a proteomic fingerprint of dissolved organic carbon and of soil particles

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METHODS Waltraud X. Schulze Gerd Gleixner Klaus Kaiser Georg Guggenberger Matthias Mann Ernst-Detlef Schulze A proteomic fingerprint of dissolved organic carbon and of soil particles Received: 17 March 2004 / Accepted: 20 July 2004 / Published online: 22 September 2004 Ó Springer-Verlag 2004 Abstract Mass spectrometry-based proteomics was ap- plied to analyze proteins isolated from dissolved or- ganic matter (DOM). The focal question was to identify the type and biological origin of proteins in DOM, and to describe diversity of protein origin at the level of higher taxonomic units, as well as to detect extracellular enzymes possibly important in the carbon cycle. Identified proteins were classified according to their phylogenetic origin and metabolic function using the National Center for Biotechnology Information (NCBI) protein and taxonomy database. Seventy-eight percent of the proteins in DOM from the lake but less than 50% in forest soil DOM originated from bacteria. In a deciduous forest, the number of identified proteins decreased from 75 to 28 with increasing soil depth and decreasing total soil organic carbon content. The number of identified proteins and taxonomic groups was 50% higher in winter than in summer. In spruce forest, number of proteins and taxonomic groups de- creased by 50% on a plot where trees had been girdled a year before and carbohydrate transport to roots was terminated. After girdling, proteins from four taxo- nomic groups remained as compared to nine taxonomic groups in healthy forest. Enzymes involved in degra- dation of organic matter were not identified in free soil DOM. However, cellulases and laccases were found among proteins extracted from soil particles, indicating that degradation of soil organic matter takes place in biofilms on particle surfaces. These results demonstrate a novel application of proteomics to obtain a ‘‘prote- omic fingerprint’’ of presence and activity of organisms in an ecosystem. Introduction Loss of biodiversity has become not only a scientific but also a political issue since the Rio Convention on Biodiversity. The science of biodiversity has centered mainly on the functional role of species in ecosystems and the understanding of the genetics and evolution of species, their habitats and distribution (Heywood and Watson 1995). Nevertheless, it remains fairly open how best to describe the organisms present in an ecosystem, and which component of diversity is important in the broader context of ecosystem func- tioning (Purvis and Hector 2000). Is it the spotted owl of the redwoods; is it the plant cover, a group of in- sects, the structural diversity of ecosystems, or the entirety of all organisms that matters (Loreau et al. 2002)? In this exploratory study, we determine the proteome of samples of dissolved organic matter (DOM) and of soil particles derived from lake and forest ecosystems. The identity of the proteins, their phylogenetic origin, their functions and spatial distri- bution could in future become a helpful tool to de- scribe presence and activity of organisms and their proteins in the environment. Water is a common resource for all organisms in an ecosystem. Mainly in soil, the organisms actively exchange organic material with the water phase as part of their cellular functioning, or passively, after cell death, when cell contents are released into soil water. Analysis of the organic composition of soil water should therefore allow ‘‘fingerprinting’’ of the W. X. Schulze (&) M. Mann Center for Experimental BioInformatics (CEBI), Department of Biochemistry and Molecular Biology, University of Southern Denmark, 5230 Odense, Denmark E-mail: [email protected] Fax: +45-6593-3018 G. Gleixner E.-D. Schulze Max-Planck-Institute for Biogeochemistry, P.O. Box 100164, 07743 Jena, Germany K. Kaiser G. Guggenberger Soil Biology and Soil Ecology, Institute of Soil Science and Plant Nutrition, Martin Luther University Halle-Wittenberg, Weidenplan 14, 06108 Halle, Germany Oecologia (2005) 142: 335–343 DOI 10.1007/s00442-004-1698-9

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METHODS

Waltraud X. Schulze Æ Gerd Gleixner Æ Klaus KaiserGeorg Guggenberger Æ Matthias Mann

Ernst-Detlef Schulze

A proteomic fingerprint of dissolved organic carbon and of soil particles

Received: 17 March 2004 / Accepted: 20 July 2004 / Published online: 22 September 2004� Springer-Verlag 2004

