INFECTION IN COTTON AND COFFEE PLANTSainfo.cnptia.embrapa.br/digital/bitstream/item/... · METHODOLOGICAL EVALUATION OF 2-DE TO STUDY ROOT PROTEOMICS DURING NEMATODE INFECTION IN

Full Terms & Conditions of access and use can be found athttp://www.tandfonline.com/action/journalInformation?journalCode=lpbb20

Preparative Biochemistry & Biotechnology

ISSN: 1082-6068 (Print) 1532-2297 (Online) Journal homepage: http://www.tandfonline.com/loi/lpbb20

METHODOLOGICAL EVALUATION OF 2-DE TOSTUDY ROOT PROTEOMICS DURING NEMATODEINFECTION IN COTTON AND COFFEE PLANTS

Octavio L. Franco , Jackeline L. Pereira , Paulo H. A. Costa , Thales L. Rocha ,Érika V. S. Albuquerque , Maria F. Grossi-de-Sá , Regina M. D. G. Carneiro , RuiG. Carneiro & Angela Mehta

To cite this article: Octavio L. Franco , Jackeline L. Pereira , Paulo H. A. Costa , Thales L.Rocha , Érika V. S. Albuquerque , Maria F. Grossi-de-Sá , Regina M. D. G. Carneiro , Rui G.Carneiro & Angela Mehta (2010) METHODOLOGICAL EVALUATION OF 2-DE TO STUDYROOT PROTEOMICS DURING NEMATODE INFECTION IN COTTON AND COFFEE PLANTS,Preparative Biochemistry & Biotechnology, 40:2, 152-163, DOI: 10.1080/10826060903558976

To link to this article: https://doi.org/10.1080/10826060903558976

Published online: 08 Mar 2010.

Submit your article to this journal

Article views: 129

Citing articles: 8 View citing articles

http://www.tandfonline.com/action/journalInformation?journalCode=lpbb20http://www.tandfonline.com/loi/lpbb20http://www.tandfonline.com/action/showCitFormats?doi=10.1080/10826060903558976https://doi.org/10.1080/10826060903558976http://www.tandfonline.com/action/authorSubmission?journalCode=lpbb20&show=instructionshttp://www.tandfonline.com/action/authorSubmission?journalCode=lpbb20&show=instructionshttp://www.tandfonline.com/doi/citedby/10.1080/10826060903558976#tabModulehttp://www.tandfonline.com/doi/citedby/10.1080/10826060903558976#tabModule

METHODOLOGICAL EVALUATION OF 2-DE TO STUDY ROOTPROTEOMICS DURING NEMATODE INFECTION IN COTTONAND COFFEE PLANTS

Octavio L. Franco,1,2 Jackeline L. Pereira,1 Paulo H. A. Costa,3,4

Thales L. Rocha,4 Érika V. S. Albuquerque,4 Maria F. Grossi-de-Sá,1,4

Regina M. D. G. Carneiro,4 Rui G. Carneiro,5 and Angela Mehta4

1Centro de Análises Proteômicas e Bioqu��micas, Pós-Graduação em CiênciasGenomicas e Biotecnologia, Universidade Cató lica de Bras��lia, Bras��lia, DF-Brazil2Departamento de Biologia, UFJF, MG-Brazil3UnB, Bras��lia, DF-Brazil4Embrapa Recursos Genéticos e Biotecnologia, Bras��lia, DF-Brazil5IAPAR, Londrina, PR, Brazil

& The identification of plant proteins expressed in response to phytopathogens is a remainingchallenge to proteome methodology. Proteomic methods, such as electrophoresis and mass spec-trometry have been extensively used for protein differential expression studies in several plantsincluding Arabidopsis thaliana, rice, and wheat. However, in coffee (Coffea canephora) andcotton (Gossypium hirsutum), bidimensional electrophoresis (2-DE) analysis has been rarelyemployed. Moreover, global protein expression in both agricultural plants in response to biotic stressconditions had not been reported until now. In this study, Meloidogyne paranaensis and M. incog-nita, two devastating phytonematodes for numerous crop cultures, were used to infect resistant gen-otypes of coffee and cotton plants. The protein expression of infected- and non-infected roots wereevaluated by 2-DE following in silico experiments. Additionally, gels were stained with silvernitrate and=or Coomassie brilliant blue in order to obtain an optimized method for proteomicanalysis of plant-nematode interaction. The 2-DE analysis revealed an enhanced number of proteinspots, as well as differentially expressed proteins, when Coomassie brilliant blue was used. Theresults obtained here could be extended to other plant species, providing valuable information toroot-nematode interactions.

