The Protein Journal

, Volume 36, Issue 4, pp 308–321 | Cite as

A Rapid and Reliable Method for Total Protein Extraction from Succulent Plants for Proteomic Analysis

  • Fernando Lledías
  • Felipe Hernández
  • Viridiana Rivas
  • Abisaí García-Mendoza
  • Gladys I. Cassab
  • Jorge Nieto-Sotelo


Crassulacean acid metabolism plants have some morphological features, such as succulent and reduced leaves, thick cuticles, and sunken stomata that help them prevent excessive water loss and irradiation. As molecular constituents of these morphological adaptations to xeric environments, succulent plants produce a set of specific compounds such as complex polysaccharides, pigments, waxes, and terpenoids, to name a few, in addition to uncharacterized proteases. Since all these compounds interfere with the analysis of proteins by electrophoretic techniques, preparation of high quality samples from these sources represents a real challenge. The absence of adequate protocols for protein extraction has restrained the study of this class of plants at the molecular level. Here, we present a rapid and reliable protocol that could be accomplished in 1 h and applied to a broad range of plants with reproducible results. We were able to obtain well-resolved SDS/PAGE protein patterns in extracts from different members of the subfamilies Agavoideae (Agave, Yucca, Manfreda, and Furcraea), Nolinoideae (Dasylirion and Beucarnea), and the Cactaceae family. This method is based on the differential solubility of contaminants and proteins in the presence of acetone and pH-altered solutions. We speculate about the role of saponins and high molecular weight carbohydrates to produce electrophoretic-compatible samples. A modification of the basic protocol allowed the analysis of samples by bidimensional electrophoresis (2DE) for proteomic analysis. Furostanol glycoside 26-O-β-glucosidase (an enzyme involved in steroid saponin synthesis) was successfully identified by mass spectrometry analysis and de novo sequencing of a 2DE spot from an Agave attenuata sample.


Agave CAM plants Protein extraction Protease Electrophoresis Mass spectrometry 



Bidimensional electrophoresis


Crassulacean acid metabolism


Trichloroacetic acid




Phenylmethylsulfonyl fluoride



We are thankful to Dr. Salvador Arias from Jardín Botánico, Instituto de Biología, Universidad Nacional Autónoma de México for providing O. ficus-indica, L.marginatus, and M. magnimamma samples. We also thank Unidad de Proteómica and Unidad Universitaria de Apoyo Bioinformático, Instituto de Biotecnología, Universidad Nacional Autónoma de México for all mass spectrometry analysis and the production of our local Agave fasta database, respectively. This work was supported by research grants from PAPIIT/DGAPA/UNAM IN212116 (F Lledías) and IG200515 (J Nieto-Sotelo and G Cassab), UNAM-Allied/Domecq P-150 (J Nieto-Sotelo and G Cassab), and CONACyT PN-247732 (J Nieto-Sotelo and G Cassab).

Compliance with Ethical Standards

Conflict of interest

The authors declare that they have no conflicts of interest.

Human participants and animals

This article does not contain any studies with human participants or animals performed by any of the authors.

Supplementary material

10930_2017_9720_MOESM1_ESM.pdf (801 kb)
Supplementary material 1 (PDF 801 KB)