Abstract Mass spectrometry-based proteomics was ap-plied to analyze proteins isolated from dissolved or-ganic matter (DOM). The focal question was toidentify the type and biological origin of proteins inDOM, and to describe diversity of protein origin at thelevel of higher taxonomic units, as well as to detectextracellular enzymes possibly important in the carboncycle. Identified proteins were classified according totheir phylogenetic origin and metabolic function usingthe National Center for Biotechnology Information(NCBI) protein and taxonomy database. Seventy-eightpercent of the proteins in DOM from the lake but lessthan 50% in forest soil DOM originated from bacteria.In a deciduous forest, the number of identified proteinsdecreased from 75 to 28 with increasing soil depth anddecreasing total soil organic carbon content. Thenumber of identified proteins and taxonomic groupswas 50% higher in winter than in summer. In spruceforest, number of proteins and taxonomic groups de-creased by 50% on a plot where trees had been girdleda year before and carbohydrate transport to roots wasterminated. After girdling, proteins from four taxo-nomic groups remained as compared to nine taxonomicgroups in healthy forest. Enzymes involved in degra-dation of organic matter were not identified in free soilDOM. However, cellulases and laccases were foundamong proteins extracted from soil particles, indicating

that degradation of soil organic matter takes place inbiofilms on particle surfaces. These results demonstratea novel application of proteomics to obtain a ‘‘prote-omic fingerprint’’ of presence and activity of organismsin an ecosystem.

Introduction

Loss of biodiversity has become not only a scientificbut also a political issue since the Rio Convention onBiodiversity. The science of biodiversity has centeredmainly on the functional role of species in ecosystemsand the understanding of the genetics and evolution ofspecies, their habitats and distribution (Heywood andWatson 1995). Nevertheless, it remains fairly openhow best to describe the organisms present in anecosystem, and which component of diversity isimportant in the broader context of ecosystem func-tioning (Purvis and Hector 2000). Is it the spotted owlof the redwoods; is it the plant cover, a group of in-sects, the structural diversity of ecosystems, or theentirety of all organisms that matters (Loreau et al.2002)? In this exploratory study, we determine theproteome of samples of dissolved organic matter(DOM) and of soil particles derived from lake andforest ecosystems. The identity of the proteins, theirphylogenetic origin, their functions and spatial distri-bution could in future become a helpful tool to de-scribe presence and activity of organisms and theirproteins in the environment.

Water is a common resource for all organisms inan ecosystem. Mainly in soil, the organisms activelyexchange organic material with the water phase aspart of their cellular functioning, or passively, aftercell death, when cell contents are released into soilwater. Analysis of the organic composition of soilwater should therefore allow ‘‘fingerprinting’’ of the

W. X. Schulze (&) Æ M. MannCenter for Experimental BioInformatics (CEBI),Department of Biochemistry and Molecular Biology,University of Southern Denmark, 5230 Odense, DenmarkE-mail: [email protected]: +45-6593-3018

G. Gleixner Æ E.-D. SchulzeMax-Planck-Institute for Biogeochemistry,P.O. Box 100164, 07743 Jena, Germany

K. Kaiser Æ G. GuggenbergerSoil Biology and Soil Ecology,Institute of Soil Science and Plant Nutrition,Martin Luther University Halle-Wittenberg,Weidenplan 14, 06108 Halle, Germany

Oecologia (2005) 142: 335–343DOI 10.1007/s00442-004-1698-9

major classes of the active life forms present in asystem at a given time. Such information can, inprinciple, also be obtained from the presence of spe-cific phospholipid fatty acids in ecosystem compart-ments. However, this is a low-resolution method forthe classification of soil organisms (Zelles 1997). Incontrast, PCR amplification and sequencing of con-served rDNA sequences allows precise identification oforganisms in ecosystems, but gives no informationabout their activity. Since DNA binds to clay, theorganisms may not even be present anymore. Bothmethods have largely been restricted to determinetaxonomic relationships of individual species (Harriset al. 2002), and reciprocal community hybridizationperformed on DNA samples extracted from soil en-abled the description of seasonal variations in soilmicrobial communities (Lipson et al. 2002). Sinceproteins represent the catalytic potential and reactivityof an ecosystem, they link phylogenetic presence andbiogeochemistry. Therefore, this class of macromole-cules would be ideally suited to characterize the activecomponent of phylogenetic diversity in the soils ofecosystems and to relate presence of specific proteinsto soil organic chemistry. In soils, organic compoundsof the soil solution, i.e., DOM, interact with solid soilparticles by sorptive processes. Hence, the organicsubstrate for soil microorganisms is partly removedfrom the soil solution and this may be also true forenzymes. However, catalytic processes may take placeonly in biofilms of soil particles. Thus we analyze bothproteins in DOM and in soil particles.