Keywords differential expression, Meloydogine, proteins, staining methods, two-dimensional gels

Address correspondence to Octavio L. Franco, Universidade Católica de Brası́lia, Pós-Graduaçãoem Ciências Gênomicas e Biotecnologia, SGAN 916–Av W5–Módulo C, Brası́lia, DF, Brazil. E-mail:[email protected]; Angela Mehta, Embrapa Recursos Genéticos e Biotecnologia, PqEB, Av. w=5Norte Final, Asa Norte, CEP 70770-900, Brası́lia, DF, Brazil. E-mail: [email protected]

Preparative Biochemistry & Biotechnology, 40:152–163, 2010Copyright # Taylor & Francis Group, LLCISSN: 1082-6068 print/1532-2297 onlineDOI: 10.1080/10826060903558976

INTRODUCTION

Cotton (Gossipium hirsutum) and coffee (Coffea canephora) are importantcrops cultivated worldwide that are severely attacked by various phytonema-todes such as Meloidogyne incognita and Meloidogyne paranaensis. Severalattempts have been made to control nematode infection, including theuse of chemical nematicides and crop rotation; however, the control ofthese pathogens using these methods is generally inefficient.[1]

The use of resistant genotypes and genetically modified plants withenhanced resistance has been an alternative that could contribute to asignificant reduction in economic losses.[2]

Another approach, proposed to improve nematode resistance, consistsof the identification of proteins and genes involved in plant response topathogens, focusing specially on genome and proteome analysis of infectedroot tissue in resistant genotypes.[3] Proteomic studies involvetwo-dimensional gel electrophoresis (2-DE), followed by spot identificationby mass spectrometry.[4,5] These data have revealed several proteinsinvolved in plant developmental, metabolic processe[6] and different pro-teins involved in plant response to non-biotic and biotic environmentalstresses.[7] Crop plants were chosen as prototypes and have been exten-sively used to clarify biological questions. Among these plants, we canhighlight Arabidopsis thaliana,[8] Oryza sativa,[9] Zea mays,[10] and Pisum sati-vum.[11] On the other hand, some plants are rarely studied by this kind ofapproach. For example, in Coffea, few studies using 2-DE were reported toanalyze seeds and endosperm proteins.[12–15] Moreover, no proteomicanalyses were carried out with cotton tissues (G. hirsutum). In fact, few pro-tein expression studies in coffee and cotton plants in response to bioticstress conditions have been reported until now.[16,17]

The main difficulty in studying plant-nematode interactions involvesthe development of specific protocols for root proteomic studies, sinceeach plant shows different chemical properties.[18] In this field, severalminor problems have commonly been found, such as protein concen-tration and extraction, as well as spot identification, especially in organismsthat do not have their genome at least partially elucidated.[19] Moreover,attention must also be given to the 2D protein staining techniques, whichoften involve silver nitrate, Coomassie Brilliant Blue (CBB) or fluorescentstains.[20] Each approach has advantages and disadvantages from boththe conceptual and the methodological viewpoints. Silver staining is usuallypreferred over CBB due to the higher sensitivity obtained. Nevertheless,silver-staining resolution could significantly vary according to the studiedorganism and, more specifically, to the researched tissue. Occurrence ofsmear and background after silver staining could occur due to the presenceof phenolic compounds, salts, lipids, and carbohydrates, interfering

Methodological Evaluation of 2-DE to Study 153

proteome resolution and analysis. When identification of proteins byMS=MS is intended, gels stained with CBB are more successfullyanalyzed.[21]

In view of plant-pathogen interaction, this report aims to compare rootprotein patterns from two different crop plants, cotton and coffee,non-infected and infected with the pathogen during different timepointsby proteomic techniques, which include 2-DE gels and in silico studies.Additionally, two classic staining methods, one using silver nitrate andthe other CBB, were employed in order to optimize root protein mapdevelopment.

