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Mass spectrometry for direct identification of biosignatures andmicroorganisms in Earth analogs of Mars

Laura Garcia-Descalzo a, Eva Garcıa-Lopez a, Ana Maria Moreno a, Alberto Alcazar b,Fernando Baquero a,c, Cristina Cid a,n

a Microbial Evolution Laboratory, Center for Astrobiology (CSIC-INTA), 28850 Torrejon de Ardoz, Spainb Department of Investigation, Hospital Ramon y Cajal, 28034 Madrid, Spainc Department of Microbiology, Hospital Ramon y Cajal, 28034 Madrid, Spain

a r t i c l e i n f o

Article history:

Received 11 February 2012

Received in revised form

25 July 2012

Accepted 3 August 2012

Keywords:

Mars

Biosignatures

Microorganisms

Mass spectrometry

MALDI-TOF-MS

Antarctic glaciers

33/$ - see front matter & 2012 Elsevier Ltd. A

x.doi.org/10.1016/j.pss.2012.08.009

viations: 2-DE, two-dimensional electrophore

on; GC, gas chromatography; MALDI-TOF-MS

ion/ionization-time of flight mass spectromet

; SDS-PAGE, sodium dodecyl sulfate-polyacry

espondence to: Microbial Evolution Laborat

TA), Ctra. Ajalvir, km 4, 28850 Torrejon de A

4 915206455; fax: þ34 915201074.

ail addresses: cidsc@inta.es, cidsc@cab.inta-cs

e cite this article as: Garcia-Descalzrth analogs of Mars. Planetary and S

a b s t r a c t

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.

1. Introduction

There is considerable interest in investigating other worldsand the microbial forms that thrive in extreme environments,especially under those conditions that can provide a model forMartian environments. The Martian surface environment exhibitsextremes of salinity, temperature, desiccation, and radiation(Crisler et al., 2012). By analogy with terrestrial extremophilecommunities, potential protected niches have been postulated forMars, such as sulfur-rich subsurface areas for chemoautotrophiccommunities, rocks for endolithic communities, cold environ-ments and permafrost regions (Onstott et al., 2009), hydrothermalvents (Shapiro and Schulze-Makuch, 2009) soil, or evaporitecrystals (Fig. 1) (Horneck, 2000; Benison et al., 2008). For instance,there are several similarities between the vast deposits of sulfates

ll rights reserved.

sis; ESI, electro spray

, matrix-assisted laser

ry; PCR, polymerase chain

lamide gel electrophoresis

ory, Center for Astrobiology

rdoz, Madrid, Spain.

ic.es (C. Cid).

o, L., et al., Mass spectromepace Science (2012), http:/

and iron oxides on Mars and the main sulfide-containing ironbioleaching products found in the Rio Tinto (Amils et al., 2011).

Further, on Earth, the discovery of cold-tolerant microorganismsin 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, sub-

glacial lakes and high mountains are considered representative of

these environments in which psychrophile bacteria are the unique

inhabitants. When compared to other known prokaryotes, psychro-

philic bacteria possess many unique qualities and molecular

mechanisms that allow their adaptation to cold environments

(Garcıa-Descalzo et al., 2011).In exploration of analog environments of Mars it is important

to screen rapidly for the presence of biosignatures and micro-organisms and especially to identify them accurately.

try for direct identification of biosignatures and microorganisms/dx.doi.org/10.1016/j.pss.2012.08.009

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 1General 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)

L. Garcia-Descalzo et al. / Planetary and Space Science ] (]]]]) ]]]–]]]2

Traditional bacteriological techniques, listed in Table 1 (Fig. 2),such as culture on selective medium (Fig. 2A), microscopictechniques (Fig. 2B), distinction of genotypes by DNA sequenceanalysis (Fig. 2C and D) or biochemical activity tests, are oftentime-consuming and labor-intensive.

Several methods have been reported that could be useful forMars exploration. Among them, short-wave infrared (SWIR) spec-troscopic instruments such as the Portable Infrared Mineral Analy-zer (PIMA) have been tested to investigate sites of paleobiologicalinterest (Brown et al., 2004). RAMAN spectroscopy (Breier et al.,2010) has been used to make a variety of measurements that areonly stable under in situ conditions. Gas chromatography–massspectrometry has been applied to analyze carboxylic acid mixtures(Pietrogrande et al., 2005). Further, antibody microarrays havebeen developed for life detection in planetary exploration (Parroet al., 2011). In this study, with the aim of determining whether

Please cite this article as: Garcia-Descalzo, L., et al., Mass spectromein Earth analogs of Mars. Planetary and Space Science (2012), http:/

matrix-assisted laser desorption/ionization-time of flight massspectrometry (MALDI-TOF-MS), could be an option for bacterialdifferentiation we perform the identification of bacterial speciesfrom an Antarctic glacier.

