Orig Life Evol Biosph (2011) 41:523–527 DOI 10.1007/s11084-011-9258-x BIOSIGNATURES
Detection of Peptidic Sequences in the Ancient Acidic Sediments of Río Tinto, Spain María Colín-García & Basem Kanawati & Mourad Harir & Phillippe Schmitt-Kopplin & Ricardo Amils & Victor Parro & Miriam García & David Fernández-Remolar
Received: 1 August 2011 / Accepted: 18 August 2011 / Published online: 3 December 2011 # Springer Science+Business Media B.V. 2011
Abstract Biomarkers are molecules that are produced by or can be associated with biological activities. They can be used as tracers that give us an idea of the ancient biological communities that produced them, the paleoenvironmental conditions where they lived, or the mechanism involved in their transformation and preservation. As a consequence, the preservation potential of molecules over time depends largely on their nature, but also on the conditions of the environment, which controls the decomposition kinetics. In this context, proteins and nucleic acids, which are biomolecules bearing biological information, are among the most labile molecules. In this research, we report the presence of short-chained peptides obtained from extracts of ferruginous sedimentary deposits that have been produced under the acidic and oxidizing solutions of Río Tinto, Spain. These preliminary results go against the paradigmatic idea that considers the acidic and oxidizing environments inappropriate for the preservation of molecular information. Keywords Río Tinto . Biomarkers . Peptides . Preservation
M. Colín-García : R. Amils : V. Parro : M. García : D. Fernández-Remolar (*) Centro de Astrobiología, (CSIC/INTA), Instituto Nacional de Técnica Aeroespacial. Ctra. de Torrejón a Ajalvir, km 4, 28850. Torrejón de Ardoz, Madrid, Spain e-mail:
[email protected] M. Colín-García Instituto de Geología, Universidad Nacional Autónoma de México, Ciudad Universitaria, 04510 México, DF, Mexico M. Colín-García Instituto de Ciencias Nucleares, Universidad Nacional Autónoma de México, Circuito Exterior S/N Ciudad Universitaria, 04510 México, DF, Mexico B. Kanawati : M. Harir : P. Schmitt-Kopplin Department of BioGeoChemistry and Analytics, Institute of Ecological Chemistry, Helmholtz Zentrum München, German Research Center for Environmental Health, Ingolstaedter Landstrasse 1, 85764 Neuherberg, Germany
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Introduction Biomarkers are molecules produced by or related to biological activities. Mackenzie (1984) defined them as organic compounds detected in the geosphere, whose basic structure suggests a link with a known natural product. Because of that, biomarkers can be used to interpret the past geological, physico-chemical and biological conditions of an environment. The possibility of finding life in other places of the Solar System or even the Universe has expanded the application of biomarkers now used for astrobiological purposes (Fernández Calvo et al. 2006). Theoretically, all the molecules derived from an organism could be used as biomarkers. However, in the case of simple organic compounds it is very difficult, and in some cases impossible, to determine their biological origin. This is the case of monomers such as amino acids and sugars, which have also been found in meteorites (i.e. Oró, 1961, Cronin and Pizzarello 1983, Sephton et al. 2004, Pizzarrello 2007) or can be synthesized abiotically (i.e. Pizzarello et al. 1994, Cooper et al. 2001). Molecular complexity, a main feature for life, cannot be reproduced abiotically, and biopolymers (proteins, polysaccharides, polynucleotides, etc.) are examples of that (Fernández Calvo et al. 2006). In this paper we report the preservation of large biomolecules bearing high biological information such as peptides in old sediments of the extreme environment of Rio Tinto (see Fernández-Remolar et al. 2005 for geological settings). This environment is considered as an analog for some early Mars environments that were characterized by a low pH and oxidizing conditions. Despite the extreme conditions (low pH and high metal concentration in waters), the river has a high microbial diversity (Amaral-Zettler et al. 2002) which provides a great diversity in biomolecules in the solutions and sediments of the river (Parro et al. 2011).
Methods Samples of modern and ancient deposits of three different terraces defined as young, intermediate and old terrace (Fernández-Remolar et al. 2005) ranging from 2000 years to 2.1 Ma were collected. After sample crushing, two different fractions were separated for different preparation and analysed as follows: (1) a small fraction (0.5 g approx.) was separated to be analyzed by FTIR, and (2) 50 g was treated with a PBS-EDTA solution (Phosphate-buffered saline-Ethylenediaminetetra acetic acid) for the extraction of the protein fraction. Later on, the extracts were analyzed by spectrophotometric techniques and by one of the most sensitive available techniques, Fourier Transform Ion Cyclotron Resonance Mass Spectrometry (FTICR-MS) for corroboration of the presence of peptides. For this last technique (FTICR-MS) a Bruker (Bremen, Germany) APEX 12 Qe Fourier transform ion cyclotron resonance mass spectrometer (FTICR-MS) equipped with a 12 Tesla superconducting magnet and an APOLLO II electrospray source was used to obtain ultrahigh resolution mass spectra of the samples.