Large-scale, high-sensitivity protein analysis has re-cently become possible due to advances in mass spec-trometry-based proteomics (Aebersold and Mann 2003)and bioinformatics. So far, this methodology has onlybeen used at the cellular or organism level. Here weextend proteomic analysis to the identification of pro-teins in complex media such as lake waters or waterextracted from soils and soil particles. It is the mainaim of this exploratory study to determine the phylo-genetic groups from which these proteins originated,and to assign the potential catalytic function of theseproteins. The methodology was applied to DOM ob-tained from different and contrasting environments,such as: (1) water originating from a peat bog lake,Lake Hohloh (Schulten and Gleixner 1999), soil waterpercolating through (2) the profile of an unmanageddeciduous forest, the Hainich, Thuringia, (3) a man-aged evergreen spruce forest, the Wetzstein, Thuringia,and (4) to soil particles obtained from an acid soilunder spruce at Waldstein, NE Bavaria, Germany. Wefound that the protein pools sampled were highlycomplex and varied between ecosystems, with soildepth and with season. This is a first documentationthat the environmental proteome may also reflectpresence of different taxonomic groups, and that par-ticle-bound soil enzymes possibly participating indecomposition of organic matter originate from differ-ent organisms.

Materials and methods

Site descriptions

Lake Hohloh Lake Hohloh is a brown-water peat pond(pH 3.4) in a natural preservation area at the top of amountain (1,000 m above sea level) in the Black Forest,Germany. The lake is only recharged by precipitationand the water level is controlled by natural outflow. Themean dissolved organic carbon (DOC) of the lake waterwas 25.6±3.2 mg l�1 (Kracht and Gleixner 2000).Vegetation surrounding the lake is mainly Sphagnumspp., some Eriophorum, and at greater distance to thelake also Pinus sylvestris and Picea abies.

Hainich Soil water was collected in an unmanagedforest of the National Park Hainich at 430 m elevation.It is a deciduous forest with about ten dominant treespecies, and a leaf area index of 6. Leaf-litter productionis 440 g m�2 and fine root production is 410 g m�2.Total inorganic nitrogen reaches a concentration of447 lg g�1 in the 5- to 10-cm horizon. Soil humus formwas of mull type and carbon mineralization reached amaximum of 123 g C m�2 a�1 in the top mineral soil.Both parameters indicate high soil microbial activity.Mean annual DOC content was highest at 5 cm depth(16.4±5.8 mg l�1) and decreased to 9.9±3.2 mg l�1 at10 cm depth, and to 7.6±2.7 mg l�1 at 20 cm depth(Table 1). DOC concentrations were larger in summer(July, 14 mg l�1 at 10 cm) than in winter (January,7 mg l�1 at 10 cm; Table 1).

Wetzstein This is a managed spruce forest growing atan elevation of 750 m. The stand of the study plot waseven-aged, 35 years old. In a separate experiment, partof this forest plot was girdled in spring 2001 in order to

Table 1 Summary of samples from different forest ecosystems

Season Vegetation Weight(mg)

TIC(mg/l)

DOC(mg/l)

Corg

(%)NO3

(mg/l)

