ASTROBIOLOGYVolume 8, Number 2, 2008© Mary Ann Liebert, Inc.DOI: 10.1089/ast.2006.0102

Research Paper

Glycine Identification in Natural Jarosites Using LaserDesorption Fourier Transform Mass Spectrometry:

Implications for the Search for Life on Mars

J. MICHELLE KOTLER,1 NANCY W. HINMAN,1 BEIZHAN YAN,2DAPHNE L. STONER,2 and JILL R. SCOTT3

ABSTRACT

The jarosite group minerals have received increasing attention since the discovery of jarositeon the martian surface by the Mars Exploration Rover Opportunity. Given that jarosite canincorporate foreign ions within its structure, we have investigated the use of jarosite as anindicator of aqueous and biological processes on Earth and Mars. The use of laser desorptionFourier transform mass spectrometry has revealed the presence of organic matter in severaljarosite samples from various locations worldwide. One of the ions from the natural jarositeshas been attributed to glycine because it was systematically observed in combinations ofglycine with synthetic ammonium and potassium jarosites, Na2SO4 and K2SO4. The abilityto observe these organic signatures in jarosite samples with an in situ instrumental technique,such as the one employed in this study, furthers the goals of planetary geologists to deter-mine whether signs of life (e.g., the presence of biomolecules or biomolecule precursors) canbe detected in the rock record of terrestrial and extraterrestrial samples. Key Words: Jarosite—Mars—Mass spectrometry—Organic matter—Biosignatures. Astrobiology 8, 253–266.

253

INTRODUCTION

IN THE SEARCH FOR LIFE on other planets, thedetection of organic molecules and their assign-

ment as biological evidence are key goals. Duringthe Viking mission, pyrolysis gas chromatogra-phy/mass spectrometry was used on the surfaceof Mars to detect organic molecules (Beimann etal., 1977). Although the Viking mission was un-successful in providing evidence for significantquantities of organic molecules on Mars (Benner

et al., 2000), the search continues for new ways toidentify organic molecules in geologic materials(Navarro-González et al., 2006). Jarosite is a primemineral candidate for harboring organic com-pounds. Recently, Skelley et al. (2004) reportedthe detection of amino acids associated withterrestrial jarosite samples through the use of a portable capillary electrophoresis instrumentcalled the Mars Organic Analyzer. Quantities ofamino acids were also detected by Aubrey et al.(2006) in gypsum and jarosite samples with a

1Geosciences Department, University of Montana, Missoula, Montana.2Department of Chemistry, University of Idaho, Idaho Falls, Idaho.3Chemical Sciences, Idaho National Laboratory, Idaho Falls, Idaho.

complex extraction procedure. Thermodynamicevaluation of the 2 most common jarosite groupminerals [i.e., jarosite, KFe3(SO4)2(OH)6, and na-trojarosite, NaFe3(SO4)2(OH)6] has shown thatthese 2 members should be stable on the mar-tian surface (Navrotsky et al., 2005). The mineralgroup is stable on Earth; reported occurrences ofsamples date to several million years old (Hofs-tra et al., 1999; Stoffregen et al., 2000). The jarositegroup minerals were first postulated to occur onMars by Burns (1987a, 1987b, 1989). In 2004, theMars Exploration Rover Opportunity confirmedthe presence of jarosite group minerals on themartian surface (Christensen et al., 2004; Klingel-hofer et al., 2004; Squyres et al., 2004).

Since the discovery of jarosite group mineralson Mars, interest has focused on linking the ori-gin and characteristics of the mineral group to theexistence of extant or extinct life on Mars (Squyresand Knoll, 2005). On Earth, jarosite group min-eral formation can be influenced or mediated bysulfur- and iron-metabolizing microorganisms(Eneroth and Koch, 2004; Bridge and Johnson,2000; Sasaki and Konno, 2000; Akai et al., 1999;Karamanev, 1991; Clark et al., 1993; Grishin et al.,1988). It is therefore possible that biological “fin-gerprints” left over from jarosite formation canbe used to identify extant or extinct signs of lifein the geologic record on Earth and, potentially,Mars.

The jarosite group minerals can accommodatea wide variety of substitutions in several crystal-lographic sites of the unit cell. These substitutionsoften cause a distortion in the crystal lattice thatcreates vacancies in certain crystallographic sites(Dutrizac and Jambor, 2000). The general chemi-cal formula for jarosite is XFe3(SO4)2(OH)6, wherethe X represents both monovalent and divalentcations that can occupy the axial positions in thecrystal structure. Commonly found ions includeK�, Na�, H3O�, NH4

�, and Pb2� with reports of“foreign ions” also occupying this position(Dutrizac et al., 1996; Gieré et al., 2003; Dutrizac,2004). The term foreign ion is used to describe anyatomic or molecular ion that is found associatedin an unidentified manner—either trapped, in-cluded, or possibly substituted—in the mineralstructure and that is not considered common tothe mineral chemistry. Becker and Gasharova(2001) investigated the ability of jarosite to incor-porate foreign ions and determined that jarositecould act as a storage mineral for heavy metals.