  1. 1.
    Keeley JE, Rundel PW (2003) Evolution of CAM and C4 carbon-concentrating mechanisms. Int J Plant Sci 164(3 Suppl):S55–S77CrossRefGoogle Scholar
  2. 2.
    Yamori W, Hikosaka K, Way DA (2014) Temperature response of photosynthesis in C3, C4, and CAM plants: temperature acclimation and temperature adaptation. Photosynth Res 119(1–2):101–117CrossRefGoogle Scholar
  3. 3.
    Nobel P (1996) High productivities of certain agronomic CAM species. In: Winter K, Smith JAC (eds) Crassulacean acid metabolism. Biochemistry, ecophysiology and evolution. Springer, Berlin, pp 255–265CrossRefGoogle Scholar
  4. 4.
    García-Mendoza A, Galván VR (1995) Riqueza de las familias Agavaceae y Nolinaceae en México. Bol Soc Bot México 56:7–24Google Scholar
  5. 5.
    Good-Avila SV, Souza V, Gaut BS, Eguiarte LE (2006) Timing and rate of speciation in Agave (Agavaceae). Proc Natl Acad Sci USA 103:9124–9129CrossRefGoogle Scholar
  6. 6.
    Davis AS, Dohleman F, Long SP (2011) The global potential for Agave as a biofuel feedstock. GCB Bioenergy 3:68–78CrossRefGoogle Scholar
  7. 7.
    Sparg SG, Light ME, van Staden J (2004) Biological activities and distribution of plant saponins. J Ethnopharmacol 94:219–243CrossRefGoogle Scholar
  8. 8.
    Monterrosas-Brisson N, Arenas Ocampo ML, Jiménez-Ferrer E, Jiménez-Aparicio AR, Zamilpa A, Gonzalez-Cortazar M, Tortoriello J, Herrera-Ruiz M (2013) Anti-inflammatory activity of different Agave plants and the compound cantalasaponin-1. Molecules 18:8136–8146CrossRefGoogle Scholar
  9. 9.
    Ahumada-Santosa YP, Montes-Avila J, Uribe-Beltrána M, Díaz-Camachoa SP, López-Angulo G, Rito Vega-Aviña R, López-Valenzuela JA, Heredia JB, Delgado-Vargas F (2013) Chemical characterization, antioxidant and antibacterial activities of six Agave species from Sinaloa, México. Ind Crops Prod 49:143–149CrossRefGoogle Scholar
  10. 10.
    Kee SC, Nobel PS (1986) Concomitant changes in high temperature tolerance and heat-shock proteins in desert succulents. Plant Physiol 80:596–598CrossRefGoogle Scholar
  11. 11.
    Nobel PS, Smith SD (1983) High and low temperature tolerances and their relationships to distribution of agaves. Plant Cell Environ 6:711–719Google Scholar
  12. 12.
    González-Cruz L, Jaramillo-Flores ME, Bernardino-Nicanor Mora-Escobedo R (2011) Influence of plant age on fructan content and fructosyltranserase activity in Agave atrovirens Karw leaves. Afr J Biotechnol 10:15911–15920Google Scholar
  13. 13.
    Mellado-Mojica E, López MG (2012) Fructan metabolism in A. tequilana Weber blue variety along its developmental cycle in the field. J Agric Food Chem 60:11704–11713CrossRefGoogle Scholar
  14. 14.
    Nobel PS (1976) Water relations and photosynthesis of a desert CAM plant Agave deserti. Plant Physiol 58:576–582CrossRefGoogle Scholar
  15. 15.
    Gentry HS (1982) Agaves of continental North America. University of Arizona Press, TucsonGoogle Scholar
  16. 16.
    Wattendorff J, Holloway PJ (1980) Studies on the ultrastructure and histochemistry of plant cuticles: the cuticular membrane of Agave americana L. in situ. Ann Bot 46:13–28CrossRefGoogle Scholar
  17. 17.
    North GB, Brinton EK, Garrett TY (2008) Contractile roots in succulent monocots: convergence, divergence and adaptation to limited rainfall. Plant Cell Environ 31:1179–1189CrossRefGoogle Scholar
  18. 18.
    Luján R, Lledías F, Martínez LM, Barreto R, Cassab G, Nieto-Sotelo J (2009) Small heat-shock proteins and leaf cooling capacity account for the unusual heat tolerance of the central spike leaves in Agave tequilana var Weber. Plant Cell Environ 32:1791–1803CrossRefGoogle Scholar
  19. 19.
    Carpentier SC, Witters E, Laukens K, Deckers P, Swennen R, Panis B (2005) Preparation of protein extracts from recalcitrant plant tissues: an evaluation of different methods for two-dimensional gel electrophoresis analysis. Proteomics 5:2497–2507CrossRefGoogle Scholar
  20. 20.
    Wang W, Tai F, Chen S (2008) Optimizing protein extraction from plant tissues for enhanced proteomics analysis. J Sep Sci 31:2032–2039CrossRefGoogle Scholar
  21. 21.