Falla Beeche 3.9 5.7 13.4 13.7 1.6Falla Beeche 8.6 12.1 10.8 12.0 0.5Falla Beeche 4.4 4.8 8.2 10.4 <0.50Falla Beeche 4.5 1.3 22.5 14.2 1.8Falla Beeche 4.3 5.7 9.1 8.1 0.8Falla Beeche 19.6 8.8 8.0 4.6 <0.50Winterb Beeche 4.6 2.5 17.5 20.7 2.8Winterb Beeche 3.8 4.0 7.2 12.0 3.2Summerc Beeche 5.6 2.2 22.2 20.2 14.9Summerc Beeche 5.5 2.7 14.4 15.5 24.5Springd Sprucee 10.2 1.6 32.0 27.1 4.37Springd Sprucef 9.9 1.1 24.8 38.8 6.70

a17.10.2002 and 30.10.2002.b13.12.2002 and 08.01.2003.c04.07.2001.d15.04.2003.eHainich, unmanaged forest.fWetzstein, managed forest, ungirdled.gWetzstein, managed forest, girdled.

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cut the supply of carbohydrates to the roots. Soil waterwas taken as part of this girdling experiment in April2003 and compared with an un-girdled forest stand. Soilorganic and inorganic carbon is listed in Table 1.

Waldstein This site is a 145-year-old spruce forest at anelevation of 780 m. Soil samples were collected from anacid forest soil (Podzol), developed from a graniticsolifluction layer. The organic forest floor layer wasmoder type.

Soil water and surface water collection

Surface water from lake Hohloh was collected, filtered,and freeze-dried. The water sample was taken in June1998. Percolating soil water was obtained using glassceramic suction plates. Plates were installed at 5, 10 and20 cm depth at three independent locations within thefootprint area of an Eddy covariance measuring systemat the Hainich (Knohl et al. 2003) and at 5–10 cm at theWetzstein sites (Hahn 2003). Water was collected every2 weeks and filtered through a 0.2-lm acetate filtermembrane prior to freeze-drying. The present study usedmixed samples from the two subplots. Samples weretaken in October, December and July (Hainich), and inApril (Wetzstein).

Preparation of soil microparticles

Bulk soil samples were dried in air at 20�C, and passedthrough a 2-mm sieve. Discrete particles of organicmatter were removed from subsamples by heavy liquidflotation with sodium polytungstate at a density of1.6 g cm�3 (Christense 1992). Subsamples (5 g) wereslurried in 25 ml sodium tungstate solution, shaken for24 h, then centrifuged at 10,000 g for 30 min. The su-pernatants were removed and the settled soil materialswere washed thoroughly with deionized H2O until theelectrical conductivity of the solution was <50 lS.Thereafter, samples were air-dried at 20�C. Proteinswere removed from inorganic material by dissolving themineral matrix using 10% HF. After neutralization,proteins were separated from other organic molecules bygel filtration and SDS-gel electrophoresis and subjectedto analysis by mass spectrometry as described below.

Sample preparation for LC-MS

Humic acids and other small molecules were removedfrom the soil solution by size exclusion gel filtration overSepharose 4B. Protein-containing fractions were com-bined, concentrated, and separated via SDS-polyacryl-amide gel electrophoreses. After silver staining, the gelwas cut into slices of approximately equal protein con-tent and proteins were in-gel digested using trypsin(Shevchenko et al. 1996). Tryptic peptides were

extracted from the gel particles, and desalted usingSTAGE-Tips (Rappsilber et al. 2003).

Liquid chromatography and mass spectrometry

Mixtures of tryptic peptides were separated by nanoflowliquid chromatography (LC) (Ishihama et al. 2002) priorto analysis by high mass-accuracy tandem mass spec-trometry (LC MS/MS) on a QSTAR pulsar quadrupoletime-of-flight hybrid mass spectrometer. Peptide se-quences were derived through information-dependentacquisition of fragmentation spectra of multiple-chargedpeptides (Rappsilber et al. 2002). Acquired spectra weresearched against the NCBI protein database (http://www.ncbi.nlm.nih.gov/) using the Mascot algorithm(Perkins et al. 1999). The following search parameterswere applied: maximum of one missed trypsin cleavage,cysteine carbamidomethylation, methionine oxidation,and a maximum 0.2 Da error tolerance in both the MSand MS/MS data (40 ppm after dynamic recalibration).Only fully tryptic peptides were allowed and all hits weremanually verified against the raw mass spectrometricdata using accepted rules for peptide fragmentation in aquadrupole-TOF hybrid mass spectrometer. On aver-age, 30% of the proteins were identified by a singletryptic peptide.