Under the acidic conditions (pH � 2.5) at whichjarosite forms, certain amino acids such as glycine(C2H5NO2) present in solution would be charged(pKa �-COOH � 2.3; pKa �-NH3

� � 9.6) (Math-ews et al., 1999). This could provide a charge-bal-ancing substitution, especially in rapidly precip-itating systems where the inclusion of foreignions would likely occur. Unless the system wassupersaturated with respect to these foreign ions,the amount of substitution would be below thedetection limits of standard powder diffractionmethods (generally 3–5%) and could go unde-tected without the use of sensitive microprobetechniques. Also, if the foreign ions exist as a sep-arate phase within the mineral matrix or are ad-sorbed to the mineral surface, they would not bedetected by conventional methods at low con-centrations. For this study, natural jarosite sam-ples were analyzed with a laser desorption mi-croprobe Fourier transform mass spectrometer(LD-FTMS) to determine whether this techniquecould provide the high-resolution chemical dataneeded to detect organic matter incorporated orassociated with jarosite.

The LD-FTMS used in this study is housed atthe Idaho National Laboratory and was createdfor highly reproducible laser beam scanning andcollection of sensitive high-resolution chemicaldata (Scott and Tremblay, 2002). With the newtechnique of geomatrix-assisted laser desorp-tion/ionization (GALDI) (Yan et al., 2007a), whichis based on principles established from matrix-as-sisted laser desorption/ionization (MALDI), it ispossible to use jarosite as the geomatrix to aid inorganic matter detection. Direct laser desorptionof biomolecules often does not result in produc-tion of ions; hence, the addition of a matrix thataids desorption and ionization processes is nec-essary. In MALDI, the matrices are usually aro-matic acids that result in protonation or cation-ization of biomolecules (Karas et al., 1987). Yan etal. (2007a) demonstrated that minerals or geoma-trices can also assist in the detection of biomole-cules.

Herein, we report our use of the GALDI tech-nique to identify mass spectral signatures of or-ganic matter associated with natural jarosite sam-ples collected from 7 different locations aroundthe world. Additional characterization by ran-dom powder X-ray diffraction (XRD) studies andtotal carbon analysis were performed. Synthe-sized jarosite standards were also analyzed to as-

KOTLER ET AL.254

sist in identification of organic matter present inthe natural samples.

MATERIALS AND METHODS

Natural samples

Natural jarosite samples were acquired fromthe University of Montana Dana Collection, com-mercial sources, or the authors (Table 1). Sampleswere isolated from the parent rock matrices andground to a fine powder with a corundum mortarand pestle. Synthetic samples were also groundto ensure homogeneity. Samples were mountedon glass slides for random powder XRD analysis.

Synthetic samples

Potassium jarosite [KFe3(SO4)2(OH)6] was pre-pared according to Dutrizac and Chen’s (2003)method. Potassium sulfate (0.4 M K2SO4) wasadded to a 0.4 M FeCl3 solution in a 100 ml roundbottom flask with a stoichiometric ratio of 2:3 sul-fate salt to ferric iron. The solution was stirredunder reflux conditions at 100°C for 24 hours. Tocollect the precipitate, the solutions were vacuumfiltered while hot with a Buchner funnel andWhatman #4 filter paper. The precipitates werewashed under vacuum 3 times with 1 L of deion-ized water and air dried. The synthesis of am-monium jarosite [NH4Fe3(SO4)2(OH)6] was ac-complished with an identical procedure, except

that (NH4)2SO4 was used in place of K2SO4. Ex-perimental mixtures of glycine or alanine withsodium or potassium sulfate (Sigma-Aldrich,MA) or a synthetic jarosite were prepared by mix-ing �3–5% analyte with the sulfate or jarosite ma-trix before LD-FTMS preparations.

X-ray powder diffraction

Powdered samples were mounted on glassslides for random powder XRD analysis. X-raydiffraction analyses were performed with aPhilips APD 3720 X-ray diffractometer with a stepsize of 0.02 °2� and a scan rate of 0.750 °2�/min.Patterns obtained were compared to the patternfor synthetic jarosite as published in the JointCommittee on Powder Diffraction Standards(JCPDS) file 22-0827, and unit cell dimensionswere calculated from the hkl {003} and {110} crys-tallographic reflections.

Total carbon analysis

Powdered jarosite samples underwent totalcarbon and sulfur analysis by SGS Mineral Ser-vices of Canada for total carbon and sulfur analy-sis by the furnace/infrared method (Leco SC632-Series, ASTM method E-1915-97).

