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Mass spectrometry for direct identification of biosignatures and microorganisms in Earth analogs of Mars
Planetary and Space Science 72 (2012) 138–145
Contents lists available at SciVerse ScienceDirect
Planetary and Space Science journal homepage: www.elsevier.com/locate/pss
Mass spectrometry for direct identification of biosignatures and microorganisms in Earth analogs of Mars Laura Garcia-Descalzo a, Eva Garcı´a-Lo´pez a, Ana Maria Moreno a, Alberto Alcazar b, Fernando Baquero a,c, Cristina Cid a,n a
´n de Ardoz, Spain Microbial Evolution Laboratory, Center for Astrobiology (CSIC-INTA), 28850 Torrejo Department of Investigation, Hospital Ramon y Cajal, 28034 Madrid, Spain c Department of Microbiology, Hospital Ramon y Cajal, 28034 Madrid, Spain b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 11 February 2012 Received in revised form 25 July 2012 Accepted 3 August 2012 Available online 8 September 2012
Rover missions to Mars require portable instruments that use minimal power, require no sample preparation, and provide suitably diagnostic information to an Earth-based exploration team. In exploration of analog environments of Mars it is important to screen rapidly for the presence of biosignatures and microorganisms and especially to identify them accurately. Matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF-MS) has enormously contributed to the understanding of protein chemistry and cell biology. Without this technique proteomics would most likely not be the important discipline it is today. In this study, besides ‘true’ proteomics, MALDI-TOF-MS was applied for the analysis of microorganisms for their taxonomic characterization from its beginning. An approach was developed for direct analysis of whole bacterial cells without a preceding fractionation or separation by chromatography or electrophoresis on samples of bacteria from an Antarctic glacier. Supported by comprehensive databases, MALDI-TOF-MS-based identification could be widely accepted within only a few years for bacterial differentiation in Mars analogs and could be a technique of election for Mars exploration. & 2012 Elsevier Ltd. All rights reserved.
Keywords: Mars Biosignatures Microorganisms Mass spectrometry MALDI-TOF-MS Antarctic glaciers
1. Introduction There is considerable interest in investigating other worlds and the microbial forms that thrive in extreme environments, especially under those conditions that can provide a model for Martian environments. The Martian surface environment exhibits extremes of salinity, temperature, desiccation, and radiation (Crisler et al., 2012). By analogy with terrestrial extremophile communities, potential protected niches have been postulated for Mars, such as sulfur-rich subsurface areas for chemoautotrophic communities, rocks for endolithic communities, cold environments and permafrost regions (Onstott et al., 2009), hydrothermal vents (Shapiro and Schulze-Makuch, 2009) soil, or evaporite crystals (Fig. 1) (Horneck, 2000; Benison et al., 2008). For instance, there are several similarities between the vast deposits of sulfates
Abbreviations: 2-DE, two-dimensional electrophoresis; ESI, electro spray ionization; GC, gas chromatography; MALDI-TOF-MS, matrix-assisted laser desorption/ionization-time of flight mass spectrometry; PCR, polymerase chain reaction; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis n Correspondence to: Microbial Evolution Laboratory, Center for Astrobiology (CSIC-INTA), Ctra. Ajalvir, km 4, 28850 Torrejo´n de Ardoz, Madrid, Spain. Tel.: þ34 915206455; fax: þ 34 915201074. E-mail addresses: [email protected], [email protected] (C. Cid). 0032-0633/$ - see front matter & 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.pss.2012.08.009
and iron oxides on Mars and the main sulfide-containing iron bioleaching products found in the Rio Tinto (Amils et al., 2011). Further, on Earth, the discovery of cold-tolerant microorganisms in glaciated and permanently frozen environments has broadened the known range of environmental conditions which support microbial life. These microorganisms, known as psychrophiles are defined as organisms having an optimal temperature for growth at about 15 1C or lower, a maximal temperature for growth at about 20 1C, and a minimal temperature for growth at 0 1C or below. The lowest temperature limit for life seems to be around 20 1C, which is the value reported for bacteria living in permafrost soil and in sea ice. Terrestrial models of extraterrestrial icy worlds are being intensively studied (Alcazar et al., 2010). Among them, bacterial communities from Arctic and Antarctic permafrost, subglacial lakes and high mountains are considered representative of these environments in which psychrophile bacteria are the unique inhabitants. When compared to other known prokaryotes, psychrophilic bacteria possess many unique qualities and molecular mechanisms that allow their adaptation to cold environments (Garcı´a-Descalzo et al., 2011). In exploration of analog environments of Mars it is important to screen rapidly for the presence of biosignatures and microorganisms and especially to identify them accurately.
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Fig. 1. Biosignatures and microorganisms from analog environments of Mars. (A) Cold environment in Antarctica. (B) Sulfide-containing iron bioleaching products found in the Rio Tinto (Spain). (C) Hydrothermal vents in Kamchatka peninsula (Russia). (D) Scanning electron microscopy of samples reveals the presence of distinct bacillary particles in an Antarctic glacier (red arrows), suggestive of intact microbes interspersed with mineral granules. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Table 1 General methods for identification of microorganisms. Type of method
Time required
Technique
Cultural
Several days
Morphology by optical and electronic microscopy Selective and differential media Biochemical tests
Serological
One day
Enzyme-linked immunosorbent assays (ELISA) Immunofluorescence assays
Genetic
One day
Nucleic acid hybridization Fluorescent in situ hybridization (FISH) Polymerase chain reaction (PCR) Reverse transcriptase (RT-PCR) Microarrays
Other methods
One to several days
Flow cytometry Pulsed-field gel electrophoresis (PFGE) Polyacrylamide gel electrophoresis (PAGE)
Traditional bacteriological techniques, listed in Table 1 (Fig. 2), such as culture on selective medium (Fig. 2A), microscopic techniques (Fig. 2B), distinction of genotypes by DNA sequence analysis (Fig. 2C and D) or biochemical activity tests, are often time-consuming and labor-intensive. Several methods have been reported that could be useful for Mars exploration. Among them, short-wave infrared (SWIR) spectroscopic instruments such as the Portable Infrared Mineral Analyzer (PIMA) have been tested to investigate sites of paleobiological interest (Brown et al., 2004). RAMAN spectroscopy (Breier et al., 2010) has been used to make a variety of measurements that are only stable under in situ conditions. Gas chromatography–mass spectrometry has been applied to analyze carboxylic acid mixtures (Pietrogrande et al., 2005). Further, antibody microarrays have been developed for life detection in planetary exploration (Parro et al., 2011). In this study, with the aim of determining whether
matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF-MS), could be an option for bacterial differentiation we perform the identification of bacterial species from an Antarctic glacier.