2. Material and methods

2.1. Bacterial growth and sample preparation

Glacial ice samples were collected at an Antarctic glacier inMount Pond, Deception Island. Ice samples were obtained byremoving 20–30 cm of thick debris and cutting out a square blockof 20 cm on a side. Samples were wrapped in plastic bags andstored at �20 1C until processing. Ice samples were processed byusing a surface decontamination and melting procedure consistent

try for direct identification of biosignatures and microorganisms/dx.doi.org/10.1016/j.pss.2012.08.009

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.

L. Garcia-Descalzo et al. / Planetary and Space Science ] (]]]]) ]]]–]]] 3

with previous studies (Bidle et al., 2007). All procedures wereperformed by using bleach-sterilized work areas, a UV-irradiatedlaminar flow hood, ethanol-sterilized tools and sterilized gloves. Tocontrol for laboratory contamination, 1 L of MilliQ rinse water wasfrozen and subjected to identical analytical procedures.

Ice-melt water was amended with nutrients and incubated at4 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 ofMgSO4 �7H2O/0.05 g of CaCl2/0.05 g of MnSO4 �H2O/0.01 g ofFeCl3 �6H2O 0.01; M2, 1 g of glucose/1 g of peptone/0.5 g of yeastextract/0.2 g of MgSO4 �7H2O/0.05 g MnSO4 �H2O; M3, 1 g ofglucose/0.5 g of casamino acids/0.5 g of yeast extract/1 g ofKH2PO4/0.5 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 formula-tions were made as concentrated stocks (20–200x), sterilized byautoclaving and directly added (o1/50 volume ratio) to melt-water at 1x final concentration. Colonies were isolated on agarplates supplemented with M1, M2, M3 or M4, incubated at 4 1C.

Further, bacterial cultures of Shewanella frigidimarina andPsychrobacter cryohalolentis were used as control. These bacteriawere purchased from culture collections (ATCCs) and incubatedin marine broth 2216.

2.2. Extraction of the soluble protein fraction

Bacteria from each biological replicate (four in total) wereobtained from the individual cultures. The cells were harvested,washed and lysed in buffer A (20 mM Tris–HCl, pH 7.6; 140 mMpotassium chloride; 2 mM benzamidine; 1 mM EDTA; 10 mg/mlpepstatin A, leupeptin and antipain), using a French Press at 4 1C.Cell debris was removed by centrifugation at 11,000g for 10 min

Please cite this article as: Garcia-Descalzo, L., et al., Mass spectromein Earth analogs of Mars. Planetary and Space Science (2012), http:/

to obtain a supernatant, and the protein extracts were processedusing a 2-D Clean-Up kit (GE Healthcare, Spain). The pellet wasfrozen and stored at �80 1C. The protein content was determinedin cell extracts using the BioRad protein assay based on theBradford 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 combinedwith SDS-PAGE for 2-DE by using the Multiphor II apparatus forthe first dimension and standard vertical slab gel electrophoresisfor the second dimension, according to the manufacturer’sinstructions. Samples (about 500 mg of protein) were preparedin 7 M urea/2 M thiourea and 5% b-mercaptoethanol, centrifugedand applied to pH gradient strips for IEF. Carrier ampholyte ureaIEF was carried out using immobilized pH 3–10 and pH 3–11 non-linear gradient strips (18 cm).

After the first dimension, the IEF strips were processed for thesecond dimension in SDS-PAGE carried out on 12% acrylamide(2.6% crosslinking) gels (1.0 mm thick) with IEF strips being usedas the stacking gels. The spots resolved by 2-DE from the gelswere stained with Coomassie Blue or with MALDI-MS-compatiblesilver reagent for peptide mass fingerprinting analysis and proteinidentification. Protein spots were excised manually from theCoomassie Blue or silver stained 2-DE gels, de-stained and thendigested automatically using a Proteineer DP protein digestionstation (Bruker-Daltonics, Bremen, Germany). An aliquot of theabove digestion solution was mixed with an aliquot of a-cyano-4-hydroxycinnamic acid (Bruker-Daltonics) in 33% aqueous aceto-nitrile and 0.1% trifluoroacetic acid.

try for direct identification of biosignatures and microorganisms/dx.doi.org/10.1016/j.pss.2012.08.009

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).