Laser desorption Fourier transform mass spectrometry

Samples were formed into pellets with a half-inch Beckman dye with a Carver Laboratory

GLYCINE FOUND IN JAROSITES USING LD-FTMS 255

TABLE 1. NATURAL JAROSITE SAMPLE NAMES, LOCATIONS, AND SOURCES

Sample name Location Source

Jarosite-MT West of Agency Fort Belknap UM Dana CollectionReservation, Montana

Jarosite-AZ Toughnut Mine-Tombstone, UM Dana CollectionCoochise County, Arizona

Jarosite-NM Copiapo Jarosite Mine, Dona UM Dana CollectionAna County, New Mexico

Jarosite-AUS Bolcummata, South Australia UM Dana CollectionJarosite-NZ Te Karo Bay, Coromandal Collected April 4, 2005

Peninsula, New ZealandJarosite-RUS Perm, Russia Purchased from Rubelev

Colours, Natural Pigments,USA (707-539-8215)

Jarosite-SPA El Jaroso, Spain (type locality) Purchased from De SteenenKamer, Mineralen, EdelstenenSieraden-Netherlands(www.desteenenkamer.nl)

Jarosite (synthetic) — Synthesized

UM, University of Montana.

Press at a pressure of 3.4 � 107 Pa before mount-ing onto a 316 SS probe tip with epoxy (Devcon5-minute epoxy, Danvers, MA). Epoxy was al-lowed to cure to �70% dryness before pellet wasapplied to prevent the sample from absorbing theepoxy. Mass spectra were obtained with a Fouriertransform mass spectrometer equipped with a 7-Tesla Oxford (Oxford, England) superconductingmagnet, a 2-inch cubic cell, and an Odyssey con-trol and data acquisition system (Thermo-Finni-gan FT/MS, Bremen, Germany) (Scott and Trem-blay, 2002; Yan et al., 2007a). A Nd:YAG laser(Continuum, Santa Clara, CA) operating at 355nm with a 6 ns pulse width was used for de-sorption/ionization at a laser fluence of 8600J/m2 focused to a �6 �m diameter spot, mea-sured as described previously (Scott and Trem-blay, 2002). All spectra were collected from sin-gle laser shots. The sample was positioned �0.5cm from the front electrostatic trap plate of theionization cell. During the ionization event, thepotential on the front and rear trap plates wasmaintained at 0 V. After ionization, a trappingpotential of 2 V was applied to both trap platesand maintained until the quench event at the endof the sequence. A delay of 0.5 s was imposedprior to application of a radio frequency chirp ex-citation applied to opposing plates of the cubiccell over the range of 50 Hz to 4 MHz with asweep rate of 3600 Hz/�s. The ions were detectedin direct mode with 64 to 128 K data points withresolution ranging from 5000 to 50,000 and mass

errors of less than �0.002 u for known masses.Raw data were baseline corrected, Hammingapodized, zero filled, and Fourier transformed toproduce the mass spectra. Pressure during analy-sis was �2 � 10�7 Pa (2 � 10�9 Torr). Externalcalibration of the FTMS was performed with Na-attached polyethylene glycol 1000.

RESULTS

Random powder XRD patterns indicate thatthe natural samples were dominated by a jarositemineral phase with few contaminants present. A range of jarosite compositions were indicatedin the samples by the unit cell variations. The unit cell dimensions of all the natural and syn-thetic jarosite samples in this study are plotted inFig. 1 and listed in Table 2. The reference valuesfor jarosite, natrojarosite, and hydronium jaro-site are also plotted for comparison with stan-dard compositions assigned to various jarositegroup end-members. Within any one group, theunit cell dimensions may vary, as they are de-pendent on cation substitutions and indicatedeviations from strict formula constraints be-tween jarosite [KFe3(SO4)2(OH)6] and natro-jarosite [NaFe3(SO4)2(OH)6]. Hydronium ion sub-stitution may also affect unit cell dimensions inthe jarosite group minerals by altering the lengthvalues along both a- and c-axes. Solid solutionsexist among jarosite, natrojarosite, and hydro-nium jarosite, and end-member compositions arerarely found in natural or synthetic samples(Dutrizac and Jambor, 2000). The unit cell lengthsfor the samples along the a-axis ranged between7.25 Å and 7.50 Å. Values for the c-axis rangedbetween 16.64 Å and 17.70 Å. The syntheticjarosite sample has unit cell dimensions of a �

KOTLER ET AL.256

TABLE 2. UNIT CELL DIMENSIONS OF THE NATURAL

AND SYNTHETIC JAROSITE SAMPLES

Unit cell dimensions

Sample a (Å) c (Å)

Jarosite (synthetic) 7.32 17.13Jarosite-AZ 7.45 17.11Jarosite-AUS 7.41 16.98Jarosite-MT 7.42 17.20Jarosite-NZ 7.45 17.15Jarosite-NM 7.26 17.42Jarosite-RUS 7.31 16.58Jarosite-SPA 7.39 17.58

FIG. 1. Plot of synthetic jarosite and natural jarositeunit cell dimensions (x) compared to standard referencevalues for end member compositions of jarosite, natro-jarosite, hydronium jarosite, and ammoniojarosite (�).Unit cell dimensions are listed in Table 2.

7.32 Å and c � 17.13 Å. The JCPDS reference val-ues for jarosite are the dimensions a � 7.29 Å andc � 17.16 Å.