2. Material and methods 2.1. Bacterial growth and sample preparation Glacial ice samples were collected at an Antarctic glacier in Mount Pond, Deception Island. Ice samples were obtained by removing 20–30 cm of thick debris and cutting out a square block of 20 cm on a side. Samples were wrapped in plastic bags and stored at 20 1C until processing. Ice samples were processed by using a surface decontamination and melting procedure consistent
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Fig. 2. Traditional bacteriological techniques. (A) Bacterial cultures. (B) Scanning electron microscopy. (C) Agarose gel of amplicons obtained by polymerase chain reaction (PCR). (D) Phylogenetic analysis performed by 16S rDNA analysis.
with previous studies (Bidle et al., 2007). All procedures were performed by using bleach-sterilized work areas, a UV-irradiated laminar flow hood, ethanol-sterilized tools and sterilized gloves. To control for laboratory contamination, 1 L of MilliQ rinse water was frozen and subjected to identical analytical procedures. Ice-melt water was amended with nutrients and incubated at 4 1C in the dark. Nutrient formulations were as follows in g L 1 (M1, 5 g of peptone, 0.15 g of ferric ammonium citrate, 0.2 g of MgSO4 7H2O/0.05 g of CaCl2/0.05 g of MnSO4 H2O/0.01 g of FeCl3 6H2O 0.01; M2, 1 g of glucose/1 g of peptone/0.5 g of yeast extract/0.2 g of MgSO4 7H2O/0.05 g MnSO4 H2O; M3, 1 g of glucose/0.5 g of casamino acids/0.5 g of yeast extract/1 g of KH2PO4/0.5 g of CaCl2 2H2O/0.5 g of MnCl2 4H2O; M4 (R2A) 0.5 g of yeast extract/0.5 g of peptone/0.5 g of casamino acids/ 0.5 g of glucose/0.5 g of soluble starch/0.5 g of sodium pyruvate/ 0.5 g of KH2PO4/0.05 g of MgSO4 7H2O). All nutrients formulations were made as concentrated stocks (20–200x), sterilized by autoclaving and directly added ( o1/50 volume ratio) to meltwater at 1x final concentration. Colonies were isolated on agar plates supplemented with M1, M2, M3 or M4, incubated at 4 1C. Further, bacterial cultures of Shewanella frigidimarina and Psychrobacter cryohalolentis were used as control. These bacteria were purchased from culture collections (ATCCs) and incubated in marine broth 2216. 2.2. Extraction of the soluble protein fraction Bacteria from each biological replicate (four in total) were obtained from the individual cultures. The cells were harvested, washed and lysed in buffer A (20 mM Tris–HCl, pH 7.6; 140 mM potassium chloride; 2 mM benzamidine; 1 mM EDTA; 10 mg/ml pepstatin A, leupeptin and antipain), using a French Press at 4 1C. Cell debris was removed by centrifugation at 11,000g for 10 min
to obtain a supernatant, and the protein extracts were processed using a 2-D Clean-Up kit (GE Healthcare, Spain). The pellet was frozen and stored at 80 1C. The protein content was determined in cell extracts using the BioRad protein assay based on the Bradford method using different dilutions of BSA as the standard. The samples were adjusted to a protein concentration of 5–10 mg/ml. All steps were carried out at 4 1C.
2.3. Two-dimensional electrophoresis Horizontal slab gel isoelectric focusing (IEF) was combined with SDS-PAGE for 2-DE by using the Multiphor II apparatus for the first dimension and standard vertical slab gel electrophoresis for the second dimension, according to the manufacturer’s instructions. Samples (about 500 mg of protein) were prepared in 7 M urea/2 M thiourea and 5% b-mercaptoethanol, centrifuged and applied to pH gradient strips for IEF. Carrier ampholyte urea IEF was carried out using immobilized pH 3–10 and pH 3–11 nonlinear gradient strips (18 cm). After the first dimension, the IEF strips were processed for the second dimension in SDS-PAGE carried out on 12% acrylamide (2.6% crosslinking) gels (1.0 mm thick) with IEF strips being used as the stacking gels. The spots resolved by 2-DE from the gels were stained with Coomassie Blue or with MALDI-MS-compatible silver reagent for peptide mass fingerprinting analysis and protein identification. Protein spots were excised manually from the Coomassie Blue or silver stained 2-DE gels, de-stained and then digested automatically using a Proteineer DP protein digestion station (Bruker-Daltonics, Bremen, Germany). An aliquot of the above digestion solution was mixed with an aliquot of a-cyano-4hydroxycinnamic acid (Bruker-Daltonics) in 33% aqueous acetonitrile and 0.1% trifluoroacetic acid.
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Fig. 3. Components of a mass spectrometer and structural formulae of three typical matrix compounds. (A) The three primary components of a mass spectrometer: an ion source to vaporize and ionize samples, a mass analyzer to separate ions based on their mass to charge ratio and a detector to measure the separated ions. (B) Matrix compounds: 2,5dihydroxy-benzoic acid (DHBA), a-cyano-4-hydroxy-cinnamic acid (HCCA) and sinapicnic acid (SA).
2.4. Direct analysis of whole cell bacteria The identification protocol used was based on the method previously described by Cid et al. (2010) with minor variations. Frozen cell pellets (about 100 mg) were thawed on ice and resuspended in 400 mL cold deionized water. One microlitre (about 0.20 mg) of sample was diluted with 19 mL of 0.1% TFA aqueous buffer. An aliquot of the above solution was mixed with an aliquot of a-cyano-4-hydroxycinnamic acid (Bruker-Daltonics) in 33% aqueous acetonitrile and 0.1% trifluoroacetic acid. This mixture was deposited onto a MALDI probe (600 mm AnchorChip for Bruker-Daltonics) and allowed to dry at room temperature. When the matrix for MALDI-TOF-MS was changed to sinapinic acid or 2,5-dihydroxybenzoic acid, or the preparation of solvents for matrix was changed, the resulting MS spectra were affected. As such, we used the protocol described here for matrix preparation consistently throughout this study. MALDI-TOF-MS (/MS) data were obtained using an Ultraflex TOF-TOF mass spectrometer equipped with a LIFT-MS/MS device (Bruker-Daltonics) and a 4800 MALDI-TOF-TOF mass spectrometer (Applied Biosystems). Spectra were acquired in the positive-ion mode at 50 Hz laser frequency, and 100–1500 individual spectra were averaged. When available, for fragment ion analysis in the tandem timeof-flight (TOF/TOF) mode, precursors were accelerated to 8 kV and selected in a timed ion gate. Spectral data were analyzed through
MS BioTools program (Bruker-Daltonics) or ProteinPilot software (Applied Biosystems) to search the NCBInr database using the Mascot database search algorithm (Matrix Science, London, UK).
3. Theory Mass spectrometry is not a new technique. The separation of charged particles based on mass, charge and flight path has been known since 1897, when Thomson measured the charge-to-mass ratio of electron. This technique, associated to others, has been employed for detection of fatty acids, sugar monomers and peptides. It is able to analyze with high accuracy the composition of different chemical elements and atomic isotopes splitting their atomic nuclei according to their mass–charge ratio (m/z). It can be used to identify different chemical elements that form a compound or to determine the isotopic content of different elements in the same compound. The three primary components needed to produce a mass spectrometer are relatively straightforward as shown in Fig. 3A. These have not change much since that time, and include an ion source to vaporize and ionize samples, a mass analyzer to separate ions based on their mass to charge ratio and a detector to measure the separated ions. Firstly, the material to be analyzed is ionized and ions are then transported by magnetic or electric fields to the mass analyzer.
