European Journal of Soil Science, December 2009, 60, 845–859
doi: 10.1111/j.1365-2389.2009.01184.x
Soil metaproteomics: a review of an emerging
environmental science. Significance, methodology and
perspectives
F. B A S T I D A, J. L. M O R E N O, C. NI C O L Á S, T. H E R N Á N D E Z & C. G A R C Í A
Centro de Edafologı́a y Biologı́a Aplicada del Segura (CEBAS-CSIC), Department of Soil and Water Conservation and Waste
Management, Campus Universitario de Espinardo, Espinardo, Murcia 30100, Spain
Summary
Soil is a dynamic system in which microorganisms perform important tasks in organic matter transformations
and nutrient cycles. Recently, some studies have started to focus on soil metaproteomics as a tool for
understanding the function and the role of members of the microbial community. The aim of our work
was to provide a review of soil proteomics by looking at the methodologies used in order to illustrate the
challenges and gaps in this field, and to provide a broad perspective about the use and meaning of soil
metaproteomics. The development of soil metaproteomics is influenced strongly by the extraction methods.
Several methods are available but only a few provide an identification of soil proteins, while others extract
proteins and are able to separate them by electrophoresis but do not provide an identification. The extraction
of humic compounds together with proteins interferes with the latter’s separation and identification, although
some methods can avoid these chemical interferences. Nevertheless, the major problems regarding protein
identification reside in the fact that soil is a poor source of proteins and that there is not enough sequencedatabase information for the identification of proteins by mass spectrometric analysis. Once these pitfalls have
been solved, the identification of soil proteins may provide information about the biogeochemical potential
of soils and pollutant degradation and act as an indicator of soil quality, identifying which proteins and
microorganisms are affected by a degradation process. The development of soil metaproteomics opens the
way to proteomic studies in other complex substrates, such as organic wastes. These studies can be a source
of knowledge about the possibility of driven soil restoration in polluted and degraded areas with low organic
matter content and even for the identification of enzymes and proteins with a potential biotechnological value.
Introduction
The structure and function of soil microbial community
members
Soil is a dynamic system in which physical, chemical and
biological components interact. Within this system, microorganisms perform an important task in the decomposition and transformation of soil materials, and are involved in the cycles of carbon,
nitrogen and phosphorus. However, interest in microbial functionality has grown in recent years as we seek to understand the
relationship between microbial communities and their surrounding
environment.
In this respect, microbial ecology has made new advances
thanks to the development of molecular techniques that permit
the study and characterization of uncultured organisms. Such
Correspondence: F. Bastida. E-mail: [email protected]
Received 14 October 2008; revised version accepted 30 June 2009
© 2009 The Authors
Journal compilation © 2009 British Society of Soil Science
methodology, in contrast to ‘classical’ methods (to determine
soil respiration, enzyme activities, microbial biomass, etc.), which
provide an approximate measurement of soil dynamics, has
enabled us to identify microbial diversity and to clarify the
microbial community structure (Nannipieri & Smalla, 2006). The
genome became the key for soil in the 1990s and metagenomics
(the collective DNA from all the microorganisms present in a
community) is the tool used to identify genes that could be
involved in soil processes. Molecular techniques based on DNA
enabled the microbial community structure to be studied at the
DNA level and have allowed the abundance of genes related
to a given microbial group involved in a metabolic pathway to
be quantified (Figure 1). As well as these approaches, methods
based on RNA have permitted such analyses in the context of
molecular transcription and have provided some information on
gene activity. Metatranscriptomics (defined as the collective RNA
from all the microorganisms in a community) constitutes the
second step in the attempt to reveal soil-microbial functionality.
845
846 F. Bastida et al.
Figure 1 Strategies of soil microbial functionality (modified from Graves & Haystead, 2002).
Some methods involving DNA and RNA have been proposed
in order to link microbial biodiversity and functionality. The
combination of fluorescent, in situ hybridization and microautoradiography is a tool for the structural-functional analysis
of microbial ecology that allows the simultaneous determination
of the identities, activities and specific substrate-uptake profiles
of individual bacterial cells within complex microbial communities (Lee et al., 1999). Stable isotope probing (SIP) allows the
identification of species that degrade isotopically labelled compounds (Radajewski et al., 2002, 2003; Friedrich, 2006), through
DNA or RNA studies. The use of bromodeoxy uridine (BrdU) by
microorganisms makes it possible to detect and ‘immuno-capture’
the DNA of soil microorganisms that grow in the presence of
labelled compounds (Borneman, 1999). However, the main problem of these techniques is that they involve modified conditions
rather than real ones. In the last few years, effort has been devoted
to the study of the real executors of the genome at molecular
scales; that is, the proteins (Wilmes & Bond, 2004). Metaproteomics studies the collective proteins from all the microorganisms
in a community (Maron et al., 2007) and provides information
about the actual functionality in relation to metabolic pathways
and regulation cascades, more specifically so than functional genes
and the corresponding RNAs (Wilmes & Bond, 2006; Benndorf
et al., 2007). Therefore, proteomics is an ideal supplement to
functional genomics (Benndorf et al., 2007). The combination of
genomic techniques with genomic products may provide the link
between microbial community-structure and soil function (Nannipieri et al., 2003) (Figure 1).