Figure 2 shows the LD-FTMS positive modespectra of synthetic potassium jarosite. The pri-mary mass-to-charge (m/z) peaks correspond topotassium (K�, m/z 38.96) and iron (Fe�, m/z55.93) ions. Small isotope peaks of potassium andiron are also visible in the spectrum. Relativeabundances for potassium correspond to stan-dard isotopic distributions for the 2 stable potas-sium isotopes (39K � 93.25% with mass � 38.96 uand 41K � 6.73% with mass � 40.96 u). For iron,the visible isotope peaks correspond to standarddistributions for the 2 most abundant stable iso-topes (54Fe � 5.84% with mass � 53.93 u and56Fe � 91.75% with mass � 55.93 u). No high-mass cluster ions were produced in the positive

mode spectra of synthetic jarosite, which indi-cates that the only positive ions formed werethose expected from the formula unit with a laserwavelength of 355 nm.

No attempt was made to quantify relationshipsbetween the various types of cations present inthe jarosite samples in this study because the sig-nal intensities, expressed as relative peak abun-dances, are dependent on both the ionization ef-ficiencies and the concentrations of the elementsor compounds in the sample (Yan et al., 2006). Thevariation in ionization efficiencies can be signifi-cant not only for molecules but also for elements.For example, the unit formula indicates that ironoccurs at higher concentrations than potassiumin potassium jarosite [3:1, KFe3(SO4)2(OH)6], butthis is not evident in the synthetic potassiumjarosite spectra (Fig. 1) because potassium ionizes

GLYCINE FOUND IN JAROSITES USING LD-FTMS 257

FIG. 2. LD-FTMS positive mode spectrum of synthetic jarosite. Potassium and iron ions observed at m/z 38.963and m/z 55.934, respectively.

more efficiently at a wavelength of 355 nm thandoes iron. Therefore, no attention was focused onthe quantitative relationships between one iontype and another. However, the isotopic abun-dances for a particular element or compound aremore quantitative than abundances between dif-ferent species because the ionization energies forthe isotopes are very close to each other. There-fore, relative isotopic abundances can be veryuseful for assisting in the identification of ele-mental compositions; however, the accuracy ofisotope ratios does vary with mass analyzer and

ionization method. For FTMS, electron ionizationprovides isotope ratio accuracy of �1%, whilewith laser desorption ionization the accuracy canbe as poor as �6% even for abundant element iso-topes (Spell et al., 1993). Laser desorption or ab-lation produces less accurate isotope ratios be-cause the slightly different ionization energies ofthe isotopes and the wider range of kinetic ener-gies cause fractionation that obscures the results.

Similar to the case of synthetic jarosite, the LD-FTMS positive mode spectrum of the natural min-eral contains potassium and iron peaks at m/z ra-

KOTLER ET AL.258

TABLE 3. MAJOR LD-FTMS POSITIVE PEAKS (m/z) OBSERVED IN THE NATURAL JAROSITE SAMPLES

Sample name

Jarosite-AZ 38.96 55.93 212.84 275.09 317.13Jarosite-AUS 38.96 55.93 84.91 196.87 275.09 317.13Jarosite-MT 38.96 55.93 259.11Jarosite-NM 38.96 55.93 128.41 195.60 275.09 373.22Jarosite-NZ 38.96 55.93 128.42 259.11 275.08 317.13Jarosite-RUS 38.96 55.93 301.86 317.11Jarosite-SPA 38.96 55.93 128.42 195.40

aMasses reported are averages from all spectra acquired for that sample.

Major m/z positive mode peaks*

FIG. 3. LD-FTMS positive mode spectra of jarosites from (A) Arizona (AZ), (B) Australia (AUS), (C) New Mexico(NM), and (D) New Zealand (NZ), showing a common peak at m/z 275. All other major peaks are listed in Table 3.

tios of 38.96 and 55.93, respectively. In addition,the spectra of all natural samples contain peaksat higher masses, which indicates the presence ofother ions or molecules in the samples (Table 3).One particular peak at m/z 275 was observed inall the spectra obtained from jarosite samples

from Arizona, Australia, New Mexico, and NewZealand (Fig. 3). Expanding the spectral regionfrom m/z 270 to m/z 280 revealed that this peakhad a set of associated isotope peaks that wereless abundant than the primary peak (Fig. 4 andTable 4).