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Techniques for ionization have been key to determine what type of samples can be analyzed by mass spectrometry. Two techniques are often used with liquid and solid biological samples: electro spray ionization (ESI) and laser matrix-assisted laser desorption/ionization (MALDI). In the MALDI ionization analytes co-crystallized with a suitable matrix are converted into ions by the action of a laser. This source of ionization is usually associated with a time of flight analyzer (TOF) in which the ions are separated according to their mass–charge after being accelerated in an electric field. At last, a mass spectrometer detector records the charge induced or current produced when an ion passes by or hits a surface. The choice of matrix is a critical step in MALDI-MS because it can promote ionization of specific families of compounds (e.g., phospholipids, peptides and proteins). Different types of matrix used in the experiments are shown in Fig. 3B. Profiling of fatty acid monomers, released from membrane phospholipids of whole cells, using gas chromatography (GC) with
flame ionization detector has been one of the most widely used analytical method for bacterial speciation (after isolation and growth of individual bacterial species). When necessary, the identity of these fatty acids is confirmed by GC–MS. Additionally, other small molecules are used extensively to assess the microbial content in environmental samples. Hydroxy fatty acids (components of the lipid A region of lipopolysaccharides) are used to quantitate the levels of Gram-negative bacteria and muramic acid (that takes part of the glycan backbone of peptidoglycan) as a marker for total bacterial load. Phospholipids can be ionized as intact entities for MS or MS–MS analysis. On MS–MS analysis, in the negative ion mode, individual fatty acids are identified from the product ion spectra. The class of phospholipid can be determined by summing the masses of individual fatty acids and subtracting the value from the mass of the parent ion. Dramatic improvements in the sensitivity of phospholipid analysis have been achieved using ESI–MS and MS–MS. However, phospholipids are widely distributed in
Fig. 4. Identification of bacteria in a mixture. The representative MALDI-TOF-MS spectra of two bacterial species of a mixture are displayed. (A) Shewanella frigidimarina, (B) Psychrobacter frigidicola.
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Table 2 Identification of proteins. ACcesion code
Locus code
Protein description
Organism
Score
MW
PI
Matched peptides
gi9114563931 gi993005146
YP_751445 YP_579583
Phosphate binding protein Isocitrate dehydrogenase, NADPdependent
Shewanella frigidimarina NCIMB400 Psychrobacter chryohalolentis K5
377 160
34,834 83,153
8.78 5.63
10 8
Fig. 5. Identification of whole bacteria. (A, B) Representative direct mass spectra of whole cells isolated from glacial ice. (C) 2-DE gels were used to check the identification of S. oneidensis.
nature that species level detection in complex matrices is not afforded. Further, MALDI-TOF-MS has enormously contributed to the identification of peptides. Without this technique proteomics would most likely not be the important discipline it is today. Besides ‘true’ proteomics, MALDI-TOF-MS is being applied for the analysis of microorganisms for their taxonomic characterization from its beginning.
4. Results 4.1. Analysis of proteins by MALDI-TOF-MS Two bacterial isolates of S. frigidimarina and P. cryohalolentis were used to examine the minimum number of bacterial cells needed for identification by MALDI-TOF-MS. We did a series of bacterial dilutions. The minimum number for correct identification was determined to be 5.9 10 3 for S. frigidimarina and 5.5 10 3 for P. cryohalolentis (not shown). On the other hand, we examined the capability of identifying bacteria from bacterial mixtures. We mixed an equal amount of S. frigidimarina and P. cryohalolentis. The bacterial species in the mixture were also identified (Fig. 4). Table 2 summarizes the characteristics of the two identified proteins that obtained best scores in the analysis of these bacterial species.
4.2. Direct analysis of whole cell bacteria Ice-melt water from glacial ice samples was amended with nutrients and incubated at 4 1C in the dark. Colonies were isolated on agar plates supplemented with M1, M2, M3 or M4, incubated at 4 1C. Frozen cell pellets were analyzed by MALDI-TOF-MS. Fig. 5 shows the direct identification of proteins of Shewanella oneidensis from whole cells. Data of two representative identified proteins are shown in Table 3 (Fig. 5A and B). In order to check this result, cell extracts of this culture were analyzed by two-dimensional electrophoresis (2-DE) (Fig. 5C). Several spots were excised from gels in the range between a molecular weight of 50–60 and pI 4–8. They were analyzed by MALDI-TOF-MS. Spots indicated in Fig. 5 as 1 and 2 corresponded to the same identified proteins listed in Table 3.
5. Discussion In a search for extant life beyond Earth, biomolecules and microorganisms are the most likely candidates. On Earth, life has developed strategies to cope with the so-called extreme conditions (Des Marais et al., 2008). The successful identification of unknown biosignatures and microorganisms from Earth analogs of Mars requires the intelligent use of several different techniques. Among them, some traditional methods include division into serotypes by
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Table 3 Identification of bacteria. Spot
Accesion code
Locus code
Protein description
Organism
Score
MW
PI
Matched peptides
1 2
gi924372295 gi924372557
NP_716337 NP_716599
Chaperonin GroEL Fumarate reductase flavoprotein precursor
Shewanella oneidensis MR1 Shewanella oneidensis MR1
115 213
57,101 62,865
4.84 7.27
6 14
specific antibodies, distinction of genotypes by DNA sequence analysis and biochemical tests. These methods provide high sensitivity and specificity, but their efficiency is limited by the complexity of the procedures, they are time-consuming and are completely dependent on the availability of antibodies or the knowledge of genetic sequences of the target bacteria. A survey was carried out on microorganisms from psychrophilic environments with the aim to demonstrate that directly subjecting intact bacterial colonies for protein profiling using MALDI-TOF-MS can be a simple and reliable approach to accurately identify them (Van Veen et al., 2010). The principle issue relating to the use of MALDI-TOF-MS for bacterial characterization is resolving power. This factor has profound effect in large measure because the characterization of whole bacteria by MALDI involves the analysis of a very complex mixture. This is done without any preliminary separation of components. TOF mass spectrometry can be easily coupled to MALDI ionization sources, and MALDI-TOF-MS represents a very rapid method for analyzing the proteins desorbed directly from whole cells (Lay and Liyanage, 2006). The whole cell spectra produced by MALDITOF-MS have taxonomically characteristic features that can be used to differentiate bacteria at the genus, species and strain level, even though only a very small portion of the bacterial proteome can be detected by direct analysis (Welker and Moore, 2011). 5.1. Applications to Mars exploration Searching for traces of extinct and/or extant life on Mars is one of the major objectives for remote sensing and in situ exploration of the planet. As a whole, the surface of Mars is extremely hostile due to high UV radiation, desiccation, oxidants, and low temperatures, among a variety of conditions that limit the capability to support life. But increasingly it is believed that past and present Mars has or does provide habitable conditions sufficient to support microorganismal life forms. There are on Mars some specific niches that could have been habitable during transient and local episodes. This includes environments in fluvial deposits, gullies, transient geothermal and/or hydrothermal conditions that can be triggered by large impacts or by proximity with igneous activity. Subsurface ice abounds in the polar regions as evidenced by satellite observations and the recent Phoenix mission (Smith et al., 2009). The spacecrafts sent to Mars regularly reveal new evidence suggesting that the environmental conditions on early Mars were very different than today, with liquid water flowing on the surface. In past times, life might have emerged under Martian conditions milder than the present ones, and left some remnants at the surface. Even if this did not happen, prebiotic molecules may have been preserved in the soil, and they might be similar to those that prevailed on the Earth surface some 3.5–4 billion years ago. There is increasing evidence of liquid water in ancient times when conditions were similar to those on Earth during the first emergence of life. There are also areas with specific delivery and burial of constituents including volcanic ashes spring deposits, atmospheric deposits, in addition to extraterrestrial delivery of meteoritic organics. Further, some special mineral sites can interact with transient conditions to change the habitability conditions. In upcoming years various space missions will investigate the habitability of Mars and the possibility of extinct or extant life
