group of marine organisms (see Table 1 and Results section). Specifically, the protocol uses an initial desalting step with Milli Q water – phosphate buffer in order to remove the salt from the algal tissues. Afterwards, the tissue is pulverized using liquid nitrogen and homogenized with sucrose, EDTA and CHAPS. The proteins are extracted using phenol and washed with ammonium acetate. Finally, the quality of the extracted proteins is improved by using the 2-D clean-

In another important proteomic work with macroalgae developed by Kim et al. (2008) and published contemporarily with the Contreras et al. (2008) work, using as models the red algae *Bostrychia radicans* and *B. moritziana* (Plantae, Rhodophyta), used a lysis buffer comprised principally by urea and thiourea (detailed protocol in Appendix B). Although these species belong to the same group of red algae like *G. changii*, the simplicity of this method utilized in comparison with the phenol one (Wong et al., 2006) is due to the

morphological characteristics of this species (see image in Appendix B).

easier spot detection.

Up Kit (GE Healthcare).

**2.2 Macroalgae methodology** 

Thus, this method is not fully accurate for this algal species.

after resuspending the proteins, the mixture must be left at room temperature for at least 1 h (e.g. Hippler et al., 2001; Kim et al., 2005; Tran et al., 2009). Likewise, the amount of proteins normally loaded is 500 µg, concentration enough to yield well resolved gels (e.g. Förster et al., 2006; Mahong et al., 2012; Wang et al., 2003).

The IEF profile contains several steps, which vary between the different works, so making comparisons is complicated and not very productive. Nowadays, researchers worldwide use IPG gel strips for a better protein migration, which leads to a better 2-DE pattern (e.g. Mahong et al., 2012; Wang et al., 2003). Having said that, all IPG gel strips must be first rehydrated for at least 10 h before setting the IEF profile. As an example we chose the IEF profile of Wang et al. (2003) which was initiated at 250 V for 15 min, and gradually ramped to 10,000 V over 5 h, and remained at 10,000 V for an additional 6 h.

After the IEF steps and prior to the second dimension, IPG gel strips must be incubated twice in an equilibration buffer containing Tris-HCl, urea, glycerol and SDS. The first time DTT is added to the equilibration buffer in order to denaturate proteins, whereas the second time iodoacetamide is added to alkylate the reduced cysteines and inhibit protein refolding. After equilibration, IPG gel strips are ready to be loaded on to the second dimensional SDS-PAGE for the vertical protein separation (i.e. according to their molecular weight). Gel thickness will vary in each experiment in order to allow the desired protein separation. Regardless of this, gels are run until the bromophenol blue reaches the bottom of the gel since it migrates faster than the proteins. The last step for obtaining the 2-DE gel is gel staining in which two principal stains are used: blue Coomassie and silver nitrate. Regardless of this, generally prior to staining, the gels are washed with deionised water. After staining, the excess of dye is removed with deionised water to obtain well-defined gels with minimum background noise.

Now with the gels stained, we are able to determine which protocol(s) yielded the best 2-DE gel(s) in terms of patterns quality (i.e. minimum or none streaking, spots with defined circles, a maximum spot number and high spot intensity). In the work by Kim et al. (2005) 2- DE gel images show smearing, some vertical streaking and high horizontal streaking specifically in the acidic side of the gel. Also, spots are not well-defined circles and are overlapped among them. Similar were the image gels by Tran et al. (2009), because smearing as well as vertical and horizontal streaking are present in the acidic part of the 2-DE gel. Also several spots were overlapped among them; nevertheless a few of them were well defined. These were the two protocols that yielded the worst results (e.g. poor gel resolution quality) and this must be to the simplicity of the protein extraction protocols used. The two protocols that follow in terms of 2-DE gel quality are those of Hippler et al. (2001) and Förster et al. (2006). In both works 2-DE gels are of high quality, which obviously obey more complex protein extraction protocols. In the oldest work, there are several traits that give this images high quality: i) minimum horizontal streaking, ii) well defined spots (i.e circle shaped), iii) highly stained spots and iv) high number of spots (since only thylakoid membrane proteins were extracted) (Hippler et al., 2001). The high quality of 2-DE gels is probably due to that only a portion of the cell proteins was extracted having less contaminants interfering in both IEF and second dimension. Förster et al. (2006) 2-DE gel images show a high number of spots and most of them are well define with almost no smearing. However, a lot of vertical streaking is observed in the gels, thus the problems must be found in the second dimension since minimum horizontal streaking is present.

Finally, the protocols that yielded the 2-DE images of higher quality were those developed by Wang et al. (2003) and Mahong et al. (2012). In both works, total proteins were extracted from two different microalgae, *H. pluvialis* and *C. reinhardtii* respectively. Highlight that both protocols are the most complex ones among all six analyzed. Gels from both works succeeded in having reduced streaking as well as defined, highly stained and high number of spots. Nevertheless, if one must choose between both, it is Mahong et al. (2012) protocol the one with the best results since gels in this work have minimum background allowing an easier spot detection.