L. Garcia-Descalzo et al. / Planetary and Space Science ] (]]]]) ]]]–]]]4

2.4. Direct analysis of whole cell bacteria

The identification protocol used was based on the methodpreviously described by Cid et al. (2010) with minor variations.Frozen cell pellets (about 100 mg) were thawed on ice and re-suspended in 400 mL cold deionized water. One microlitre (about0.20 mg) of sample was diluted with 19 mL of 0.1% TFA aqueousbuffer. An aliquot of the above solution was mixed with an aliquotof a-cyano-4-hydroxycinnamic acid (Bruker-Daltonics) in 33%aqueous acetonitrile and 0.1% trifluoroacetic acid. This mixturewas deposited onto a MALDI probe (600 mm AnchorChip forBruker-Daltonics) and allowed to dry at room temperature. Whenthe matrix for MALDI-TOF-MS was changed to sinapinic acid or2,5-dihydroxybenzoic acid, or the preparation of solvents formatrix was changed, the resulting MS spectra were affected. Assuch, we used the protocol described here for matrix preparationconsistently throughout this study. MALDI-TOF-MS (/MS) datawere obtained using an Ultraflex TOF-TOF mass spectrometerequipped with a LIFT-MS/MS device (Bruker-Daltonics) and a4800 MALDI-TOF-TOF mass spectrometer (Applied Biosystems).Spectra were acquired in the positive-ion mode at 50 Hz laserfrequency, and 100–1500 individual spectra were averaged.When available, for fragment ion analysis in the tandem time-of-flight (TOF/TOF) mode, precursors were accelerated to 8 kV andselected in a timed ion gate. Spectral data were analyzed through

Please cite this article as: Garcia-Descalzo, L., et al., Mass spectromein Earth analogs of Mars. Planetary and Space Science (2012), http:/

MS BioTools program (Bruker-Daltonics) or ProteinPilot software(Applied Biosystems) to search the NCBInr database using theMascot database search algorithm (Matrix Science, London, UK).

3. Theory

Mass spectrometry is not a new technique. The separation ofcharged particles based on mass, charge and flight path has beenknown since 1897, when Thomson measured the charge-to-massratio of electron. This technique, associated to others, has beenemployed for detection of fatty acids, sugar monomers andpeptides. It is able to analyze with high accuracy the compositionof different chemical elements and atomic isotopes splitting theiratomic nuclei according to their mass–charge ratio (m/z). It can beused to identify different chemical elements that form a com-pound or to determine the isotopic content of different elementsin the same compound. The three primary components needed toproduce a mass spectrometer are relatively straightforward asshown in Fig. 3A. These have not change much since that time,and include an ion source to vaporize and ionize samples, a massanalyzer to separate ions based on their mass to charge ratio anda detector to measure the separated ions.

Firstly, the material to be analyzed is ionized and ions are thentransported by magnetic or electric fields to the mass analyzer.

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L. Garcia-Descalzo et al. / Planetary and Space Science ] (]]]]) ]]]–]]] 5

Techniques for ionization have been key to determine what typeof samples can be analyzed by mass spectrometry. Two techni-ques are often used with liquid and solid biological samples:electro spray ionization (ESI) and laser matrix-assisted laserdesorption/ionization (MALDI). In the MALDI ionization analytesco-crystallized with a suitable matrix are converted into ions bythe action of a laser. This source of ionization is usually associatedwith a time of flight analyzer (TOF) in which the ions areseparated according to their mass–charge after being acceleratedin an electric field. At last, a mass spectrometer detector recordsthe charge induced or current produced when an ion passes by orhits a surface.

The choice of matrix is a critical step in MALDI-MS because itcan promote ionization of specific families of compounds (e.g.,phospholipids, peptides and proteins). Different types of matrixused in the experiments are shown in Fig. 3B.

Profiling of fatty acid monomers, released from membranephospholipids of whole cells, using gas chromatography (GC) with

Fig. 4. Identification of bacteria in a mixture. The representative MALDI-TOF-MS spectr

(B) Psychrobacter frigidicola.