existing in the Red Planet. Preliminary analyses of Mars analog soils on Earth provide crucial information to determine whether signatures of past and/or present life may still exist in the Martian regolith and these analyses also help when choosing target locations for molecular signatures of life on Mars. These multidisciplinary findings help in the preparation phase for future Mars missions, and are crucial to successfully target locations that may host organic matter, as well as extract and detect biosignatures on Mars. Rover missions to Mars require portable instruments that use minimal power, require no sample preparation, and provide suitably diagnostic mineralogical information to an Earth-based exploration team (Brown et al., 2004). In exploration of analog environments of Mars it is important to screen rapidly for the presence of biosignatures and especially to identify them accurately. Traditional biodetection techniques are often time-consuming and labor-intensive. MALDI-TOF-MS has become a popular and versatile method of analyzing a broad range of macromolecules (proteins, DNA, oligonucleotides, oligosaccharides) from biological origin. The accuracy and speed with which data can be obtained by MALDI-TOF-MS could make this a powerful tool for environmental monitoring in Earth analogs of Mars (Jimmy et al., 1994; Perkins et al., 1999; Ruelle et al., 2004). In the present work we apply our analysis to the identification of biosignatures and microorganisms in samples from an Antarctic glacier as an Earth analog of Mars. We show that our method is actually reliable for biodetection, and therefore it is a powerful tool for the search of life on Mars in the next generation of space missions to the planet.
6. Conclusion Scientists disagree when it comes to evaluating the chances for detecting life on Mars. There is scientific consensus that past conditions on Mars may have allowed life to develop and that the search for extinct life may be more fruitful than looking for extant life. Life on Earth originated approximately 3.5–4 billion years ago and has adapted to nearly every explored environment, including hydrothermal vents, arid deserts, and ice lakes in Antarctica. Immense progress in the study of extreme life has changed our view of habitability beyond Earth. Recent data suggest that microorganisms can survive in cold and dry climates and inside salt deposits. These and other ecological niches could harbor extremophiles in the subsurface of Mars. However, a wide distribution of life as observed on our planet is not expected on Mars; an overall low amount of biomass, restricted to localized areas, is more probable. The search for organic material and biosignatures on Mars is a highly complex endeavor. Instruments on future Mars missions are limited to searching for signs of life that conform to our preconceived notions of biomarkers. A combination of solar ultraviolet radiation and oxidation processes in the soil are destructive to organic material and life on and close to the surface. Galactic cosmic rays can penetrate the surface and effectively destroy organic and biological materials over geological timescales. The successful hunt for extant biosignatures will be a tradeoff between multiple parameters, including accessibility, biomarker concentration, the preservation potential, extractability, and instrument performance. When deploying organic detection instruments on Mars, consideration only of the geological context and the history of regional aqueous processes for
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landing site selection may be insufficient. The host microenvironment of putative microbes on Mars must be compatible with the capabilities of the instrumentation payload. Earth-based field research in extreme environments (such as dry deserts and permafrost regions) that investigates metabolic processes of microbes and their geological environments will therefore be a vital research activity in the preparation phases of future space missions. The detection of extant life prior to Mars sample return missions will depend strongly on instrumentation that can distinguish biological from non-biological organic matter and fossil organic matter from recent remains. With respect to the previous in situ exploration of Mars, the astrobiological relevance of the upcoming Mars missions will be greatly improved with the capability to thoroughly study samples at a microscopic scale, in a combined protocol, with a suite of highly performing instruments. Analog environments on Earth can provide us a deeper understanding of where and how to look for life on Mars. In this paper we have compared the different techniques applied for the identification of biomarkers in these regions and identify the implications to Astrobiology and the search for habitable environments on Mars. Several methods have been developed for the identification of biomarkers and microorganisms. These include, among others, cultural, serological and genetic methods. These methods require obtaining pure cultures. Microscopic observation requires confirmation of the suspected identity by other methods with specific antibodies or molecular probes, which should be previously synthesized. Although PCR, arrays and other techniques using antibodies or probes are sometimes employed with mixed samples, if none of the probes or antibodies corresponds to any of the bacteria in the sample, then other tests with pure cultures should be necessary. Therefore, mass spectrometry presents an attractive alternative for bacterial differentiation in the exploration of identification of biosignatures and microorganisms in Earth analogs of Mars. In this study, we combined the analysis of proteins by MALDI-TOF-MS with direct analysis of whole cell bacteria to identify the microbial populations associated with Antarctic glaciers. Based on results obtained with cultured bacteria, and also on tests performed with environmental samples, we give a view of the analytical capability for the experiments to detect and identify a broad range of molecules, including proteins and whole cells. Firstly, we performed bacterial cultures of both environmental ice samples from Antarctic glaciers and bacteria from cultured collections (Shewanella frigidimarina and Psychrobacter cryohalolentis), used as control. Secondly, bacterial isolates and mixtures of several bacterial species were identified by MALDI-TOF-MS. Further, environmental samples of ice were subjected to direct identification of whole cell bacteria. Classical proteomic analysis with 2-DE gels demonstrated that the same proteins and bacterial species could be detected by both techniques. The data acquired with this instrumentation should thus be of primary importance to give an insight into the potential existence of a present or past life on Mars, and to determine the influence of the environmental surface conditions on the current potential habitability of Mars.
Acknowledgment We are indebted to Drs. M. Martinez-Gomariz and C. Gil from Proteomic Unit of the Parque Cientı´fico de Madrid. We thank also Mr. A. Casals and Mr. J. Barba for their skillful assistance at the Antarctic Research Station Gabriel de Castilla. This research was supported by Grants CTM/2008-00304/ANT and CTM2010-12134-E /ANT from the Spanish Ministerio de Economı´a y Competitividad.