#### **2.2 Macroalgae methodology**

28 Gel Electrophoresis – Advanced Techniques

after resuspending the proteins, the mixture must be left at room temperature for at least 1 h (e.g. Hippler et al., 2001; Kim et al., 2005; Tran et al., 2009). Likewise, the amount of proteins normally loaded is 500 µg, concentration enough to yield well resolved gels (e.g. Förster et

The IEF profile contains several steps, which vary between the different works, so making comparisons is complicated and not very productive. Nowadays, researchers worldwide use IPG gel strips for a better protein migration, which leads to a better 2-DE pattern (e.g. Mahong et al., 2012; Wang et al., 2003). Having said that, all IPG gel strips must be first rehydrated for at least 10 h before setting the IEF profile. As an example we chose the IEF profile of Wang et al. (2003) which was initiated at 250 V for 15 min, and gradually ramped

After the IEF steps and prior to the second dimension, IPG gel strips must be incubated twice in an equilibration buffer containing Tris-HCl, urea, glycerol and SDS. The first time DTT is added to the equilibration buffer in order to denaturate proteins, whereas the second time iodoacetamide is added to alkylate the reduced cysteines and inhibit protein refolding. After equilibration, IPG gel strips are ready to be loaded on to the second dimensional SDS-PAGE for the vertical protein separation (i.e. according to their molecular weight). Gel thickness will vary in each experiment in order to allow the desired protein separation. Regardless of this, gels are run until the bromophenol blue reaches the bottom of the gel since it migrates faster than the proteins. The last step for obtaining the 2-DE gel is gel staining in which two principal stains are used: blue Coomassie and silver nitrate. Regardless of this, generally prior to staining, the gels are washed with deionised water. After staining, the excess of dye is removed with deionised water to obtain well-defined gels

Now with the gels stained, we are able to determine which protocol(s) yielded the best 2-DE gel(s) in terms of patterns quality (i.e. minimum or none streaking, spots with defined circles, a maximum spot number and high spot intensity). In the work by Kim et al. (2005) 2- DE gel images show smearing, some vertical streaking and high horizontal streaking specifically in the acidic side of the gel. Also, spots are not well-defined circles and are overlapped among them. Similar were the image gels by Tran et al. (2009), because smearing as well as vertical and horizontal streaking are present in the acidic part of the 2-DE gel. Also several spots were overlapped among them; nevertheless a few of them were well defined. These were the two protocols that yielded the worst results (e.g. poor gel resolution quality) and this must be to the simplicity of the protein extraction protocols used. The two protocols that follow in terms of 2-DE gel quality are those of Hippler et al. (2001) and Förster et al. (2006). In both works 2-DE gels are of high quality, which obviously obey more complex protein extraction protocols. In the oldest work, there are several traits that give this images high quality: i) minimum horizontal streaking, ii) well defined spots (i.e circle shaped), iii) highly stained spots and iv) high number of spots (since only thylakoid membrane proteins were extracted) (Hippler et al., 2001). The high quality of 2-DE gels is probably due to that only a portion of the cell proteins was extracted having less contaminants interfering in both IEF and second dimension. Förster et al. (2006) 2-DE gel images show a high number of spots and most of them are well define with almost no smearing. However, a lot of vertical streaking is observed in the gels, thus the problems must be found in the second dimension since minimum horizontal streaking is present.

al., 2006; Mahong et al., 2012; Wang et al., 2003).

with minimum background noise.

to 10,000 V over 5 h, and remained at 10,000 V for an additional 6 h.

The difficulty in obtaining high quality 2-DE gels from macroalgae was first highlighted by Wong et al. (2006), who obtained algal proteins from *Gracilaria changii* (Plantae, Rhodophyta) using four different extraction methods: 1) direct precipitation by trichloroacetic acid/acetone, 2) direct lysis using urea buffer, 3) tris buffer and 4) phenol/chloroform. However, only methods 3) and 4) were compared for their suitability to generate *G. changii* proteins for two-dimensional gel electrophoresis. It was stated in this work that the phenol/chloroform method (detailed protocol in Appendix B) was Ideal for obtaining well resolved 2-DE patterns. Nevertheless, the quality of the 2-DE profiles was poor due to the presence of high amounts of interfering substances accompanied by low protein yield and horizontal and vertical streaking along gels regardless the pH gradient. Thus, this method is not fully accurate for this algal species.

As part of an on-going work focused on unravelling the metabolic processes occurring in physiologically stressed brown macroalgae, a new method for protein extraction that minimizes the co-extraction of non-protein compounds using two structurally distinct brown algal species *Scytosiphon gracilis* (Chromista, Ochrophyta) (Contreras et al., 2007b) and *Ectocarpus siliculosu*s (Chromista, Ochrophyta) (Contreras et al., 2008) was developed. In order to do this, several protein extraction methods available in the literature were tested. However, neither of the previous protocols was ideal for obtaining a good quality algal protein extraction, due to high background noise, band distortion, and more importantly, very low protein dissolution. The protocol developed in this work allowed the use of a highly resolving 2-DE protein analyses, providing the opportunity to unravel potentially novel physiological processes unique to this group of marine organisms (see Table 1 and Results section). Specifically, the protocol uses an initial desalting step with Milli Q water – phosphate buffer in order to remove the salt from the algal tissues. Afterwards, the tissue is pulverized using liquid nitrogen and homogenized with sucrose, EDTA and CHAPS. The proteins are extracted using phenol and washed with ammonium acetate. Finally, the quality of the extracted proteins is improved by using the 2-D clean-Up Kit (GE Healthcare).