To determine the elemental composition of themain peak at m/z 275, elemental and isotopic com-position searches were performed with theOdyssey Interpretation software (Thermo-Finni-gan FT/MS, Bremen, Germany). Potential ele-mental compositions with all possible combina-tions of inorganic and organic elements that werelikely to be present in the samples were identi-fied (i.e., C, N, O, S, H, Si, Na, K, Fe, Pb, P, Cu,and Zn). Carbon was included because analysesindicated that all natural samples had levels ofcarbon that were above the detection limits of theLeco carbon analyzer (Table 5). Higher-qualityspectra revealed that the mass of the peak was275.087 u, where the non-integer portion repre-sents the total mass defect from the combinedmass defects of the elements present [the massdefects of elemental isotopes are a result of using12C (12.00000000 u) as the standard]. To ensurethat a sufficiently wide range of likely candidateswas identified, the computer was queried for anycombinations of element isotopes within �0.005u of 275.087 u. Several compositions that con-tained carbon, nitrogen, sulfur, oxygen, and hy-drogen met this requirement. Some of these com-binations were easily dismissed by followingstandard mass spectral interpretation procedures(McLafferty and Turecek, 1993; Sack et al., 1984;Kim et al., 2006). The remaining possibilities werefurther subjected to the requirements that themass of the compound be very close to the targetvalue (within �0.005 u or less) and the calculated

GLYCINE FOUND IN JAROSITES USING LD-FTMS 259

TABLE 4. MASSES OF ISOTOPIC PEAKS FOR EXPERIMENTAL DATA AND POTENTIAL THEORETICAL COMPOSITION ASSIGNMENTS

Nominal isotope mass (u) 275 276 277 278 279

Observed isotope massa 275.087 276.091 277.084 278.088Theoretical isotope mass 275.088 276.092 277.084 278.087

[C11H19N2O2S2]�

Theoretical isotope mass 277.104 278.107 279.010[C11H19N2O2S2 � 2H]�

Theoretical isotope mass 275.087 276.090 277.085b 278.088[C14H20OSK]�

Theoretical isotope mass 277.102 278.106 279.100b

[C14H20OSK � 2H]�

aLD-FTMS mass error range � 0.002 u for given experimental conditions.bDominant isotope reported, as there are 2 lower-abundance isotopes with the same nominal mass.

FIG. 4. Expanded region (A) LD-FTMS positive modespectrum from m/z 270 to m/z 280 of jarosite-NewZealand sample that shows the isotopic distribution ofthe major peak at m/z 275 and (B) the theoretical isotopicdistribution of compound C11H19N2O2S2 predicted toform during gas-phase ionization of the jarosite samples.

isotopic abundance patterns be similar to thoseobserved.

Based on these criteria, the most likely identityof the peak at m/z 275 was determined to be acluster ion with the composition C11H19N2O2S2(Fig. 4 and Table 4). The mass for this cluster ionis 275.088 u, which would give a mass accuracyof �3 ppm. To illustrate that a small change incomposition can dramatically affect the mass de-fect and, therefore, the mass accuracy, the sub-stitution of a combination of NH2 for one Owould create a cluster (C11H21N3OS2) that wouldhave a mass of 275.112 u, while a substitution oftwo O’s for one S would create a cluster(C11H19N2O4S) with a mass of 275.106 u. Both al-ternative clusters have masses that are too highfor the reported accuracy of the instrument.

The possibility was considered that potassiumcould play a role in the cluster composition, re-sulting in a cluster with an elemental composi-tion of C14H20OSK with a mass of 275.087 u alongwith associated isotopes given in Table 4. An-other reason for assigning a composition for m/z275 was to account for the peak intensity for theisotope peak at m/z 277 ([M � 2]�1), which ishigher than would be expected for an organiccompound composed of only C, H, O, and N.Two S atoms or an SK combination would ac-count for the increased abundance of the [M �2]�1 peak. However, the difference betweenisotopic abundances for C11H19N2O2S2 andC14H20OSK is that the abundance of the [M �1]�1 peak for the C14H20OSK cluster is 20% higherthan that for C11H19N2O2S2. One caveat to usingisotopic distribution patterns is that they can becorrupted by the presence of other ions, espe-cially those that differ in number of hydrogenatoms present (Ham et al., 2003; Yan et al., 2007b).The possibility of such interferences can be ruled

out in this case because of the mass difference be-tween the [M]�1 and [M � 2]�1 isotope peaks(Table 4), which is 1.997 u. If the [M � 2]�1 peakwas due to the addition of 2 hydrogens (i.e., [M �2H]�1), then the mass difference would be 2.016u based on the theoretical masses given in Table4. The “contraction” in the mass difference be-tween the [M]�1 and [M � 2]�1 peaks is due tothe presence of a heteroatom, such as S or K. Theexperimental mass difference between the [M]�1

and [M � 2]�1 peaks of 1.997 u is between thetheoretical mass differences of the isotopes for thetwo clusters, which are 1.996 u and 1.998 u forC11H19N2O2S2 and C14H20OSK, respectively.While this mass difference does not help to dis-tinguish between these 2 composition choices, it does rule out interference from [M � 2H]�1.Participation of cations (e.g., K or Na) in thecluster formation was ruled out based on exper-iments with glycine combined with synthetic am-monium jarosite, synthetic potassium jarosite,Na2SO4, or K2SO4 as discussed below. Therefore,C11H19N2O2S2 was believed to be the best fit forthe elemental composition for m/z 275, and com-parison of the experimental spectrum with thetheoretical spectrum of C11H19N2O2S2 revealedthat the 2 spectra were virtually identical (Fig. 4).