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flame ionization detector has been one of the most widely usedanalytical method for bacterial speciation (after isolation andgrowth of individual bacterial species). When necessary, theidentity of these fatty acids is confirmed by GC–MS. Additionally,other small molecules are used extensively to assess the microbialcontent in environmental samples. Hydroxy fatty acids (compo-nents of the lipid A region of lipopolysaccharides) are used toquantitate the levels of Gram-negative bacteria and muramic acid(that takes part of the glycan backbone of peptidoglycan) as amarker for total bacterial load.

Phospholipids can be ionized as intact entities for MS orMS–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 themasses of individual fatty acids and subtracting the value fromthe mass of the parent ion. Dramatic improvements in the sensi-tivity of phospholipid analysis have been achieved using ESI–MSand MS–MS. However, phospholipids are widely distributed in

a of two bacterial species of a mixture are displayed. (A) Shewanella frigidimarina,

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Table 2Identification of proteins.

ACcesion code Locus code Protein description Organism Score MW PI Matched peptides

gi9114563931 YP_751445 Phosphate binding protein Shewanella frigidimarina NCIMB400 377 34,834 8.78 10

gi993005146 YP_579583 Isocitrate dehydrogenase, NADPdependent Psychrobacter chryohalolentis K5 160 83,153 5.63 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.

L. Garcia-Descalzo et al. / Planetary and Space Science ] (]]]]) ]]]–]]]6

nature that species level detection in complex matrices is notafforded.

Further, MALDI-TOF-MS has enormously contributed to theidentification of peptides. Without this technique proteomicswould most likely not be the important discipline it is today.Besides ‘true’ proteomics, MALDI-TOF-MS is being applied for theanalysis of microorganisms for their taxonomic characterizationfrom 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 cellsneeded for identification by MALDI-TOF-MS. We did a series ofbacterial dilutions. The minimum number for correct identifica-tion was determined to be 5.9�10�3 for S. frigidimarina and5.5�10�3 for P. cryohalolentis (not shown).

On the other hand, we examined the capability of identifyingbacteria from bacterial mixtures. We mixed an equal amount of S.

frigidimarina and P. cryohalolentis. The bacterial species in themixture were also identified (Fig. 4). Table 2 summarizes thecharacteristics of the two identified proteins that obtained bestscores in the analysis of these bacterial species.

Please cite this article as: Garcia-Descalzo, L., et al., Mass spectromein Earth analogs of Mars. Planetary and Space Science (2012), http:/

4.2. Direct analysis of whole cell bacteria

Ice-melt water from glacial ice samples was amended withnutrients and incubated at 4 1C in the dark. Colonies were isolatedon agar plates supplemented with M1, M2, M3 or M4, incubated at4 1C. Frozen cell pellets were analyzed by MALDI-TOF-MS. Fig. 5shows the direct identification of proteins of Shewanella oneidensis

from whole cells. Data of two representative identified proteins areshown in Table 3 (Fig. 5A and B).

In order to check this result, cell extracts of this culture wereanalyzed by two-dimensional electrophoresis (2-DE) (Fig. 5C).Several spots were excised from gels in the range between amolecular weight of 50–60 and pI 4–8. They were analyzed byMALDI-TOF-MS. Spots indicated in Fig. 5 as 1 and 2 correspondedto the same identified proteins listed in Table 3.

5. Discussion

In a search for extant life beyond Earth, biomolecules andmicroorganisms are the most likely candidates. On Earth, life hasdeveloped strategies to cope with the so-called extreme conditions(Des Marais et al., 2008). The successful identification of unknownbiosignatures and microorganisms from Earth analogs of Marsrequires the intelligent use of several different techniques. Amongthem, some traditional methods include division into serotypes by

try for direct identification of biosignatures and microorganisms/dx.doi.org/10.1016/j.pss.2012.08.009

Table 3Identification of bacteria.