EXPERIMENTAL

Plant Material

Resistant genotypes of cotton (G. hirsutum L.) and coffee (C. canephora)plants were grown in plastic bags containing sterile soil and maintained ingreen houses. After three and six months, cotton and coffee roots wereused for nematode infection. Non-infected plant roots were collected,washed, and frozen in liquid nitrogen for further protein extraction andwere considered as the control condition.

Nematode Culture and Inoculation

Meloidogyne paranaensis and Meloidogyne incognita were used to infect cof-fee and cotton roots, respectively. Roots were washed and homogenized in ablender with 0.5% (v=v) sodium hypochlorite in order to obtain nematodes.The resulting material was sifted and the eggs were removed. After eclosion,juveniles were collected by centrifugation for 30 min at 2,500� g andcounted for plant infection. Each plant was infected with approximately10,000 juveniles of M. paranaensis and 20,000 of M. incognita, respectively.Infected coffee and cotton roots were collected 6 and 10 days after inocu-lation. Plant material was stored at �80�C until use.

Protein Extraction

For coffee root analysis, proteins were extracted according to de Motand Vanderleyden.[22] Cotton root tissues were resuspended (1:3 w:v) with40 mM Tris-HCl (pH 7.0) containing 250 mM sucrose, 1% Triton X-100,10 mM EDTA, 1 mM PMSF and 1 mM DTT. Extractions were carried outat 4�C for 2 h following a precipitation with TCA=acetone, accordingDamerval et al.[23]

154 O. L. Franco et al.

Gel Electrophoresis Analyses

Isoelectric focusing of coffee proteins was performed according to de Motand Vanderleiden. Polyacrylamide gels containing 3.6% acrylamide, 0.21%bis-acrylamide, 7.2% ampholyte pH 5–7 and 3–10 in the proportion of 5=1(v=v); 2% Nonidet P-40 and 55% urea were used. Approximately 150mg ofproteins were loaded onto the gel after a pre-run. Electrophoresis was per-formed at 400 V for 18 h, using NaOH 20 mM in the upper compartmentof the chamber and H3PO4 10 mM in the lower. Isoelectric focusing for cot-ton proteins was conducted according to Gorg et al.[24] using 18-cm immobi-lized pH gradient (IPG) strips with a pH range of 3–10 and a Multiphor IIelectrophoresis system from General Electric. Strips containing 200mg of pro-tein samples were rehydrated with 2% CHAPS, 8 M urea, 7 mg dithiothreitol(DTT) and 2% IPG buffer for 16 h following isoelectric focusing, which wascarried out for 380 min at 3 kV, 2 mA and 5 W. After the first dimension, stripswere equilibrated in a solution containing 6 M urea, 1% DTT and 2% SDS for15 min and then applied to slab gels. The second dimension was performedin 18� 24 cm SDS-PAGE 12.5% gels as described by Laemmli,[25] as well SDS-PAGE minigel 12.5%. In both, bromophenol blue was used as tracking dye.Electrophoresis was performed at 250 V, 80 mA and 10 W for 7 h. Molecularweight markers here utilized were bought from Sigma Co. Each gel analysiswas carried out in biological and technical triplicates.

Staining Methods

Two dimensional gels were fixed overnight in a solution containing30% ethanol and 10% acetic acid. Silver staining was carried out accordingto Blum et al.[26] Silver stained gels were destained with a solution contain-ing 100 mM sodium thiosulphate and 30 mM potassium ferricyanate andfurther CBB stained. For CBB staining, a Colloidal Coomassie solution(0.1% Coomassie G250; 2% phosphoric acid; 10% ammonium sulphateand 20% methanol) was used.

In Silico Analyses

Gels were digitized using the scanner HP Scanjet Model 8290 andfurther analyzed with the Bionumerics software v. 4.5 (Applied-maths).First, the calibration with a gray scale was necessary to transform gray levelsinto values for each pixel of the gel picture. The calibration method used acalibration curve from Bionumerics Software. All gel pictures were analyzedas .tiff files. The six gel images were placed in one folder and the wizarddetection method proposed by the software was used for spot detection.


Automatically detected spots were manually checked, and some of themmanually added or removed according size (>0.2 cm), format (circular),and density (>2 pixel.cm�1). Following the detection procedure, the nor-malization step was carried out to attribute a common protein identity foridentical spots derived from different images. For this procedure, a refer-ence gel was constructed and automatically matching options of Bionu-merics Software were used. For each sample, when a protein was detectedin all gel images, this protein was automatically added to the reference gel.