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Systematic and Applied Microbiology 34, 2–11.
Contents lists available at SciVerse ScienceDirect
Planetary and Space Science journal homepage: www.elsevier.com/locate/pss
Mass spectrometry for direct identification of biosignatures and microorganisms in Earth analogs of Mars Laura Garcia-Descalzo a, Eva Garcı´a-Lo´pez a, Ana Maria Moreno a, Alberto Alcazar b, Fernando Baquero a,c, Cristina Cid a,n a
´n de Ardoz, Spain Microbial Evolution Laboratory, Center for Astrobiology (CSIC-INTA), 28850 Torrejo Department of Investigation, Hospital Ramon y Cajal, 28034 Madrid, Spain c Department of Microbiology, Hospital Ramon y Cajal, 28034 Madrid, Spain b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 11 February 2012 Received in revised form 25 July 2012 Accepted 3 August 2012 Available online 8 September 2012
Rover missions to Mars require portable instruments that use minimal power, require no sample preparation, and provide suitably diagnostic information to an Earth-based exploration team. In exploration of analog environments of Mars it is important to screen rapidly for the presence of biosignatures and microorganisms and especially to identify them accurately. Matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF-MS) has enormously contributed to the understanding of protein chemistry and cell biology. Without this technique proteomics would most likely not be the important discipline it is today. In this study, besides ‘true’ proteomics, MALDI-TOF-MS was applied for the analysis of microorganisms for their taxonomic characterization from its beginning. An approach was developed for direct analysis of whole bacterial cells without a preceding fractionation or separation by chromatography or electrophoresis on samples of bacteria from an Antarctic glacier. Supported by comprehensive databases, MALDI-TOF-MS-based identification could be widely accepted within only a few years for bacterial differentiation in Mars analogs and could be a technique of election for Mars exploration. & 2012 Elsevier Ltd. All rights reserved.
Keywords: Mars Biosignatures Microorganisms Mass spectrometry MALDI-TOF-MS Antarctic glaciers
1. Introduction There is considerable interest in investigating other worlds and the microbial forms that thrive in extreme environments, especially under those conditions that can provide a model for Martian environments. The Martian surface environment exhibits extremes of salinity, temperature, desiccation, and radiation (Crisler et al., 2012). By analogy with terrestrial extremophile communities, potential protected niches have been postulated for Mars, such as sulfur-rich subsurface areas for chemoautotrophic communities, rocks for endolithic communities, cold environments and permafrost regions (Onstott et al., 2009), hydrothermal vents (Shapiro and Schulze-Makuch, 2009) soil, or evaporite crystals (Fig. 1) (Horneck, 2000; Benison et al., 2008). For instance, there are several similarities between the vast deposits of sulfates
Abbreviations: 2-DE, two-dimensional electrophoresis; ESI, electro spray ionization; GC, gas chromatography; MALDI-TOF-MS, matrix-assisted laser desorption/ionization-time of flight mass spectrometry; PCR, polymerase chain reaction; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis n Correspondence to: Microbial Evolution Laboratory, Center for Astrobiology (CSIC-INTA), Ctra. Ajalvir, km 4, 28850 Torrejo´n de Ardoz, Madrid, Spain. Tel.: þ34 915206455; fax: þ 34 915201074. E-mail addresses: [email protected], [email protected] (C. Cid). 0032-0633/$ - see front matter & 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.pss.2012.08.009
and iron oxides on Mars and the main sulfide-containing iron bioleaching products found in the Rio Tinto (Amils et al., 2011). Further, on Earth, the discovery of cold-tolerant microorganisms in glaciated and permanently frozen environments has broadened the known range of environmental conditions which support microbial life. These microorganisms, known as psychrophiles are defined as organisms having an optimal temperature for growth at about 15 1C or lower, a maximal temperature for growth at about 20 1C, and a minimal temperature for growth at 0 1C or below. The lowest temperature limit for life seems to be around 20 1C, which is the value reported for bacteria living in permafrost soil and in sea ice. Terrestrial models of extraterrestrial icy worlds are being intensively studied (Alcazar et al., 2010). Among them, bacterial communities from Arctic and Antarctic permafrost, subglacial lakes and high mountains are considered representative of these environments in which psychrophile bacteria are the unique inhabitants. When compared to other known prokaryotes, psychrophilic bacteria possess many unique qualities and molecular mechanisms that allow their adaptation to cold environments (Garcı´a-Descalzo et al., 2011). In exploration of analog environments of Mars it is important to screen rapidly for the presence of biosignatures and microorganisms and especially to identify them accurately.
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Fig. 1. Biosignatures and microorganisms from analog environments of Mars. (A) Cold environment in Antarctica. (B) Sulfide-containing iron bioleaching products found in the Rio Tinto (Spain). (C) Hydrothermal vents in Kamchatka peninsula (Russia). (D) Scanning electron microscopy of samples reveals the presence of distinct bacillary particles in an Antarctic glacier (red arrows), suggestive of intact microbes interspersed with mineral granules. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Table 1 General methods for identification of microorganisms. Type of method
Time required
Technique
Cultural
Several days
Morphology by optical and electronic microscopy Selective and differential media Biochemical tests
Serological
One day
Enzyme-linked immunosorbent assays (ELISA) Immunofluorescence assays
Genetic
One day
Nucleic acid hybridization Fluorescent in situ hybridization (FISH) Polymerase chain reaction (PCR) Reverse transcriptase (RT-PCR) Microarrays
Other methods
One to several days
Flow cytometry Pulsed-field gel electrophoresis (PFGE) Polyacrylamide gel electrophoresis (PAGE)
Traditional bacteriological techniques, listed in Table 1 (Fig. 2), such as culture on selective medium (Fig. 2A), microscopic techniques (Fig. 2B), distinction of genotypes by DNA sequence analysis (Fig. 2C and D) or biochemical activity tests, are often time-consuming and labor-intensive. Several methods have been reported that could be useful for Mars exploration. Among them, short-wave infrared (SWIR) spectroscopic instruments such as the Portable Infrared Mineral Analyzer (PIMA) have been tested to investigate sites of paleobiological interest (Brown et al., 2004). RAMAN spectroscopy (Breier et al., 2010) has been used to make a variety of measurements that are only stable under in situ conditions. Gas chromatography–mass spectrometry has been applied to analyze carboxylic acid mixtures (Pietrogrande et al., 2005). Further, antibody microarrays have been developed for life detection in planetary exploration (Parro et al., 2011). In this study, with the aim of determining whether
matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF-MS), could be an option for bacterial differentiation we perform the identification of bacterial species from an Antarctic glacier.