In another important proteomic work with macroalgae developed by Kim et al. (2008) and published contemporarily with the Contreras et al. (2008) work, using as models the red algae *Bostrychia radicans* and *B. moritziana* (Plantae, Rhodophyta), used a lysis buffer comprised principally by urea and thiourea (detailed protocol in Appendix B). Although these species belong to the same group of red algae like *G. changii*, the simplicity of this method utilized in comparison with the phenol one (Wong et al., 2006) is due to the morphological characteristics of this species (see image in Appendix B).

Proteomics in Seaweeds: Ecological Interpretations 31

<sup>4</sup>*pI*<sup>7</sup> **MW (kDa)** 

Fig. 1. 2-D proteome of *Porphyra columbina* under natural desiccation. First dimension was performed on a linear gradient IPG strip of pH 4-7 using 600 µg of total proteins. The 12.5%

*pI* but different molecular weight were found, indicating that the *ery-M2* gene is involved in determining the properties of that protein. Hanson et al. (1974) performed 2-DE gels in order to characterize cytoplasmic and chloroplastic ribosomal proteins. Their results showed that the number of proteins in both small and large subunits was higher in cytoplasmic than in chloroplastic ribosomes, indicating that cytoplasmic ribosomes are more complex. Another study did a comparison between the ribosomal proteins of 8 species including *C. reinhardtii*  (Götz & Arnold, 1980). The results showed that the number of proteins in both subunits was similar among all species, and that the *Polytoma papillatun* (Plantae, Chlorophyta) proteins

Piperno et al. (1977) analysed the flagella proteins of wild-type and paralyzed mutants of *C. reinhardtii* in order to identify the mutated protein that incapacitates the mobility in mutant strains. In the flagella of *pf* 14, which completely lack radial spokes and associated spokeheads, 12 polypeptides were missing. Also in *pf* 1 flagella, where spokes are clearly present but spokeheads appear to be absent, 6 polypeptides were missing. Then, protein electrophoretic studies confirmed the phenotypical characteristics displayed by both paralyzed mutants, where the missing proteins may be involved in spokes and spokeheads correct morphology. Another work in *C. reinhardtii,* used a proteomic approach to analyse photosynthetic thylakoid membrane proteins isolated from wild-type and mutant strains (Hippler et al., 2001). The two mutant strains were Δ*ycf4* (PSI-deficient) and *crd1* (which is conditionally reduced in PSI and LHCI under copper-deficiency). In this work more than 30 different LHCP spots were identified using a tandem quadrupole mass spectrometer,

were the most similar to those of *C. reinhardtii* in terms of protein homology.

SDS-PAGE gel was stained with colloidal Coomassie blue.

120 85

50

35

25

20

The work described by Yotsukura et al. (2010) presents a similar protocol to the one described by Contreras et al. (2008). Here, proteins are extracted from the brown alga *Saccharina japonica* (Chromista, Ochrophyta)*,* important kelp described principally on the coastal areas of northern Japan. In this protocol, the protein extraction was improved by using phenol as the principal component in the lysis buffer (detailed protocol in Appendix B). This protocol was also used in *Ecklonia cava* (Chromista, Ochrophyta), other important kelp found on the coast along the Sea of Japan, and also good quality 2-D patterns were obtained (Yotsukura et al., 2012). The use of phenol in the protein extraction described by Contreras et al. (2008) has been recently used in the red alga *Porphyra columbina* in order to identify the proteins that are overinduced during desiccation stress tolerance responses. A highly resolved 2-DE protein was obtained using this method (Fig. 1), with minor modifications (detailed protocol in Appendix B), such as an important rinse of the protein pellet due principally to the over-production of phycocyanin and phycoerythrin. Thus, the phenol protocol developed by Contreras et al. (2008) could be used in macroalgae species from different taxonomic groups.

The first dimension of the 2-DE in the works mentioned above used approximately 200-500 µg of extracted proteins. However, for the isoelectric focusing (IEF) the protocols varied depending on the algal species used. For example, in *Bostrychia radicans* and *B. moritziana*, the voltage was linearly increased from 150-3,500 V during 3 h, followed by a constant 3,500 V, with focusing complete after 96 V. In *Scytosiphon gracilis* and *Ectocarpus siliculosu*s, on the other hand, the strips are actively rehydrated for 15 h in IEF buffer containing the proteins and focused at 20ºC with the following successive steps: a liner increase from 0 to 250 V for 15 min, a gradient phase from 250 V to 10,000 V for 4 h, and the a hold phase at 10,000 V for a total of 60 kVh. Using this protocol, the IEF for *Porphyra columbina* has some modifications, principally in a total operational voltage of 70 kVh.