RESULTS AND DISCUSSION

In the coffee root analysis, the 2-DE maps stained with CBB revealed anenhanced number of protein spots as well as differentially expressed

FIGURE 1 2D gels of Coffea canephora and Gossypium hirsutum non-inoculated and infected roots withnematodes stained with silver nitrate. The pH of the coffee and cotton gels ranged from 4 to 8 and3–10, respectively.


FIGURE 2 2D gels of Coffea canephora non-inoculated roots (A) and infected roots with M. paranaensis at6 (B) and 10 days (C) after inoculation, stained with CBB. The spot numbers indicate differentiallyexpressed proteins present in all three gels.


proteins. A total of approximately 100 spots were observed in CBB gels asopposed to 70 in silver stained gels (Fig. 1 and Fig. 2). The lower quantityof spots observed in silver stained gels could be generated by an increase ofcolor background caused by a side reaction of non proteinaceouscompounds synthesized in root tissues.

In order to identify differentially expressed proteins, the protein mapsof the 6th and 10th day after nematode infection were determined andfurther compared to maps obtained from non-infected roots (Fig. 2).2D gels revealed numerous differentially expressed proteins at 6 days afterinoculation including 7 up-, 3 down-regulated and several totally novel ones(Figs. 2 and 3). These same proteins, when analyzed at 10 days after inocu-lation, showed different patterns, suggesting that the period of nematodeinfection also could be related to protein pattern modification (Fig. 3).Moreover, correlation lines by the Image analysis superposition was createdin Bionumerics Software of 2-DE, in order to improve reliability. Thesecurves showed that both staining methods were extremely reproducible,indicating a correlation rate higher than 0.90 (data not shown). When2D gels from different dates were compared, a correlation rate lower than0.4 was obtained, clearly indicating those after different periods of plantexposition to nematodes, different protein patterns were observed.

An unexpected result obtained was that most differentially expressedproteins identified in the silver stained gels were not observed in theCBB gels. In this specific case, only 2 differential spots were common tothe gels colored with both methods, indicating that differential stainingmethods could lead to different conclusions. Vediyappan et al.[27] obtainedsimilar results in the comparison of different staining methods for theanalysis of Candida albicans proteins. It is possible that differences inthe staining capacities of silver nitrate and CBB are responsible for the

FIGURE 3 Histogram representing expression levels of protein spots present in all three conditionsdescribed for Coffea canephora roots. The differential expression pattern was calculated according tothe volume of each protein.


revelation of different proteins. In order to standardize this procedure,higher protein amounts were loaded onto the 2-D gel stained with silver.However, this modification led to the presence of more pronounced smearand background (data not shown), clearly reducing gel visibility and spotidentification.

Cotton has been considered a very recalcitrant plant to biochemistryand molecular analyses. In this report, we have applied an efficient proto-col for protein extraction for proteomic analysis by 2-DE of cotton rootsinfected with phytonematodes. As obtained for coffee roots, protein mapswere determined by 2-DE and stained with both techniques (silver nitrateand CBB). The 2D protein maps of cotton roots (Fig. 4) revealed approxi-mately 150 spots in the gels stained with CBB. Interestingly, at least50 additional spots were observed in the silver stained gels with molecularmasses ranging between 15 and 25 kDa, despite intense background(Fig. 1). Furthermore, differentially expressed proteins were analyzed,comparing infected to non-infected roots. Statistical analyses were also gen-erated by the image analysis superposition in Bionumerics software of 2-DE.These curves showed that both staining methods were extremely reproduc-ible, indicating a correlation rate higher than 0.92 (data not shown).However, when 2D gels from different dates were compared, a correlationrate lower than 0.35 was obtained, indicating that, as observed in coffeeroots, after different periods of cotton exposition to nematodes, differentexpression protein patterns were observed.

After six days of M. incognita inoculation, numerous proteins weredifferentially expressed, as observed in the CBB stained gels, including 4up and 5 down-regulated (Fig. 5). At 10 days after inoculation, 14 proteinsshowed differential expression, including 13 up and 2 down-regulated,showing once more that time after nematode infection is extremely impor-tant for root-pathogen interaction studies (Fig. 5).