Interestingly, a peak at m/z 259 appears occa-sionally in the spectra and may be related to them/z 275 peak. The nominal mass difference be-tween these two peaks is 16 u. Such a differenceis common for peaks that differ only in the pres-ence of Na (22.989 u) and K (38.963 u), which havea m of 15.974 u. Another alternative to accountfor this difference is the substitution of O for S,which gives a m of 15.977 u that is close to thedifference between the two peaks observed at m/z259 and 275 (m of 15.976 u). Therefore, the peakat m/z 259 is most likely to be the cluster ionC11H19N2O3S, which also matches the observedisotopic distribution. While unequivocal charac-terization of these cluster ions requires extensivetandem (MS/MS) and isotope exchange studies,the cluster ions observed at m/z 275 and m/z 259were clearly organic in nature.

Glycine, the smallest amino acid, was physi-cally mixed with synthetic ammonium or potas-sium jarosite to investigate how jarosite inter-acted with biomolecules in the laser desorptionplume. Interestingly, the physical mixtures pro-duced the same cluster ion at m/z 275, as can beseen in the spectra from the natural samples (Fig.5). In a traditional MALDI experiment, one would

KOTLER ET AL.260

TABLE 5. TOTAL CARBON ANALYSIS OF

SYNTHETIC AND NATURAL SAMPLES

Sample % total carbon

Synthetic jarosite BDLJarosite-AZ 0.10Jarosite-AUS 0.07Jarosite-MT 0.78Jarosite-NZ 0.05Jarosite-NM 0.06Jarosite-RUS 0.02Jarosite-SPA 0.06

BDL, below detection limit.

have expected to see a peak at m/z 76 that corre-sponded to the protonated version of glycine(C2H5O2NH�) or a peak that corresponded to acationized glycine molecule (e.g., C2H5O2NX�,where X � Na or K), neither of which was ob-served. In addition to the set of peaks around m/z275, which have the same isotopic distributionpattern as seen in the spectra of the natural sam-ples and the theoretical isotopic distribution ofC11H19N2O2S2 (Figs. 3 and 4), the spectrum in Fig.5 also shows the same distribution of potassiumand iron present in the standard synthetic jarositespectrum. To test whether the peak at m/z 275 isdistinct for glycine or general to the presence ofany amino acid, the experiments were run with

alanine in place of glycine. Alanine was chosenbecause it is the second smallest amino acid com-pared to glycine; in alanine one additional methylgroup replaces the hydrogen side chain of glycinein the chemical structure. When synthetic jarositewas mixed homogeneously with the amino acidalanine (Fig. 6), a complex mass spectrum was ob-served with the most abundant peak in the spec-trum at m/z 266 and no peak observed at m/z 275.The additional methyl group affects the molecu-lar mass of alanine, which suggests that the de-tection of alanine in the mass spectrometer wouldbe different than glycine by a net change of 14 u(–CH2). Since a net change of 14 u (or multiplesthereof) was not observed in the alanine-jarosite

GLYCINE FOUND IN JAROSITES USING LD-FTMS 261

FIG. 5. LD-FTMS positive modespectrum of synthetic jarosite mixedwith glycine showing the presence ofa major peak at m/z 275. The expandedview shows the presence of the clus-ter ion at m/z 275.

FIG. 6. LD-FTMS positive modespectrum of synthetic jarosite mixedwith alanine showing a complex dis-tribution of cluster ion peaks with amajor peak at m/z 266.

spectrum, the formation of the cluster ions is acomplex gas-phase phenomenon whose mecha-nism is not yet understood. Comparison of theglycine and alanine results does suggest that theidentity of the peak at m/z 275 is not likely to berelated to any amino acid other than glycine;however, not all amino acids have been tested.

Because the LD-FTMS spectrum of glycinealone did not show any peaks (data not shown)and because there is sulfur in the cluster ion(C11H19N2O2S2) of samples with jarosite, it is pos-sible that the sulfur comes from the sulfate in thejarosite crystal lattice and is responsible for for-mation of the cluster ion in the laser-inducedplume created during LD-FTMS analysis. Whenglycine was mixed homogeneously with potas-sium sulfate (K2SO4), a series of high-mass frag-mentation patterns were observed (Fig. 7). Theexpanded region of this spectrum from m/z 270to m/z 280 showed the same peaks that were pre-sent in the natural jarosite samples, syntheticglycine-jarosite mixture, and theoretical isotopicdistribution of C11H19N2O2S2 (m/z 275). Thepotassium sulfate–glycine mixture sample alsoproduced a strong peak at m/z 259. Similar peakpatterns were observed when glycine was mixedwith Na2SO4, which suggests that neither Na orK participate in formation of the ions. However,the most convincing evidence that Na or Kcations in jarosite were not participating in theformation of the cluster ion at m/z 275 is that thecombination of glycine with synthetic ammo-nium jarosite, which should be free from Na orK, also produced this peak.