    Marker RE, López J (1947) Biogenesis of the steroidal sapogenins in Agave, Manfreda and Hesperaloe. J Am Chem Soc 69:2403–2404CrossRefGoogle Scholar
  22. 22.
    Tipton KF (1964) Agavain: a new plant proteinase. Biochim Biophys Acta 92:341–350Google Scholar
  23. 23.
    Srinivasan M, Bratia IS (1953) The Carbohydrates of Agave vera cruz. Mill Biochem 55:286–289CrossRefGoogle Scholar
  24. 24.
    Du Toit PJ (1976) Isolation and partial characterization of a protease from Agave americana variegata. BBA-Enzymol 429:895–911Google Scholar
  25. 25.
    Schaller A (2004) A cut above the rest: the regulatory function of plant proteases. Planta 220:183–197CrossRefGoogle Scholar
  26. 26.
    Damerval C, de Vienne D, Zivy M, Thiellement H (1986) The technical improvements in two-dimensional electrophoresis increase the level of genetic variation detected in wheat-seedling proteins. Electrophoresis 7:52–54CrossRefGoogle Scholar
  27. 27.
    Martínez-García JF, Monte E, Quail PH (1999) A simple, rapid and quantitative method for preparing Arabidopsis protein extracts for immunoblot analysis. Plant J 20(2):251–257CrossRefGoogle Scholar
  28. 28.
    Isaacson T, Damasceno CMB, Saravanan RS, He Y, Catalá C, Saladié M, Rose JKC (2006) Sample extraction techniques for enhanced proteomic analysis of plant tissues. Nat Protoc 1:769–774CrossRefGoogle Scholar
  29. 29.
    Wei W, Vignani R, Scali M, Cresti M (2006) A universal and rapid protocol for protein extraction from recalcitrant plant tissues for proteomic analysis. Electrophoresis 27:2782–2786CrossRefGoogle Scholar
  30. 30.
    Dubravko Pavoković D, Križnik B, Krsnik-Rasol M (2012) Evaluation of protein extraction methods for proteomic analysis of non-model recalcitrant plant tissues. Croat Chem Acta 85:177–183CrossRefGoogle Scholar
  31. 31.
    Charney J, Tomarelli RM (1947) A colorimetric method for the determination of the proteolytic activity of duodenal juice. J Biol Chem 171:501–505Google Scholar
  32. 32.
    Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193:265–275Google Scholar
  33. 33.
    Heussen C, Dowdle EB (1980) Electrophoretic analysis of plasminogen activators in polyacrylamide gels containing sodium dodecyl sulfate and co-polymerized substrates. Anal Biochem 102:196–202CrossRefGoogle Scholar
  34. 34.
    Shapiro AL, Viñuela E, Maizel JV (1967) Molecular weight estimation of polypeptide chains by electrophoresis in SDS-polyacrylamide gels. Biochem Biophys Res Commun 28:815–20CrossRefGoogle Scholar
  35. 35.
    Horvath A, Riezman H (1994) Rapid protein extraction from Saccharomyces cerevisiae. Yeast 10:1305–1310CrossRefGoogle Scholar
  36. 36.
    Balen B, Krsnik-Rasol M, Zadro I, Simeon-Rudolph V (2004) Esterase activity and isoenzymes in relation to morphogenesis in Mammillaria gracillis Pfeiff tissue culture. Acta Bot Croat 63:83–91Google Scholar
  37. 37.
    O’Farrell PH (1975) High resolution two-dimensional electrophoresis of proteins. J Biol Chem 250:4007–4021Google Scholar
  38. 38.
    Rajalingam D, Loftis C, Xu JJ, Kumar TKS (2009) Trichloroacetic acid-induced protein precipitation involves the reversible association of a stable partially structured intermediate. Protein Sci 18:980–993CrossRefGoogle Scholar
  39. 39.
    Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685CrossRefGoogle Scholar
  40. 40.
    Blum H, Beier H, Gross HJ (1986) Improved silver staining of plant proteins, RNA and DNA in polyacrylamide gels. Electrophoresis 8:93–99CrossRefGoogle Scholar
  41. 41.
    Makkar H, Sharma O, Negi S (1980) Assay of proteins by Lowry’s method in the presence of high concentrations of beta-mercaptoethanol. Anal Biochem 104:124–116CrossRefGoogle Scholar
  42. 42.
    Gross S, Martin JA, Simpson J, Abraham-Juarez MJ, Wang Z, Visel A (2013) De novo transcriptome assembly of drought tolerant CAM plants Agave deserti and Agave tequilana. BMC Genom 14:563CrossRefGoogle Scholar
  43. 43.
    Reimerdes EH, Klostermeyer H (1976) Determination of proteolytic activities on casein substrates. Method Enzymol 45:26–28CrossRefGoogle Scholar
  44. 44.