2. Material and methods 2.1. Bacterial growth and sample preparation Glacial ice samples were collected at an Antarctic glacier in Mount Pond, Deception Island. Ice samples were obtained by removing 20–30 cm of thick debris and cutting out a square block of 20 cm on a side. Samples were wrapped in plastic bags and stored at 20 1C until processing. Ice samples were processed by using a surface decontamination and melting procedure consistent
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Fig. 2. Traditional bacteriological techniques. (A) Bacterial cultures. (B) Scanning electron microscopy. (C) Agarose gel of amplicons obtained by polymerase chain reaction (PCR). (D) Phylogenetic analysis performed by 16S rDNA analysis.
with previous studies (Bidle et al., 2007). All procedures were performed by using bleach-sterilized work areas, a UV-irradiated laminar flow hood, ethanol-sterilized tools and sterilized gloves. To control for laboratory contamination, 1 L of MilliQ rinse water was frozen and subjected to identical analytical procedures. Ice-melt water was amended with nutrients and incubated at 4 1C in the dark. Nutrient formulations were as follows in g L 1 (M1, 5 g of peptone, 0.15 g of ferric ammonium citrate, 0.2 g of MgSO4 7H2O/0.05 g of CaCl2/0.05 g of MnSO4 H2O/0.01 g of FeCl3 6H2O 0.01; M2, 1 g of glucose/1 g of peptone/0.5 g of yeast extract/0.2 g of MgSO4 7H2O/0.05 g MnSO4 H2O; M3, 1 g of glucose/0.5 g of casamino acids/0.5 g of yeast extract/1 g of KH2PO4/0.5 g of CaCl2 2H2O/0.5 g of MnCl2 4H2O; M4 (R2A) 0.5 g of yeast extract/0.5 g of peptone/0.5 g of casamino acids/ 0.5 g of glucose/0.5 g of soluble starch/0.5 g of sodium pyruvate/ 0.5 g of KH2PO4/0.05 g of MgSO4 7H2O). All nutrients formulations were made as concentrated stocks (20–200x), sterilized by autoclaving and directly added ( o1/50 volume ratio) to meltwater at 1x final concentration. Colonies were isolated on agar plates supplemented with M1, M2, M3 or M4, incubated at 4 1C. Further, bacterial cultures of Shewanella frigidimarina and Psychrobacter cryohalolentis were used as control. These bacteria were purchased from culture collections (ATCCs) and incubated in marine broth 2216. 2.2. Extraction of the soluble protein fraction Bacteria from each biological replicate (four in total) were obtained from the individual cultures. The cells were harvested, washed and lysed in buffer A (20 mM Tris–HCl, pH 7.6; 140 mM potassium chloride; 2 mM benzamidine; 1 mM EDTA; 10 mg/ml pepstatin A, leupeptin and antipain), using a French Press at 4 1C. Cell debris was removed by centrifugation at 11,000g for 10 min
to obtain a supernatant, and the protein extracts were processed using a 2-D Clean-Up kit (GE Healthcare, Spain). The pellet was frozen and stored at 80 1C. The protein content was determined in cell extracts using the BioRad protein assay based on the Bradford method using different dilutions of BSA as the standard. The samples were adjusted to a protein concentration of 5–10 mg/ml. All steps were carried out at 4 1C.
2.3. Two-dimensional electrophoresis Horizontal slab gel isoelectric focusing (IEF) was combined with SDS-PAGE for 2-DE by using the Multiphor II apparatus for the first dimension and standard vertical slab gel electrophoresis for the second dimension, according to the manufacturer’s instructions. Samples (about 500 mg of protein) were prepared in 7 M urea/2 M thiourea and 5% b-mercaptoethanol, centrifuged and applied to pH gradient strips for IEF. Carrier ampholyte urea IEF was carried out using immobilized pH 3–10 and pH 3–11 nonlinear gradient strips (18 cm). After the first dimension, the IEF strips were processed for the second dimension in SDS-PAGE carried out on 12% acrylamide (2.6% crosslinking) gels (1.0 mm thick) with IEF strips being used as the stacking gels. The spots resolved by 2-DE from the gels were stained with Coomassie Blue or with MALDI-MS-compatible silver reagent for peptide mass fingerprinting analysis and protein identification. Protein spots were excised manually from the Coomassie Blue or silver stained 2-DE gels, de-stained and then digested automatically using a Proteineer DP protein digestion station (Bruker-Daltonics, Bremen, Germany). An aliquot of the above digestion solution was mixed with an aliquot of a-cyano-4hydroxycinnamic acid (Bruker-Daltonics) in 33% aqueous acetonitrile and 0.1% trifluoroacetic acid.
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Fig. 3. Components of a mass spectrometer and structural formulae of three typical matrix compounds. (A) The three primary components of a mass spectrometer: an ion source to vaporize and ionize samples, a mass analyzer to separate ions based on their mass to charge ratio and a detector to measure the separated ions. (B) Matrix compounds: 2,5dihydroxy-benzoic acid (DHBA), a-cyano-4-hydroxy-cinnamic acid (HCCA) and sinapicnic acid (SA).
2.4. Direct analysis of whole cell bacteria The identification protocol used was based on the method previously described by Cid et al. (2010) with minor variations. Frozen cell pellets (about 100 mg) were thawed on ice and resuspended in 400 mL cold deionized water. One microlitre (about 0.20 mg) of sample was diluted with 19 mL of 0.1% TFA aqueous buffer. An aliquot of the above solution was mixed with an aliquot of a-cyano-4-hydroxycinnamic acid (Bruker-Daltonics) in 33% aqueous acetonitrile and 0.1% trifluoroacetic acid. This mixture was deposited onto a MALDI probe (600 mm AnchorChip for Bruker-Daltonics) and allowed to dry at room temperature. When the matrix for MALDI-TOF-MS was changed to sinapinic acid or 2,5-dihydroxybenzoic acid, or the preparation of solvents for matrix was changed, the resulting MS spectra were affected. As such, we used the protocol described here for matrix preparation consistently throughout this study. MALDI-TOF-MS (/MS) data were obtained using an Ultraflex TOF-TOF mass spectrometer equipped with a LIFT-MS/MS device (Bruker-Daltonics) and a 4800 MALDI-TOF-TOF mass spectrometer (Applied Biosystems). Spectra were acquired in the positive-ion mode at 50 Hz laser frequency, and 100–1500 individual spectra were averaged. When available, for fragment ion analysis in the tandem timeof-flight (TOF/TOF) mode, precursors were accelerated to 8 kV and selected in a timed ion gate. Spectral data were analyzed through
MS BioTools program (Bruker-Daltonics) or ProteinPilot software (Applied Biosystems) to search the NCBInr database using the Mascot database search algorithm (Matrix Science, London, UK).
3. Theory Mass spectrometry is not a new technique. The separation of charged particles based on mass, charge and flight path has been known since 1897, when Thomson measured the charge-to-mass ratio of electron. This technique, associated to others, has been employed for detection of fatty acids, sugar monomers and peptides. It is able to analyze with high accuracy the composition of different chemical elements and atomic isotopes splitting their atomic nuclei according to their mass–charge ratio (m/z). It can be used to identify different chemical elements that form a compound or to determine the isotopic content of different elements in the same compound. The three primary components needed to produce a mass spectrometer are relatively straightforward as shown in Fig. 3A. These have not change much since that time, and include an ion source to vaporize and ionize samples, a mass analyzer to separate ions based on their mass to charge ratio and a detector to measure the separated ions. Firstly, the material to be analyzed is ionized and ions are then transported by magnetic or electric fields to the mass analyzer.