An important difference observed between the 2D gels colored withsilver and CBB is that this last method caused a minor background, allow-ing the visualization of a higher number of protein spots in the regions ofhigher protein concentration (pH 6 to 4 and 20–50 kDa in coffee and pH 4to 7 and 25 to 70 kDa in cotton) (Figs. 2 and 4). On the other hand, in cot-ton roots, silver staining showed a higher sensitivity in protein detection,allowing the visualization of low abundant proteins (Fig. 1). The stainingmethod chosen for protein analysis of a given tissue or organism is ofextreme importance since it can directly affect the number and type of pro-teins analyzed. When a global screen is performed in order to identify spe-cific proteins involved in a given process, such as resistance, this feature iseven more important since different coloring methods can revealadditional proteins. These proteins could play a crucial role in theprocesses under investigation. With this aim, several studies have been


FIGURE 4 2D gels of Gossypium hirsutum non-inoculated roots (A) and infected roots with M. incognitaat 6 (B) and 10 days (C) after inoculation, stained with CBB. The spot numbers indicate differentiallyexpressed proteins present in all three gels.


performed to improve staining techniques.[28,29–31] Some authors considerthat CBB is a time-consuming technique that shows a higher background.[32]

On the other hand, when identification by mass spectrometry is intended,CBB stained proteins are more successfully sequenced than silver stainedspots. Qin et al.[33] have shown that, although silver staining is more sensitivethan CBB, a protein identification rate of only 10% was obtained.

In the coffee and cotton protein profile analyses, a quite different plantresponse to pathogen infection was obtained when the sampling period wasmodified (Figs. 3 and 5). This result may be due to differences in the level ofresistance to the specific nematode species. Although several conditions suchas extraction methods and electrophoresis parameters were different in eachexperiment, the results obtained with the staining methods were consistent.The data obtained also clearly showed the up- and down-regulation of severalproteins at the different sampling times in both cultures. Two proteins fromcoffee and one from cotton 2-DE profiles were identified as a chitinase (spot64) and a pathogenesis related protein (spot 3) (Fig. 2) and a quinonereductase 2 (spot 8) (Fig. 4), respectively.[34] Further identification of ahigher number of protein spots may reveal commonly expressed proteinsin coffee and cotton in response to nematode infection.

In summary, this report suggests several methodologies for rooteomicanalyses of coffee and cotton, showing that, for each kind of tissue, a differ-ent combo of techniques must be used. Among them, methodologicalevaluation also included three variables such as plant material and period,as well as staining methods. Parameters here presented could help plantproteomic analyses and complement severe efforts of numerous scientists.After a clear definition of methodology for each plant tissue, proteins couldbe studied and possibly could be utilized as possible candidates in thedevelopment of resistant plants against phytopathogens.

FIGURE 5 Histogram representing expression levels of protein spots present in all three conditionsdescribed for Gossypium hirsutum roots. The differential expression pattern was calculated accordingto the volume of each protein.


ACKNOWLEDGMENTS

This research was supported by Consórcio Brasileiro de Pesquisa eDesenvolvimento do Café, UCB, CAPES and CNPq.

REFERENCES

1. Randig, O.; Bongiovanni, M.; Carneiro, R.M.; Castagnone-Sereno, P. Genetic diversity of root-knotnematodes from Brazil and development of SCAR markers specific for the coffee-damaging species.Genome 2002, 45, 862–870.

2. Starr, J.L.; Simpson, C.E. Improving the utility of nematode resistance in groundnut. Commun.Agric. Appl. Biol. Sci. 2006, 71, 647–651.

3. Djian-Caporalino, C.; Fazari, A.; Arguel, M.J.; Vernie, T.; VandeCasteele, C.; Faure, I.; Brunoud, G.;Pijarowski, L.; Palloix, A.; Lefebvre, V.; Abad, P. Root-knot nematode (Meloidogyne spp) resistancegenes in pepper (Capsicum annuum L) are clustered on the P9 chromosome. Theor. Appl. Genet.2007, 114, 473–486.

4. Chevalier, F.; Rofidal, V.; Vanova, P.; Bergoin, A.; Rossignol, M. Proteomic capacity of recent fluor-escent dyes for protein staining. Phytochemistry 2004, 65, 1499–1506.