Spot Accesion code Locus code Protein description Organism Score MW PI Matched peptides

1 gi924372295 NP_716337 Chaperonin GroEL Shewanella oneidensis MR1 115 57,101 4.84 6

2 gi924372557 NP_716599 Fumarate reductase flavoprotein precursor Shewanella oneidensis MR1 213 62,865 7.27 14

L. Garcia-Descalzo et al. / Planetary and Space Science ] (]]]]) ]]]–]]] 7

specific antibodies, distinction of genotypes by DNA sequenceanalysis and biochemical tests. These methods provide high sensi-tivity and specificity, but their efficiency is limited by the complexityof the procedures, they are time-consuming and are completelydependent on the availability of antibodies or the knowledge ofgenetic sequences of the target bacteria.

A survey was carried out on microorganisms from psychro-philic environments with the aim to demonstrate that directlysubjecting intact bacterial colonies for protein profiling usingMALDI-TOF-MS can be a simple and reliable approach to accu-rately identify them (Van Veen et al., 2010).

The principle issue relating to the use of MALDI-TOF-MS forbacterial characterization is resolving power. This factor has pro-found effect in large measure because the characterization of wholebacteria 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 ioniza-tion sources, and MALDI-TOF-MS represents a very rapid methodfor analyzing the proteins desorbed directly from whole cells (Layand Liyanage, 2006). The whole cell spectra produced by MALDI-TOF-MS have taxonomically characteristic features that can be usedto differentiate bacteria at the genus, species and strain level, eventhough only a very small portion of the bacterial proteome can bedetected 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 oneof the major objectives for remote sensing and in situ exploration ofthe planet. As a whole, the surface of Mars is extremely hostile dueto high UV radiation, desiccation, oxidants, and low temperatures,among a variety of conditions that limit the capability to supportlife. But increasingly it is believed that past and present Mars has ordoes provide habitable conditions sufficient to support micro-organismal life forms. There are on Mars some specific niches thatcould have been habitable during transient and local episodes. Thisincludes environments in fluvial deposits, gullies, transient geother-mal and/or hydrothermal conditions that can be triggered by largeimpacts or by proximity with igneous activity. Subsurface iceabounds in the polar regions as evidenced by satellite observationsand the recent Phoenix mission (Smith et al., 2009). The spacecraftssent to Mars regularly reveal new evidence suggesting that theenvironmental conditions on early Mars were very different thantoday, with liquid water flowing on the surface. In past times, lifemight have emerged under Martian conditions milder than thepresent ones, and left some remnants at the surface. Even if this didnot happen, prebiotic molecules may have been preserved in thesoil, and they might be similar to those that prevailed on the Earthsurface some 3.5–4 billion years ago.

There is increasing evidence of liquid water in ancient timeswhen conditions were similar to those on Earth during the firstemergence of life. There are also areas with specific delivery andburial of constituents including volcanic ashes spring deposits,atmospheric deposits, in addition to extraterrestrial delivery ofmeteoritic organics. Further, some special mineral sites can interactwith transient conditions to change the habitability conditions.

In upcoming years various space missions will investigate thehabitability of Mars and the possibility of extinct or extant life

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existing in the Red Planet. Preliminary analyses of Mars analogsoils on Earth provide crucial information to determine whethersignatures of past and/or present life may still exist in the Martianregolith and these analyses also help when choosing target loca-tions for molecular signatures of life on Mars. These multidisciplin-ary findings help in the preparation phase for future Mars missions,and are crucial to successfully target locations that may hostorganic matter, as well as extract and detect biosignatures on Mars.

Rover missions to Mars require portable instruments that useminimal power, require no sample preparation, and provide suitablydiagnostic mineralogical information to an Earth-based explorationteam (Brown et al., 2004). In exploration of analog environments ofMars it is important to screen rapidly for the presence of biosigna-tures and especially to identify them accurately. Traditional biode-tection techniques are often time-consuming and labor-intensive.MALDI-TOF-MS has become a popular and versatile method ofanalyzing a broad range of macromolecules (proteins, DNA, oligo-nucleotides, oligosaccharides) from biological origin. The accuracyand speed with which data can be obtained by MALDI-TOF-MScould make this a powerful tool for environmental monitoring inEarth analogs of Mars (Jimmy et al., 1994; Perkins et al., 1999; Ruelleet al., 2004). In the present work we apply our analysis to theidentification of biosignatures and microorganisms in samples froman 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 inthe next generation of space missions to the planet.