Taxonomic and functional classification

The protein sequence derived from MS/MS spectra oftryptic peptides bears taxonomic information of theorigin of the protein. In most cases, the sequences ob-tained from tryptic peptides were unique to a specificgroup of organisms, or even one species. Since fullproteomic information in the database is available onlyfor a limited number of organisms, the identified pro-teins were grouped according to their taxonomic originon broader taxonomic levels following the nomencla-ture of the NCBI taxonomy browser (http://www.ncbi.nlm.nih.gov/Taxonomy/taxonomyhome.html/).Proteins originating from bacteria, archaea, and viruseswere not separated into further subgroups. Proteinsfrom eucaryotes were sorted by their origin from greenplants, metazoa, fungi, and ‘‘unicellular eucaryota’’holding all those taxa that do not belong to the threemajor groups of Eucaryota. Proteins from plants werefurther grouped into algae and vascular plants. Proteinsfrom metazoa were classified into Platyhelminthes,Protostomia (annelid worms, insects, and mollusks),Nematoda, and Vertebrata (mammals, birds, reptilesamphibia, fish). In some cases, sequenced peptidesidentified highly conserved regions of proteins commonto different taxonomic groups. These proteins weredesignated as ‘‘not classified’’. Identified proteinsequences were tested for redundancy by BLAST allagainst all sequences (Altschul et al. 1990). Proteinsequences with identity greater than 98% were consid-

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ered identical. The functional attributes of the identifiedproteins were classified following the enzyme commis-sion classification scheme.

Results

Proteins in aquatic and terrestrial ecosystems

A total of 148 proteins were detected in the surface waterof a black-water lake (Fig. 1). Seventy-eight percent ofthe observed proteins in the lake water originated frombacteria. Viruses and vertebrates were the next twolargest groups of organisms contributing to the proteinpool. A total of ten taxonomic groups were distinguished.

In contrast, in water derived from forest soil of anatural deciduous forest, only 75 proteins were identifiedat 5 cm depth, and less than 50% of the identified pro-teins were bacterial (Fig. 2). The fraction of proteinsoriginating from plants, fungi and vertebrates was abouttwice as high as in lake water. A total of 16 taxonomicgroups were distinguished in the soil-water solution ofthis terrestrial forest ecosystem. An analysis of theprotein composition along a depth profile of the forestsoil revealed a higher fraction of nematodal proteins insoil water taken from 20 cm depth, whereas the fractionof vertebrate proteins was largest in water from the toplayer (0–5 cm). Plant proteins were most abundant at5–10 cm depth. Total number of proteins decreased withincreasing soil depth. This correlates with decreasingcontent of DOC with increasing soil depth (Table 1). Itshould be made clear that this method does not quantifythe number of species, but it reflects the presence andactivity of different taxonomic groups, and the turnoverof proteins. For the focus of this study, it is not the aimto detect the full proteome of any organism, but to de-tect the presence of the taxonomic groups present in thesoil by at least one stable protein.

Analysis of the seasonal variation of protein contentin soil water showed that the number of detected pro-teins, and the fractions of bacterial proteins were higherin winter than in summer (Fig. 3). The seasonal differ-ences were consistent along the depth profile, and thelower number of proteins at deeper soil layers wasmaintained throughout the seasons (see Fig. 2 for fallseason). The unexpected finding of higher proteinnumber in winter was not due to increased DOC, whichwas lower in winter than in summer (Table 1). It ispossible that the humidity conditions in the winter sea-son are more favorable to presence of bacteria. Inaddition, the degradation of organic matter may be re-duced in the colder season leading to higher number ofproteins in the water samples. A reduced biologicalactivity in winter is obvious from the lower nitrateconcentrations in winter than in summer (Table 1),indicating lower rates of mineralization in winter. Inaddition, smaller organic molecules contribute to DOCin summer, but to a lesser extent in winter (Grahamet al. 1994).