5. Marques, K.; Sarazin, B.; Chane-Favre, L.; Zivy, M.; Thiellement, H. Comparative proteomics toestablish genetic relationships in the Brassicaceae family. Proteomics 2001, 1, 1457–1462.

6. Gallardo, K.; Job, C.; Groot, S.P.; Puype, M.; Demol, H.; Vandekerckhove, J.; Job, D. Proteomics ofArabidopsis seeds. Plant Physiol. 2002, 129, 823–837.

7. Bestel-Corre, G.; Dumas-Gaudot, E.; Poinsot, V.; Dieu, M.; Dierick, J.F.; van Tuinen, D.; Remacle, J.;Gianinazzi-Pearson, V.; Gianinazzi, S. Proteome analysis and identification of symbiosis related pro-teins from Medicago truncatula Gaertn by two-dimensional electrophoresis and mass spectrometry.Electrophoresis 2002, 223, 122–137.

8. Baginsky, S.; Gruı́ssem, W. Arabidopsis thaliana proteomics: from proteome to genome. J. Exp. Bot.2006, 57, 1485–1491.

9. Komatsu, S.; Zang, X.; Tanaka, N. Comparison of two proteomics techniques used to identifyproteins regulated by gibberellin in rice. J. Proteome Res. 2006, 5, 270–276.

10. Alvarez, S.; Goodger, J.Q.; Marsh, E.L.; Chen, S.; Asirvatham, V.S.; Schachtman, D.P.Characterization of the maize xylem sap proteome. J. Proteome Res. 2006, 5, 963–972.

11. Curto, M.; Camafeita, E.; Lopez, J.A.; Maldonado, A.M.; Rubiales, D.; Jorrin, J.V. A |proteomicapproach to study pea (Pisum sativum) responses to powdery mildew (Erysiphe pisi). Proteomics2006, 6, 163–174.

12. Yuffa, A.M.; De Garcia, E.G.; Nieto, M.S. Comparative study of protein electrophoretic patternsduring embryogenesis in Coffea arabica cv. Catimor. Plant Cell Rep. 1994, 13, 197–202.

13. Franco, O.L.; Pelegrini, P.B.; Gomes, C.P.; Souza, A.; Costa, F.T.; Domont, G.; Quirino, B.F.;Eira, M.T.; Mehta, A. Proteomic evaluation of coffee zygotic embryos in two different stages of seeddevelopment. Plant Physiol Biochem. 2009, 47 (11–12), 1046–1050.

14. Rogers, W.J.; Bezard, G.; Deshayes, A.; Meyer, I.; Petiard, V.; Marraccini, P. Biochemical and molecu-lar characterization and expression of the 11S-type storage protein from Coffea arabica endosperm.Plant Physiol. Biochem. 1999, 37, 261–272.

15. Gil-Agusti, M.T.; Campostrini, N.; Zolla, L.; Ciambella, C.; Invernizzi, C.; Righetti, P.G.Two-dimensional mapping as a tool for classification of green coffee bean species. Proteomics2005, 5, 710–718.

16. Coumans, J.V.; Poljak, A.; Raftery, M.J.; Backhouse, D.; Pereg-Gerk, L. Analysis of cotton (Gossypiumhirsutum) root proteomes during a compatible interaction with the black root rot fungus Thielaviop-sis basicola.. Proteomics 2008, 9 (2), 335–349.

17. Yang, Y.W.; Bian, S.M.; Yao, Y.; Liu, J.Y. Comparative proteomic analysis provides new insights intothe fiber elongating process in cotton. J. Proteome Res. 2008, 7 (11), 4623–4637.

18. Lehesranta, S.J.; Davies, H.V.; Shepherd, L.V.; Koistinen, K.M.; Massat, N.; Nunan, N.; McNicol,J.W.; Karenlampi, S.O. Proteomic analysis of the potato tuber life cycle. Proteomics 2006, 6,6042–6052.


19. Plomion, C.; Lalanne, C.; Claverol, S.; Meddour, H.; Kohler, A.; Bogeat-Triboulot, M.B.; Barre, A.;Le Provost, G.; Dumazet, H.; Jacob, D.; Bastien, C.; Dreyer, E.; de Daruvar, A.; Guehl, J.M.;Schmitter, J.M.; Martin, F.; Bonneu, M. Mapping the proteome of poplar and application to the dis-covery of drought-stress responsive proteins. Proteomics 2006, 6, 6509–6527.