DISCUSSION

The XRD results shown in Fig. 1 indicate thatthe unit cell dimensions of different naturaljarosite samples varied. These variations indicatethat an end-member composition in the naturalsamples was unlikely. A definitive classificationof the natural samples as jarosite, natrojarosite,hydronium jarosite, or ammoniojarosite based onthe XRD unit cell calculations is problematicwithout the addition of complete elementalanalysis to determine the ratios of substitutingcations relative to each other. This additionalanalysis would not have assisted in the determi-nation of hydronium or ammonium ratios due tothe inability of inductively coupled plasma massspectrometry techniques to determine these ele-mental compositions. These analyses were notperformed due to sample quantity limitations.Therefore, the samples were tentatively classifiedonly by the random powder XRD data. Based onXRD results, the samples from Arizona, Montana,and New Mexico are most likely jarosite. Thesamples from Australia and Russia are natro-jarosite, while the samples from Spain and NewZealand produced XRD spectra most similar tothat of ammoniojarosite. The classification of theNew Zealand and Spain samples as ammonio-jarosite is speculative, given the rarity of this min-eral subclass within the jarosite group minerals.The large unit cell dimensions could be attributedto the presence of foreign ions in the structure,whose identities were not determined in thisstudy. Potassium ions were observed in all of the

KOTLER ET AL.262

FIG. 7. LD-FTMS positive modespectrum of potassium sulfate (K2SO4)with glycine showing a complex dis-tribution of cluster ions and an ex-panded view of the region betweenm/z 273 and m/z 278. The expandedview shows the presence of the clus-ter ion at m/z 275.

LD-FTMS spectra (see Table 3), which indicatesthat all of the samples contained at least somequantity of potassium in their structures.

The LD-FTMS results demonstrate that glycinewas present in jarosite samples from Arizona,Australia, New Mexico, and New Zealand as ev-idenced by the presence of the spectral peak atm/z 275 and its associated isotopes. The determi-nation that the peak at m/z 275 resulted from thepresence of glycine was based on a variety of mix-ture experiments that indicated that this peak isa systematic signature when glycine is present inan ionization matrix that contains sulfate. The na-ture of association between glycine and the min-eral structure cannot be determined with the datawe measured in this study. However, becauseglycine could not be ionized alone by LD-FTMS,the presence of the geomatrix (i.e., jarosite) wasnecessary to ionize the biomolecule, as observedby Yan et al. (2007a). This study further validatesthe GALDI technique’s ability to detect biomole-cule impurities in geologic samples and, if ap-plied to relevant minerals of interest to planetarygeology, to aid in the search for signs of life onother planets. The ability to detect biomoleculesin situ without extensive sample preparation is akey advantage of this technique because the min-eral itself acts as the ionizing matrix. This wasclearly demonstrated when glycine was analyzedalone by LD-FTMS and no peaks were observedin the spectrum until a matrix (jarosite) was pre-sent (Figs. 5 and 7).

Of equal importance is the identification ofminerals or mineral groups that are capable ofstoring biomolecules in the geologic record andan understanding of the signatures they produce.This study has shown that jarosite samples fromvarious locations around the world produced ionsignatures representative of organic matter, andthat one of these signatures can be produced byglycine. Sulfur that is present as sulfate seems tobe responsible for the ionization and detection of glycine-related peaks in the samples, as evi-denced by its presence in the cluster ion observedat m/z 275 in the LD-FTMS spectra of all glycine-and sulfate-containing samples (Figs. 3, 5, and 7).While the formation pathway is currently not un-derstood, it is possible that the rather high laserfluence used means that the laser-matter interac-tion represents an ablation, as opposed to a des-orption, regime where recombination processesare more likely to take place (Aubriet et al., 2005).

While the other high-mass peaks seen in thejarosite LD-FTMS spectra have yet to be identi-fied, it is likely that they are organic, because theirmasses and isotopic distributions are consistentwith combinations of carbon and hydrogen.Searches for purely inorganic combinations ofions did not result in any matches for the massesor isotopic distributions. The corresponding iso-tope peaks that surround these additional high-mass peaks have similar distributions as thoseobserved for glycine and likely represent combi-nations of the less abundant isotopes in the or-ganic compounds, such as 13C, 15N, and 18O,which combine to form the smaller isotope peaks(such as those in observed in the m/z 275 clusterion). The parent molecules responsible for thecharged clusters can be difficult to determinewithout undertaking a variety of mixture exper-iments like those performed in this study, whichaided in the determination that the m/z 275 peakis a product of glycine ionization. Cluster ions canbe unique to specific molecules and geomatrixcombinations as demonstrated by the spectrumof the synthetic jarosite-alanine mixture (Fig. 6),which did not produce the same m/z ions as thesynthetic jarosite-glycine mixture (Fig. 5).