Proteomic diversity in response to tree girdling

To study the effect of aboveground plant cover andplant activity on phylogenetic protein composition, soil-water extracts from a naturally growing old spruce for-est and a girdled plot were analyzed at the Wetzstein(Fig. 4). On the girdled plot, phloem sap flow of treeswas interrupted leading to dying trees. In the undis-turbed forest, proteins from nine distinct taxonomicgroups were identified; only 30% of the proteins were ofbacterial origin. Most striking was the dramatic decreaseof the protein pool from the girdled plot of the sameforest. The number of proteins significantly decreased by50% and only four contributing phylogenetic groupswere detected with a large fraction of bacterial proteins(60%). The dying forest demonstrates not only that thecarbohydrate supply of the vegetation affects below-ground protein composition, but also that proteomicfingerprints are a sensitive tool to detect changes in thebiology of ecosystems.

Enzymes in free water

The largest fraction of the observed proteins werecytosolic and nuclear proteins, and 13% of the proteinsdetected in soil water of the Hainich at 5 cm depth and19% of the proteins found in the brown-water lakeHohloh were well-known anabolic and catabolic en-zymes (Fig. 5). Nevertheless, differences between thelake and the deciduous forest became apparent: perox-idases, which use molecular oxygen and are involved indegradation of complex molecules, were only found inforest-soil water, whereas transferases of one carbonresidue, possibly involved in the methane production,were exclusively detected in the water of the peat bog.

Fig. 1 Distribution of phylogenetic groups contributing to theprotein pool of surface water of black-water lake Hohloh. A totalof 157 proteins were identified in the lake water

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However, currently it still remains unclear if any of theenzymes remain functional in the soil water.

Enzymes bound to particles

Enzymes involved in degradation of biological material,such as invertases, laccases, or cellulases were not

detected in percolating soil water or surface water ofthese sites. Thus, proteins attached to mineral clay andsilt-sized soil particles (particle size <2 mm afterremoval of particulate organic debris) were investigated.Tryptic peptides of cellulase, glucosidases, collagenases,and a ligniolytic phenoloxidase were identified as beingbound to soil microparticles (Table 2). MS/MS frag-mentation spectra, which bear sequence information, are

Fig. 2 Distribution ofphylogenetic groupscontributing to the protein poolof forest-soil water along adepth gradient taken in October(‘‘fall’’). Area and number nextto the pie charts represent thenumber of proteins identified.Black bars represent the contentof DOC in the water samples of16.4±5.8, 9.9±3.2, and7.6±2.7 mg l�1 at 5, 10, and20 cm depth, respectively).Each pie chart is the average ofanalysis of three differentsampling sites

Fig. 3 Seasonal variation oforganisms contributing to theprotein pool of forest-soil waterat two different depths. Sampleswere taken in June (‘‘summer’’)and December (‘‘winter’’). Areaand number next to the piecharts represent the number ofproteins

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shown for a tryptic peptide of a fungal lignin-degradinglaccase and a fungal cellulase (Fig. 6a,b). There is a largediversity of enzymes with similar function: cellulases weredetected as originating from three different bacteria andfrom one fungus, collagenase originated from two bac-terial strains, and lignin penoloxidases and laccasesoriginated from fungi only. The direct observation oflaccase closes the gap between DNA sequencing studies(Luis et al. 2004) and biological tests (Kjoller et al. 2000).Thus, the functional diversity of enzymes within eachtaxonomic group can possibly be distinguished in future.

Discussion

Proteomics is one of the fastest-developing researchareas, and contributes substantially to our understand-ing of organisms at the cellular level (Aebersold andMann 2003; Tyers and Mann 2003). Dissolved organicnitrogen (DON) and DOC are significant for C and Ncycles of terrestrial ecosystems and undergo variations inseason and depth profile (Kaiser et al. 2001; Michalzikand Matzner 1999). DOC and DON have so far beenwell studied with respect to their d13C and d15N originand basic chemical structure (Gleixner et al. 2001; Kai-ser et al. 2001; Michalzik and Matzner 1999). Although15N NMR analysis has demonstrated that most nitrogenis present in the amide form (Almendros et al. 1991), i.e.,possibly as protein, not much is actually known aboutthese proteins, their phylogenetic origin, or enzymaticfunctions within the pool of DOM. In fact, up to nowone would have assumed that proteins degrade so fast innatural ecosystems, that detection in soil water wouldnot even be possible.