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Techniques for ionization have been key to determine what type of samples can be analyzed by mass spectrometry. Two techniques are often used with liquid and solid biological samples: electro spray ionization (ESI) and laser matrix-assisted laser desorption/ionization (MALDI). In the MALDI ionization analytes co-crystallized with a suitable matrix are converted into ions by the action of a laser. This source of ionization is usually associated with a time of flight analyzer (TOF) in which the ions are separated according to their mass–charge after being accelerated in an electric field. At last, a mass spectrometer detector records the charge induced or current produced when an ion passes by or hits a surface. The choice of matrix is a critical step in MALDI-MS because it can promote ionization of specific families of compounds (e.g., phospholipids, peptides and proteins). Different types of matrix used in the experiments are shown in Fig. 3B. Profiling of fatty acid monomers, released from membrane phospholipids of whole cells, using gas chromatography (GC) with
flame ionization detector has been one of the most widely used analytical method for bacterial speciation (after isolation and growth of individual bacterial species). When necessary, the identity of these fatty acids is confirmed by GC–MS. Additionally, other small molecules are used extensively to assess the microbial content in environmental samples. Hydroxy fatty acids (components of the lipid A region of lipopolysaccharides) are used to quantitate the levels of Gram-negative bacteria and muramic acid (that takes part of the glycan backbone of peptidoglycan) as a marker for total bacterial load. Phospholipids can be ionized as intact entities for MS or MS–MS analysis. On MS–MS analysis, in the negative ion mode, individual fatty acids are identified from the product ion spectra. The class of phospholipid can be determined by summing the masses of individual fatty acids and subtracting the value from the mass of the parent ion. Dramatic improvements in the sensitivity of phospholipid analysis have been achieved using ESI–MS and MS–MS. However, phospholipids are widely distributed in
Fig. 4. Identification of bacteria in a mixture. The representative MALDI-TOF-MS spectra of two bacterial species of a mixture are displayed. (A) Shewanella frigidimarina, (B) Psychrobacter frigidicola.
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Table 2 Identification of proteins. ACcesion code
Locus code
Protein description
Organism
Score
MW
PI
Matched peptides
gi9114563931 gi993005146
YP_751445 YP_579583
Phosphate binding protein Isocitrate dehydrogenase, NADPdependent
Shewanella frigidimarina NCIMB400 Psychrobacter chryohalolentis K5
377 160
34,834 83,153
8.78 5.63
10 8
Fig. 5. Identification of whole bacteria. (A, B) Representative direct mass spectra of whole cells isolated from glacial ice. (C) 2-DE gels were used to check the identification of S. oneidensis.
nature that species level detection in complex matrices is not afforded. Further, MALDI-TOF-MS has enormously contributed to the identification of peptides. Without this technique proteomics would most likely not be the important discipline it is today. Besides ‘true’ proteomics, MALDI-TOF-MS is being applied for the analysis of microorganisms for their taxonomic characterization from its beginning.
4. Results 4.1. Analysis of proteins by MALDI-TOF-MS Two bacterial isolates of S. frigidimarina and P. cryohalolentis were used to examine the minimum number of bacterial cells needed for identification by MALDI-TOF-MS. We did a series of bacterial dilutions. The minimum number for correct identification was determined to be 5.9 10 3 for S. frigidimarina and 5.5 10 3 for P. cryohalolentis (not shown). On the other hand, we examined the capability of identifying bacteria from bacterial mixtures. We mixed an equal amount of S. frigidimarina and P. cryohalolentis. The bacterial species in the mixture were also identified (Fig. 4). Table 2 summarizes the characteristics of the two identified proteins that obtained best scores in the analysis of these bacterial species.
4.2. Direct analysis of whole cell bacteria Ice-melt water from glacial ice samples was amended with nutrients and incubated at 4 1C in the dark. Colonies were isolated on agar plates supplemented with M1, M2, M3 or M4, incubated at 4 1C. Frozen cell pellets were analyzed by MALDI-TOF-MS. Fig. 5 shows the direct identification of proteins of Shewanella oneidensis from whole cells. Data of two representative identified proteins are shown in Table 3 (Fig. 5A and B). In order to check this result, cell extracts of this culture were analyzed by two-dimensional electrophoresis (2-DE) (Fig. 5C). Several spots were excised from gels in the range between a molecular weight of 50–60 and pI 4–8. They were analyzed by MALDI-TOF-MS. Spots indicated in Fig. 5 as 1 and 2 corresponded to the same identified proteins listed in Table 3.
5. Discussion In a search for extant life beyond Earth, biomolecules and microorganisms are the most likely candidates. On Earth, life has developed strategies to cope with the so-called extreme conditions (Des Marais et al., 2008). The successful identification of unknown biosignatures and microorganisms from Earth analogs of Mars requires the intelligent use of several different techniques. Among them, some traditional methods include division into serotypes by
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Table 3 Identification of bacteria. Spot
Accesion code
Locus code
Protein description
Organism
Score
MW
PI
Matched peptides
1 2
gi924372295 gi924372557
NP_716337 NP_716599
Chaperonin GroEL Fumarate reductase flavoprotein precursor
Shewanella oneidensis MR1 Shewanella oneidensis MR1
115 213
57,101 62,865
4.84 7.27
6 14
specific antibodies, distinction of genotypes by DNA sequence analysis and biochemical tests. These methods provide high sensitivity and specificity, but their efficiency is limited by the complexity of the procedures, they are time-consuming and are completely dependent on the availability of antibodies or the knowledge of genetic sequences of the target bacteria. A survey was carried out on microorganisms from psychrophilic environments with the aim to demonstrate that directly subjecting intact bacterial colonies for protein profiling using MALDI-TOF-MS can be a simple and reliable approach to accurately identify them (Van Veen et al., 2010). The principle issue relating to the use of MALDI-TOF-MS for bacterial characterization is resolving power. This factor has profound effect in large measure because the characterization of whole bacteria by MALDI involves the analysis of a very complex mixture. This is done without any preliminary separation of components. TOF mass spectrometry can be easily coupled to MALDI ionization sources, and MALDI-TOF-MS represents a very rapid method for analyzing the proteins desorbed directly from whole cells (Lay and Liyanage, 2006). The whole cell spectra produced by MALDITOF-MS have taxonomically characteristic features that can be used to differentiate bacteria at the genus, species and strain level, even though only a very small portion of the bacterial proteome can be detected by direct analysis (Welker and Moore, 2011). 5.1. Applications to Mars exploration Searching for traces of extinct and/or extant life on Mars is one of the major objectives for remote sensing and in situ exploration of the planet. As a whole, the surface of Mars is extremely hostile due to high UV radiation, desiccation, oxidants, and low temperatures, among a variety of conditions that limit the capability to support life. But increasingly it is believed that past and present Mars has or does provide habitable conditions sufficient to support microorganismal life forms. There are on Mars some specific niches that could have been habitable during transient and local episodes. This includes environments in fluvial deposits, gullies, transient geothermal and/or hydrothermal conditions that can be triggered by large impacts or by proximity with igneous activity. Subsurface ice abounds in the polar regions as evidenced by satellite observations and the recent Phoenix mission (Smith et al., 2009). The spacecrafts sent to Mars regularly reveal new evidence suggesting that the environmental conditions on early Mars were very different than today, with liquid water flowing on the surface. In past times, life might have emerged under Martian conditions milder than the present ones, and left some remnants at the surface. Even if this did not happen, prebiotic molecules may have been preserved in the soil, and they might be similar to those that prevailed on the Earth surface some 3.5–4 billion years ago. There is increasing evidence of liquid water in ancient times when conditions were similar to those on Earth during the first emergence of life. There are also areas with specific delivery and burial of constituents including volcanic ashes spring deposits, atmospheric deposits, in addition to extraterrestrial delivery of meteoritic organics. Further, some special mineral sites can interact with transient conditions to change the habitability conditions. In upcoming years various space missions will investigate the habitability of Mars and the possibility of extinct or extant life