20. Monteoliva, L.; Albar, J.P. Differential proteomics: an overview of gel and non-gel based approaches.Brief Funct. Genomic Proteomic 2004, 3, 220–239.

21. Humphery-Smith, I.; Cordwell, S.J.; Blackstock, W.P. Proteome research: complementarity and lim-itations with respect to the RNA and DNA worlds. Electrophoresis 1997, 18, 1217–1242.

22. de Mot, R.; Vanderleyn, J. Application of two dimensional protein analysis for strain fingerprintingand mutant analysis of Azospirillum species. Can. J. Microbiol. 1989, 35, 960–967.

23. Damerval, C.; De Vienne, D.; Zivy, M.; Thiellement, H. Technical improvements in two-dimensionalelectrophoresis increase the level of genetic variation detected in wheat-seedling proteins. Electro-phoresis 1986, 7, 52–54.

24. Gorg, A.; Postel, W.; Gubther, S. The current state of two-dimensional electrophoresis with immo-bilized pH gradients. Electrophoresis 1988, 9, 531–546.

25. Laemmli, U.K. Cleavage of structural proteins during the assembly of the head of the bacterio-phage T4. Nature 1970, 227, 680–685.

26. Blum, H.; Beier, H.; Gross, H.J. Improved silver staining of plant proteins, RNA and DNA in poly-acrylamide gels. Electrophoresis 1987, 8, 93–99.

27. Vediyappan, G.; Bikandi, J.; Braley, R.; Chaffin, W.L. Cell surface proteins of Candida albicans: prep-aration of extracts and improved detection of proteins. Electrophoresis 2000, 21, 956–961.

28. Candiano, G.; Bruschi, M.; Musante, L.; Santucci, L.; Ghiggeri, G.M.; Carnemolla, B.; Orecchia, P.;Zardi, L.; Righetti, P.G. Blue silver: a very sensitive colloidal Coomassie G-250 staining for proteomeanalysis. Electrophoresis 2004, 25, 1327–1333.

29. Silva, J.; Carvalho, A.S.; Vitorino, R.; Domingues, P.; Teixeira, P.; Gibbs, P. Protein extraction andcomparison of stain protocols for analysis of two-dimensional electrophoresis gels. Protein Pep.Lett. 2004, 11, 189–194.

30. Smejkal, G.B.; Robinson, M.H.; Lazarev, A. Comparison of fluorescent stains: relative photostabilityand differential staining of proteins in two-dimensional gels. Electrophoresis 2004, 25, 2511–2509.

31. Lanne, B.; Panfilov, O. Protein staining influences the quality of mass spectra obtained by peptidemass fingerprinting after separation on 2D gels A comparison of staining with coomassie brilliantblue and sypro ruby. J. Proteome Res. 2005, 4, 175–179.

32. Chmelı́k, J.; Mazanec, K.; Slais, K. Novel staining-free proteomic method for simultaneousidentification of proteins and determination of their pI values by using low-molecular-mass pImarkers. Electrophoresis 2007, 28 (18), 3315–3323.

33. Qin, H.; Liu, T.; Liu, B.; Song, X.; Huang, X.; Yang, J.L.; Zhao, X.; Wei, Y.Q. Comparison betweenprotein detection methods for two-dimensional gel electrophoresis Zhongguo. Shi. Yan. Xue. Ye.Xue. Za. Zhi. 2006, 14, 168–172.

34. Mehta, A.; Magalhães, B.S.; Souza, D.S.L.; Vasconcelos, E.A.R.; Silva, L.P.; Grossi-de-Sá, M.F.; Franco,O.L.; da Costa, P.H.A. Rocha TL Rooteomics: the challenge of discovering plant defence-relatedproteins in roots. Curr. Prot. Pep. Sci. 2008, 9, 108–116.


Documents

INFECTION IN COTTON AND COFFEE PLANTSainfo.cnptia.embrapa.br/digital/bitstream/item/... · METHODOLOGICAL EVALUATION OF 2-DE TO STUDY ROOT PROTEOMICS DURING NEMATODE INFECTION IN