Our results show that proteomic analysis in DOM ispossible, and that DOM from different ecosystemsclearly differ in their proteomic composition. The con-tribution of different types of organisms to the proteinpool varies between ecosystems and with soil depth andseason. Strikingly, we are even able to detect effects ofchanges in ecosystem biology (i.e., after girdling of trees)on the phylogenetic spectrum of organisms contributingto the soil protein pool. The girdling experiment and thecomparison of broad-leaf versus coniferous forests alsoreveal that ecosystem biology is related to soil proteinbiology. The girdling experiment suggests that the plantcover does influence the soil protein pool, possiblythrough changes in presence and activity of differentorganisms.

Fig. 4 Comparison of thephylogenetic contribution inprotein pools derived from anatural and a girdled spruceforest. Area and number next tothe pie charts represent thenumber of proteins

Fig. 5 Classification of detected enzymes according to the interna-tional enzyme commission classification scheme. On average, 16%of the detected proteins were known enzymes

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The seasonal variation of the protein concentrationand composition in the soil waters suggests that theprotein pool is subject to changes in the activity andturnover of the contributing organisms, and is not onlyinfluenced by the abundance of specific organisms. Thehigh percentage of bacterial proteins found in all thesamples analyzed here may thus reflect higher bacterialactivity and turnover of Protozoa compared to multi-cellular organisms like plants or animals. However, atthis stage, care needs to be taken with such interpreta-

tions, as the NCBI database also has a higher content ofbacterial proteins compared to proteins from Eu-karyota. Also, at this point we do not yet know the rateconstant of protein degradation. The high proteinnumbers detected in winter samples indicate that deg-radation is an important process which, together withprotein production from lysing cells, will influence thetotal number of detected proteins. Degradation of pro-teins in the environment is assumed to occur indepen-dently of the source organism of proteins, but proteinsof scarce organisms may be harder to detect as they maydisappear in the bulk of proteins from more abundantorganisms. It is obvious that the dynamics of proteinproduction and degradation need further studies, pos-sibly in controlled experiments.

Although the actual function of enzymes identified inthis study can only be conclusively demonstrated bymeasurements of enzymatic activities, our data indicatethat degradation of soil organic matter does obviouslynot take place in the percolation soil water. Enzymesthat are important for the turnover of organic matter insoils (Gleixner et al. 2001) were all found to be closelyassociated with soil particles. This is in agreement withthe notion that in-vitro measurements of invertase, andcellulase activity can only be detected in soil particlefractions (Kjoller et al. 2000; Stemmer et al. 1998). Foraquatic ecosystems, recent studies indicated that bio-geochemical activity is located in so-called matrix en-closed biofilms that function in storage and degradationof organic matter (Battin et al. 2003). Sorption of DOMin percolating soil waters into biofilms on soil mineralparticles and subsequent enzymatic decomposition isalso hypothesized as an important pathway in the min-eralization of DOC (Guggenberger and Kaiser 2003).

Our proteomic approach so far mainly identifies veryabundant and stable proteins. In this study, rather lownumbers of proteins were identified in some of thesamples and this imposes limitations on the interpreta-tion of proteomic fingerprints. We have found that thenumber of taxonomic groups that were distinguishedwas independent of the number of proteins identifiedonce a threshold of eight proteins was exceeded. Withimproving sensitivity of protein mass spectrometry, andimproved methods to purify proteins from environ-mental sources, a more detailed picture may emerge. Theability to unambiguously identify the proteins derivedfrom a complex mixture is a prerequisite for the kind of

Fig. 6 MS/MS fragmentation spectra of tryptic peptides repre-senting biodegradative enzymes. a Fragmentation spectrum of afungal laccase. b Fragmentation spectrum of a fungal cellulase.Peptide sequences were derived from ‘‘sequence tags’’ containinginformation of m/z values of b- and y-ion fragmentation series aswell as the mass to charge ratio of the parent ion