existing in the Red Planet. Preliminary analyses of Mars analog soils on Earth provide crucial information to determine whether signatures of past and/or present life may still exist in the Martian regolith and these analyses also help when choosing target locations for molecular signatures of life on Mars. These multidisciplinary findings help in the preparation phase for future Mars missions, and are crucial to successfully target locations that may host organic matter, as well as extract and detect biosignatures on Mars. Rover missions to Mars require portable instruments that use minimal power, require no sample preparation, and provide suitably diagnostic mineralogical information to an Earth-based exploration team (Brown et al., 2004). In exploration of analog environments of Mars it is important to screen rapidly for the presence of biosignatures and especially to identify them accurately. Traditional biodetection techniques are often time-consuming and labor-intensive. MALDI-TOF-MS has become a popular and versatile method of analyzing a broad range of macromolecules (proteins, DNA, oligonucleotides, oligosaccharides) from biological origin. The accuracy and speed with which data can be obtained by MALDI-TOF-MS could make this a powerful tool for environmental monitoring in Earth analogs of Mars (Jimmy et al., 1994; Perkins et al., 1999; Ruelle et al., 2004). In the present work we apply our analysis to the identification of biosignatures and microorganisms in samples from an Antarctic glacier as an Earth analog of Mars. We show that our method is actually reliable for biodetection, and therefore it is a powerful tool for the search of life on Mars in the next generation of space missions to the planet.
6. Conclusion Scientists disagree when it comes to evaluating the chances for detecting life on Mars. There is scientific consensus that past conditions on Mars may have allowed life to develop and that the search for extinct life may be more fruitful than looking for extant life. Life on Earth originated approximately 3.5–4 billion years ago and has adapted to nearly every explored environment, including hydrothermal vents, arid deserts, and ice lakes in Antarctica. Immense progress in the study of extreme life has changed our view of habitability beyond Earth. Recent data suggest that microorganisms can survive in cold and dry climates and inside salt deposits. These and other ecological niches could harbor extremophiles in the subsurface of Mars. However, a wide distribution of life as observed on our planet is not expected on Mars; an overall low amount of biomass, restricted to localized areas, is more probable. The search for organic material and biosignatures on Mars is a highly complex endeavor. Instruments on future Mars missions are limited to searching for signs of life that conform to our preconceived notions of biomarkers. A combination of solar ultraviolet radiation and oxidation processes in the soil are destructive to organic material and life on and close to the surface. Galactic cosmic rays can penetrate the surface and effectively destroy organic and biological materials over geological timescales. The successful hunt for extant biosignatures will be a tradeoff between multiple parameters, including accessibility, biomarker concentration, the preservation potential, extractability, and instrument performance. When deploying organic detection instruments on Mars, consideration only of the geological context and the history of regional aqueous processes for
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landing site selection may be insufficient. The host microenvironment of putative microbes on Mars must be compatible with the capabilities of the instrumentation payload. Earth-based field research in extreme environments (such as dry deserts and permafrost regions) that investigates metabolic processes of microbes and their geological environments will therefore be a vital research activity in the preparation phases of future space missions. The detection of extant life prior to Mars sample return missions will depend strongly on instrumentation that can distinguish biological from non-biological organic matter and fossil organic matter from recent remains. With respect to the previous in situ exploration of Mars, the astrobiological relevance of the upcoming Mars missions will be greatly improved with the capability to thoroughly study samples at a microscopic scale, in a combined protocol, with a suite of highly performing instruments. Analog environments on Earth can provide us a deeper understanding of where and how to look for life on Mars. In this paper we have compared the different techniques applied for the identification of biomarkers in these regions and identify the implications to Astrobiology and the search for habitable environments on Mars. Several methods have been developed for the identification of biomarkers and microorganisms. These include, among others, cultural, serological and genetic methods. These methods require obtaining pure cultures. Microscopic observation requires confirmation of the suspected identity by other methods with specific antibodies or molecular probes, which should be previously synthesized. Although PCR, arrays and other techniques using antibodies or probes are sometimes employed with mixed samples, if none of the probes or antibodies corresponds to any of the bacteria in the sample, then other tests with pure cultures should be necessary. Therefore, mass spectrometry presents an attractive alternative for bacterial differentiation in the exploration of identification of biosignatures and microorganisms in Earth analogs of Mars. In this study, we combined the analysis of proteins by MALDI-TOF-MS with direct analysis of whole cell bacteria to identify the microbial populations associated with Antarctic glaciers. Based on results obtained with cultured bacteria, and also on tests performed with environmental samples, we give a view of the analytical capability for the experiments to detect and identify a broad range of molecules, including proteins and whole cells. Firstly, we performed bacterial cultures of both environmental ice samples from Antarctic glaciers and bacteria from cultured collections (Shewanella frigidimarina and Psychrobacter cryohalolentis), used as control. Secondly, bacterial isolates and mixtures of several bacterial species were identified by MALDI-TOF-MS. Further, environmental samples of ice were subjected to direct identification of whole cell bacteria. Classical proteomic analysis with 2-DE gels demonstrated that the same proteins and bacterial species could be detected by both techniques. The data acquired with this instrumentation should thus be of primary importance to give an insight into the potential existence of a present or past life on Mars, and to determine the influence of the environmental surface conditions on the current potential habitability of Mars.
Acknowledgment We are indebted to Drs. M. Martinez-Gomariz and C. Gil from Proteomic Unit of the Parque Cientı´fico de Madrid. We thank also Mr. A. Casals and Mr. J. Barba for their skillful assistance at the Antarctic Research Station Gabriel de Castilla. This research was supported by Grants CTM/2008-00304/ANT and CTM2010-12134-E /ANT from the Spanish Ministerio de Economı´a y Competitividad.
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