The fragmentation and cluster ion patterns alsoseem to be unique to the matrix-molecule combi-nation. Synthetic jarosite, mixed with glycine, pro-duced a simple mass spectrum where only thepeak at m/z 275 and its associate isotopes were de-tected (Fig. 5) along with the jarosite signature pat-tern observed without glycine (Fig. 2). However,when synthetic jarosite was mixed with alanine, acomplex fragmentation and cluster ion patternwas observed that produced a large number ofmass spectral peaks. The mass spectrum is strik-ingly dissimilar to the glycine-jarosite pattern,which supports the conclusion that the signaturesobserved in the natural samples could be attrib-uted to glycine and not a simple combination ofcarbon, nitrogen, oxygen, and sulfur atoms thatrandomly produced a peak at m/z 275. A complexmass spectrum was also observed when potassiumsulfate was mixed with glycine, which indicatesthat a wide variety of gas-phase or laser plume in-teractions occur that lead to many combinations ofions that can be detected by LD-FTMS. It is likelythat each geomatrix-molecule combination yieldsa distinctive signature that can be detected by LD-FTMS and identifies both the matrix and the ad-ditional molecules present in the samples.

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Jarosite has become a target mineral because itwas found on the surface of Mars in 2004 (Squyreset al., 2004), and jarosite was chosen for this studybased on its mineralogical characteristics and thepossible biological influences on its formation thatwould lead to biosignature detection. The resultsin this study not only validate the GALDI tech-nique and use of LD-FTMS but also show thatjarosite is an attractive target for retaining possi-ble biosignatures in the geologic record. It is un-clear from this study how glycine or the other, yetto be identified, organic molecules interact withthe jarosite mineral structure. These moleculesmay be intercalated between crystal layers duringdeposition, adsorbed as surface-related contami-nants, or incorporated into the crystal lattices. Ifthe organic molecules are somehow incorporatedinto the jarosite crystal lattices, it is possible thatthey could persist throughout geologic time.

CONCLUSIONS

Our studies indicate that glycine was present inintact natural jarosite samples from 4 out of 7 lo-cations worldwide. Additional high-mass, com-plex ions were also observed in all 7 samples.These results are complimentary to extractionanalyses that have also shown that glycine is pre-sent in natural jarosite samples (Aubrey et al.,2006). The source of the glycine or other organicmolecules detected in the jarosite samples (i.e.,whether they were biologically produced) andhow the organic molecules are associated with themineral matrix are unknown. While a biologicalorigin for the organic matter is possible, becausejarosite formation can be microbially mediated(Eneroth and Koch, 2004; Bridge and Johnson,2000; Sasaki and Konno, 2000; Akai et al., 1999;Karamanev, 1991; Clark et al., 1993; Grishin et al.,1988), it is impossible to ascertain biogenicitywithout further information. Glycine may be pro-duced abiotically (Mita et al., 2002, and referencestherein). We cannot be certain of the origin of theglycine in the natural samples used in this studywithout further information. It is difficult to dis-miss the possibility of a biological origin for thesemolecules when the ubiquity of microorganismsin the surface environments where jarosite is typ-ically found on Earth is considered, regardless ofwhether the molecules were present during de-position or were emplaced after formation. In anycase, the current experiments have demonstrated

the direct detection of organic molecules associ-ated with natural jarosite by LD-FTMS, and theresults indicate that the use of laser desorptionmass spectrometry is an appropriate approach forthe analysis of minerals returned to Earth fromother planets or for in situ exploration on Earth.

The overall advantage of our method and in-strumental approach is the ability to detect thepresence of organic molecules from fragment orcluster ions observed in the LD-FTMS spectra ina relatively short time and without extensive sam-ple preparation. This study has shown that clus-ter ion patterns are unique to the geomatrix-mol-ecule combination and that organic molecules canbe identified within terrestrial jarosite samples. Inaddition, jarosite has been shown to be an at-tractive mineral for investigating possible biosig-natures in the geologic record.

ACKNOWLEDGMENTS

Funding for this research at the University ofMontana, University of Idaho, and the Idaho Na-tional Laboratory (INL) comes from the NASAexobiology program (EXB03-0000-0054). Wewould like to thank the University of Montana Ge-ology Department for the donation of several ofthe jarosite samples used in this study. J.M.K. andN.W.H. would like to thank John Mocko and Mur-ray Baker for field assistance during the collectionof jarosite samples from the Coromandel Penin-sula, New Zealand. We would also like to thankRohn Wood and Eric Nugent at the University ofMontana as well as Tim McJunkin at the Idaho Na-tional Laboratory for technical support on this pro-ject. J.M.K would also like to thank John Macleanfor many useful discussions and advice about thismanuscript. LD-FTMS analysis was performed atthe INL under DOE/NE Idaho Operations OfficeContract DE-AC07-05ID14517.

ABBREVIATIONS

FTMS, Fourier transform mass spectrometer;GALDI, geomatrix-assisted laser desorption/ion-ization; JCPDS, Joint Committee on Powder Dif-fraction Standards; LD-FTMS, laser desorptionmicroprobe Fourier transform mass spectrome-ter; MALDI, matrix-assisted laser desorption/ionization; m/z, mass-to-charge; XRD, X-ray dif-fraction.

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Address reprint requests to:Nancy Hinman

Geosciences DepartmentUniversity of Montana

Missoula, MT 59812

E-mail: nancy.hinman@umontana.edu

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