Results and Discussion The samples were analyzed without previous preparation by FT-IR in order to identify the typical bonds of amide groups: around 1690–1630 cm−1 for the C=O stretch, and around
Peptidic Sequences in Sediments of Río Tinto, Spain 16
Reflectance (%)
Fig. 1 IR spectrum of the intermediate terrace sample, the ground sample was analyzed for amide bond detection without further treatment. The stretching band of C=O is appreciated close to 1600 cm−1. The amide N-H band around 3500–3700 is not noticed
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3700–3500 cm−1 for the amide N-H stretch. In all three samples, the C=O stretching band was identified. However, the region from 3000 to 3700 cm−1 was masked by a very wide band, so it didn’t allow the correct identification of the N-H stretching band (Fig. 1). Even if we obtained a positive response with colorimetric methods for the determination of peptides (Bradford method), the amounts in the samples were really small. So it is Fig. 2 Mass spectra of the 1053 Da peptide, obtained by FTICR-MS. The intensity of the signal diminishes over time from younger to older samples, which suggest they were produced by ancient communities inhabiting the acidic extreme environment of Río Tinto
Intens x 10 8 4
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difficult to consider them as valid ones, because they were very close to the detection limit of the technique. In the case of the FTICR-mass spectrometry analysis, the results showed a positive determination of peptides. In fact, we detected in the oldest terrace (2.1 My old) the presence of five different peptides with the next masses: 1053.140, 1065.466, 1083.105, 1086.114, and 1097.096 Da. The decreasing intensities of peptides over time (Fig. 2) suggest that the signals are not the result of external contamination, but original peptide chains preserved in the sediments. According to these results, the preservation of protein fragments is feasible under extreme conditions (low pH and oxidation conditions). Although proteins hardly survive in natural environments, there are several factors including the nature of the molecule and the environmental conditions, which determine the rate of degradation. When an organism dies its body starts a long process of decomposition, promoted by its own enzymes or by other organisms. According to Ambler and Daniel (1991) the pathways for protein alteration are: hydrolysis of peptide bonds, modification of amino acid chains, and racemization of chiral centres. It has been reported that some amino acids and peptides could be isolated from ancient shells and bones, but changes occurred with time (Abelson 1954). In fact, it has also been suggested, that the association of protein with minerals may delay the decay (Pinck and Allison 1951, Waggoner 2002). Further analyses will be undertaken to characterize the peptides found and to have a better idea of the preservation mechanisms that allow their existence in this kind of environment. From another perspective, the persistence of a molecular record in this acidic habitat opens new possibilities for the search of traces of life along the extensive acidic deposits of Mars. Acknowledgments Part of this research is being supported by the research project AYA 2009–11681. One of us (MCG) thanks the CONACyT (82937) and PAPIIT (IN104109-3) for financial support and CONACyT for a postdoctoral fellowship.
References Abelson PH (1954) Organic constituents of fossils. Carnegie Institute of Washington Yearbook 53:97–101 Amaral-Zettler LA, Gómez F, Zettler E, Keenan BG, Amils R, Sogin ML (2002) Microbiology: Eukaryotic Diversity in Spain's River of Fire. Nature 417:137 Ambler RP, Daniel M (1991) Proteins and Molecular Palaeontology. Philos Trans Biol Sci 333:381–389 Cooper G, Kimmich N, Belisle W, Sarinana J, Brabham K, Garrel L (2001) Carbonaceous meteorites as a source of sugar-related organic compounds for the early earth. Nature 414:879–883 Cronin JR, Pizzarello S (1983) Amino acids in meteorites. Adv Space Res 3:5–18 Fernández-Remolar D, Morris RV, Gruener JE, Amils R, Knoll AH (2005) The Río Tinto Basin, Spain: Mineralogy, sedimentary geobiology, and implications for interpretation of outcrop rocks at Meridiani Planum, Mars. Earth and Planet Sci Lett 240:149–167 Fernández Calvo P, Näke C, Rivas LA, García Villadangos M, Gómez-Elvira J, Parro V (2006) A multi-array competitive immunoassay for the detection of broad-range molecular size organic compounds relevant for astrobiology. Planet Space Sci 54:1612–1621 Mackenzie AS (1984) Applications of biological markers in petroleum geochemistry. In: Books J, Welte D (eds) Advances in petroleum geochemistry, Vol 1. Academic Press, London, pp 115–214 Oró J (1961) Comets and the formation of biochemical compounds on the primitive earth. Nature 190:389–390
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Parro V, de Diego-Castilla G, Rodríguez-Manfredi JA, Rivas LA, Blanco-López Y, Sebastián E, Romeral J, Compostizo C, Herrero PL, García-Marín A, Moreno-Paz M, García-Villadangos M, Cruz-Gil P, Peinado V, Martín-Soler J, Pérez-Mercader J, Gómez-Elvira J (2011) SOLID3: a multiplex antibody microarray-based optical sensor instrument for in situ life detection in planetary exploration. Astrobiology 11:15–28 Pinck LA, Allison FE (1951) Resistance of a Protein-Montmorillonite Complex to decomposition by Soil Microorganism. Science 114:130–131 Pizzarrello S (2007) The chemistry that preceded life’s origin: a study guide from meteorites. Chem Biodivers 4:680–693 Pizzarello S, Feng X, Epstein S, Cronin JR (1994) Isotopic analyses of nitrogenous compounds from the Murchison meteorite: ammonia, amines, amino acids, and polar hydrocarbons. Geochi Cosmochim Acta 58:5579–5587 Sephton MA, Love GD, Watson JS, Verchovsky AB, Wright IP, Snape CE, Gilmour I (2004) Hydropyrolysis of Insoluble Carbonaceous Matter in the Murchison Meteorite: New Insights into its Macromolecular Structure. Geochimica et Cosmochimica Acta 68:1385–1393 Waggoner B (2002) Molecular palaeontology. Encyclopedia of Life Sciences 12:227–232