6. Conclusion

Scientists disagree when it comes to evaluating the chances fordetecting life on Mars. There is scientific consensus that pastconditions on Mars may have allowed life to develop and that thesearch for extinct life may be more fruitful than looking for extantlife. Life on Earth originated approximately 3.5–4 billion years agoand has adapted to nearly every explored environment, includinghydrothermal vents, arid deserts, and ice lakes in Antarctica.Immense progress in the study of extreme life has changed ourview of habitability beyond Earth. Recent data suggest that micro-organisms can survive in cold and dry climates and inside saltdeposits. These and other ecological niches could harbor extremo-philes in the subsurface of Mars. However, a wide distribution of lifeas observed on our planet is not expected on Mars; an overall lowamount of biomass, restricted to localized areas, is more probable.

The search for organic material and biosignatures on Mars is ahighly complex endeavor. Instruments on future Mars missions arelimited to searching for signs of life that conform to our preconceivednotions of biomarkers. A combination of solar ultraviolet radiationand oxidation processes in the soil are destructive to organic materialand life on and close to the surface. Galactic cosmic rays canpenetrate the surface and effectively destroy organic and biologicalmaterials over geological timescales. The successful hunt for extantbiosignatures will be a tradeoff between multiple parameters,including accessibility, biomarker concentration, the preservationpotential, extractability, and instrument performance. When deploy-ing organic detection instruments on Mars, consideration only of thegeological context and the history of regional aqueous processes for

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landing site selection may be insufficient. The host microenviron-ment of putative microbes on Mars must be compatible with thecapabilities of the instrumentation payload.

Earth-based field research in extreme environments (such asdry deserts and permafrost regions) that investigates metabolicprocesses of microbes and their geological environments willtherefore be a vital research activity in the preparation phases offuture space missions. The detection of extant life prior to Marssample return missions will depend strongly on instrumentationthat can distinguish biological from non-biological organic matterand fossil organic matter from recent remains.

With respect to the previous in situ exploration of Mars, theastrobiological relevance of the upcoming Mars missions will begreatly improved with the capability to thoroughly study samples at amicroscopic scale, in a combined protocol, with a suite of highlyperforming instruments. Analog environments on Earth can provideus a deeper understanding of where and how to look for life on Mars.In this paper we have compared the different techniques applied forthe identification of biomarkers in these regions and identify theimplications to Astrobiology and the search for habitable environ-ments on Mars. Several methods have been developed for the identi-fication of biomarkers and microorganisms. These include, amongothers, cultural, serological and genetic methods. These methodsrequire obtaining pure cultures. Microscopic observation requiresconfirmation of the suspected identity by other methods with specificantibodies or molecular probes, which should be previously synthe-sized. Although PCR, arrays and other techniques using antibodies orprobes are sometimes employed with mixed samples, if none of theprobes 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 alternativefor bacterial differentiation in the exploration of identification ofbiosignatures and microorganisms in Earth analogs of Mars.

In this study, we combined the analysis of proteins byMALDI-TOF-MS with direct analysis of whole cell bacteria toidentify the microbial populations associated with Antarcticglaciers. Based on results obtained with cultured bacteria, andalso on tests performed with environmental samples, we give aview of the analytical capability for the experiments to detect andidentify a broad range of molecules, including proteins and wholecells. Firstly, we performed bacterial cultures of both environmentalice samples from Antarctic glaciers and bacteria from culturedcollections (Shewanella frigidimarina and Psychrobacter cryohalolentis),used as control. Secondly, bacterial isolates and mixtures of severalbacterial species were identified by MALDI-TOF-MS. Further, envir-onmental samples of ice were subjected to direct identification ofwhole cell bacteria. Classical proteomic analysis with 2-DE gelsdemonstrated that the same proteins and bacterial species couldbe detected by both techniques. The data acquired with thisinstrumentation should thus be of primary importance to give aninsight into the potential existence of a present or past life on Mars,and to determine the influence of the environmental surface condi-tions on the current potential habitability of Mars.

Acknowledgment

We are indebted to Drs. M. Martinez-Gomariz and C. Gil fromProteomic Unit of the Parque Cientıfico de Madrid. We thank alsoMr. A. Casals and Mr. J. Barba for their skillful assistance at theAntarctic Research Station Gabriel de Castilla. This research wassupported by Grants CTM/2008-00304/ANT and CTM2010-12134-E/ANT from the Spanish Ministerio de Economıa y Competitividad.

Please cite this article as: Garcia-Descalzo, L., et al., Mass spectromein Earth analogs of Mars. Planetary and Space Science (2012), http:/

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