Table 2 Enzymes involved in degradation of soil organic matter

Protein name (taxonomic origin) Accession no. (enzyme no.) Tryptic peptides

Cellulase CelE ortholog (bacterial) gil15893851 (EC 3.2.1.4) VTSFPVESNKCellulose binding protein A precursor (bacterial) gil584895 (EC 3.2.1.4) VATVNGSVKExtracellular endocellulase (bacterial) gil6606317 (EC 3.2.1.4) SILFYEAQRCellulase (fungal) gil4586347 (EC 3.2.1.4) FVTGSNVGSR FYVQNGKCollagenase and related proteases (bacterial) gil23112072 (EC 3.4.24.3) QSFQEGKERCollagenase precursor (bacterial) gil464477 (EC 3.4.24.3) YGSGRTRLigninolytic phenoloxidase 2 precursor (fungal) gil101946 (EC 1.10.3.2) QAVVVNNVTPGPLVAGNKLaccase (fungal) gil2147619 (EC 1.10.3.2) NKLSDPTMR

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investigations carried out in this work. The informationnecessary is generated by the combination of trypticprotein digests with high mass-accuracy tandem mass-spectrometry (Rappsilber and Mann 2002). The se-quences derived for individual tryptic peptides in themajority of cases are unique to a specific protein, or atleast protein family. Thus, it is certain whether a giventryptic peptide, for example, is from a choride channelor a glycolytic enzyme. However, it is a current limita-tion of existing protein libraries that still restrict thepresent analysis to the distinction of broader taxonomicunits only. Nevertheless, there are indications thatdiversity of larger taxonomic entities correlates withspecies diversity (Baldi 2003), supporting the idea thatour rough taxonomic classification may in future de-velop into an approach to describe the more complextaxonomic composition of an ecosystem.

Current efforts of sequencing DNA samples extractedfrom environments (Venter et al. 2004) are encouragingand will provide a basis for more accurate proteinidentifications. It could be demonstrated using anexperimental dataset that cross-species protein identifi-cation by mass spectrometry (e.g., MS-BLAST) suc-cessfully identifies over 80% of the proteins by sequencesimilarity searches, because orthologue proteins sharesubstantial sequence identity (Habermann et al. 2004).Despite taxonomic distinction of specific organisms stillbeing the most accurate and most widely applicableusing rDNA analysis, peptide mass fingerprinting oftryptic digests of bacterial spores (Dickinson et al.2004a, b) or mass spectrometric analysis of whole cells(Arnold and Reilly 1998) emerge as a novel and morerapid tool to specifically distinguish microorganisms atthe sub-species level. The advantage of protein analysisis in the rapid identification of taxonomic units over abroad range of the phylogenetic tree. Furthermore,proteins are potentially active components of DOM andthus protein identification may improve our under-standing of soil organic chemistry. Once methods aremore established for proteomic analysis of environ-mental samples, it may be more rapid than DNA se-quence analysis or identification of microorganismsthrough cell culture (Dickinson et al. 2004a, b). How-ever, environmental fingerprinting on both DNA andprotein levels are needed to obtain a complete picture ofthe taxonomic groups present in an environment. Withfuture and ongoing efforts of DNA sequencing multi-cellular organisms or environments (Tyson et al. 2004;Venter et al. 2004), as well as with de-novo-sequencingof protein MS/MS spectra, a more reliable interpreta-tion of abundance and presence of organisms in anenvironment may be possible. In addition, more detailedfuture work is required to compare how our taxonomicclassification of proteomic or DNA fingerprints can re-flect actual soil biodiversity at the species level. It shouldbe made clear that the method described here does notquantify the number of species, but rather integrates thepresence and activity of different taxonomic groups, andthe turnover of proteins.

Despite their limitation, the results demonstrate that:(1) the methods of proteomics can well be applied toimprove our understanding of the functional proteincomposition of dissolved and solid organic carbon andnitrogen in environments, and (2) at the same time de-pict the presence of different taxonomic groups in theenvironment. With more refined databases in future, weexpect that proteomics will be a suitable tool to identifycontributing organisms also at finer taxonomic levels. Asa follow-up to genomics and proteomics of individualorganisms, the science of ‘‘environmental proteomics’’emerges as a powerful tool to describe changes in biol-ogy of ecosystems in a new functional context and torelate these changes to environmental parameters.

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