    Dufield DR, Wilson GS, Glass RS, Schöneich (2004) Selective site-specific Fenton oxidation of methionine in model peptides: evidence for a metal-bound oxidant. J Pharm Sci 93:1122–1130CrossRefGoogle Scholar
  45. 45.
    Guerrero C, Martín-Rufián M, Reina JJ, Heredia (2006) Isolation and characterization of a cDNA encoding a membrane bound acyl-CoA binding protein from Agave americana L epidermis. Plant Physiol Biochem 44:85–90CrossRefGoogle Scholar
  46. 46.
    Casas-Godoy L a, Arrizon J, Arrieta-Baez D, Plou FJ, Sandoval G (2016) Synthesis and emulsifying properties of carbohydrate fatty acid esters produced from Agave tequilana fructans by enzymatic acylation. Food Chem 204:437–443CrossRefGoogle Scholar
  47. 47.
    Eskander J, Catherine L, Harakat D (2011) Steroidal saponins from the leaves of Beaucarnea recurvata. Phytochemistry 72:946–951CrossRefGoogle Scholar
  48. 48.
    Amid M, Yazid M, Manap ABD, Zohdi NK (2014) Purification and characterization of alkaline-thermostable protease enzyme from pitaya (Hylocereus polyrhizus) waste: a potential low cost of the enzyme. BioMed Res Int 2014:1–8CrossRefGoogle Scholar
  49. 49.
    Perez-Pimienta JA, Flores-Gómez CA, Ruiz HA, Sathitsuksanoh N, Balan N, da Costa Sousa L, Dale BE, Singh S, Simmons BA (2016) Evaluation of agave bagasse recalcitrance using AFEX™, autohydrolysis, and ionic liquid pretreatments. Bioresource Technol 211:216–223CrossRefGoogle Scholar
  50. 50.
    Kilcoyne M, Gerlach JQ, Farrell MP, Bhavanandan VP, Joshi L (2011) Periodic acid–Schiff’s reagent assay for carbohydrates in a microtiter plate format. Anal Biochem 416:18–26CrossRefGoogle Scholar
  51. 51.
    Ku Y, Jansen O, Oles CJ, Lazar EZ, Rader JI (2003) Precipitation of inulins and oligoglucoses by ethanol and other solvents. Food Chem 81:125–132CrossRefGoogle Scholar
  52. 52.
    Puzstai A (1966) Interactions of proteins with other polyelectrolytes on a two-phase system containing phenol and aqueous buffer at various pH values. Biochem J 99:93–101CrossRefGoogle Scholar
  53. 53.
    Pinos-Rodríguez JM, Zamudio M, González SS (2008) The effect of plant age on the chemical composition of fresh and ensiled Agave salmiana leaves. S Afr J Anim Sci 38:43–50CrossRefGoogle Scholar
  54. 54.
    Potter S, Jimenez-Flores R, Pollack J, Timothy A, Lone TA, Berber-Jimenez MD (1993) Protein-saponin interaction and its influence on blood lipids. J Agric Food Chem 41:1287–1291CrossRefGoogle Scholar
  55. 55.
    Francis G, Kerem Z, Makkar HPS, Becker K (2002) The biological action of saponins in animal systems: a review. Brit J Nutr 88:587–605CrossRefGoogle Scholar
  56. 56.
    Shimoyada M, Ootsubo R, Naruse T, Watanabe K (2000) Soybean saponin on protease hydrolyses of β-lactoglobulin and α-lactalbumin. Biosci Biotechnol Biochem 64:891–893CrossRefGoogle Scholar
  57. 57.
    Liu PJ, Chena Q, Wu SS, Shen J, Lin SC (2010) Surface modification of cellulose membranes with zwitterionic polymers for resistance to protein adsorption and platelet adhesion. J Membr Sci 350:387–394CrossRefGoogle Scholar
  58. 58.
    Kaya M, Mulerčikas P, Sargin I, Kazlauskaitė S, Baublys V, Akyuz B, Bulut E, Tubelyté V (2016) Three-dimensional chitin rings from body segments of a pet diplopod species: Characterization and protein interaction studies. Mater Sci Eng C 68:716–22CrossRefGoogle Scholar
  59. 59.
    Inoue K, Shibuya M, Yamamoto K, Ebizuka Y (1996) Molecular cloning and bacterial expression of a cDNA encoding furostanol glycoside 26-O-β-glucosidase of Costus speciosus. FEBS Lett 389:273–277CrossRefGoogle Scholar
  60. 60.
    Kohara A, Nakajima C, Hashimoto K, Ikenaga T, Tanaka H, Shoyama Y, Yoshida S, Muranaka T (2005) A novel glucosyltransferase involved in steroid saponin biosynthesis in Solanum aculeatissimum. Plant Mol Biol 57:225–239CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2017

Authors and Affiliations

  1. 1.Departamento de Biología Molecular de Plantas, Instituto de BiotecnologíaUniversidad Nacional Autónoma de MéxicoCuernavacaMexico
  2. 2.Jardín Botánico, Instituto de BiologíaUniversidad Nacional Autónoma de MéxicoCiudad de MéxicoMexico

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