However, while some barriers to our knowledge have been
removed, others remain. The distribution of proteins in soil is
variable and their location depends on the soil matrix being studied. There are intracellular proteins (cytoplasmic, ectoenzymes,
periplasmic in Gram-negative bacteria) and extracellular proteins
(that is, free in soil solution or sorbed to organic matter or soil
minerals) (Gianfreda & Bollag, 1994; Nannipieri & Smalla, 2006)
(Figure 2). Such variability does not ensure that the proteins in
soil are present in sufficient amounts to be detected and much
research has tried to stimulate the proliferation of microorganisms in order to analyse the proteins (Singleton et al., 2003;
Benndorf et al., 2007). Unlike in the case of DNA, there is no
way to amplify the number of copies of a protein, so the analysis
of low-abundance proteins remains a major challenge (Graves &
Haystead, 2002). A second hindrance is the small yield of proteins
extracted from soil in which the interaction with organic matter
interferes with the resolution and identification of the extracts
(Criquet et al., 2002).
Once proteins have been extracted, another challenge is their
identification and characterization. Mass spectrometry combined
with database searches has become the preferred method for
identifying the proteins present in cells or tissues (Ishino et al.,
2007), and has been used also to identify the soil metaproteome
(Schulze et al., 2005; Benndorf et al., 2007). This technique
makes it possible to execute large-scale proteome analyses of
species whose genomes have been sequenced (Ishino et al.,
2007). The problem resides in the fact that many sequences
of soil microorganisms have not been included in databases
and no significant match is found when they are studied. In
this sense, de novo sequencing could be used to assign protein
functions, as has proved possible for complex matrices (Wilmes
& Bond, 2006).
© 2009 The Authors
Journal compilation © 2009 British Society of Soil Science, European Journal of Soil Science, 60, 845–859
Soil metaproteomics: an emerging science
Interaction with
organic matter
and minerals
847
Complexes formation
Organic matter-protein
Free-protein
Protein excretion or release
from cell lysis
Extracellular space
Mineral-protein
Cell walls
Cytoplasm
Non-proliferating cells (microbial
spores, resting vegetable cells,
protozoan cysts)
Intracellular space
Living cells
Periplasmic space
Figure 2 Soil protein sources and interaction with organic matter and minerals.
Proteomics in microorganisms, especially yeasts, and in humans
is a well-developed science (King et al., 2006). Fungal proteomics
has become an active area in recent years (Kim et al., 2007).
Plant proteomics has also developed but has tended to focus on
just five species, although plant scientists are gaining confidence
in this area (Jorrı́n et al., 2006). However, the field of soil
proteomics has only just started and substantial technological and
methodological handicaps remain. Nevertheless, the information
obtained from the soil metaproteome could be of paramount
importance because soil-mediated processes are related strongly
to issues of sustainability. In this sense, the significance of
metaproteomics resides in the identification of proteins and
microbial species involved in soil processes, as indicators of soil
quality and functionality. As in other fields (microbiology and
human proteomics), the initial steps are always difficult, and for
this reason the aim of this work is to provide a review of soil
proteomics by looking at the methodologies used so far, in order
to illustrate the challenges and pitfalls in this field and to provide
a broad perspective of the use of metaproteomics as a tool to
understand better the function and role of soil microorganisms.
Developing extraction methods and analysis of soil
proteins
The development of soil metaproteomics is strongly influenced
by the extraction method (Nannipieri & Smalla, 2006; Ogunseitan, 2006; Benndorf et al ., 2007). The way in which extractions
of soil proteins are performed is of fundamental importance for
their subsequent analysis and this has long been the key to why
proteomics has been restricted to the study of proteins of microorganisms isolated from growth cultures. The main difficulty in
developing extraction methods has been the separation of the
proteins from humic substances. These compounds interfere both
in the colorimetric quantification of proteins (Ogunseitan, 1993;
Criquet et al ., 2002) and in protein analysis by sodium dodecyl
sulphate polyacrylamide gel-electrophoresis (SDS-PAGE). Moreover, humic substances could complicate the separation of peptides obtained by triptic digestion.
Recently, Roberts & Jones (2008) analysed the sensitivity of
different protein quantification kits in the presence of humic
compounds and concluded that all the kits (Bradford reagent,
Coomassie, FluoroProfile® (Sigma-Aldrich, St.Louis, MO, USA),
NanoOrange® (Invitrogen, Paisley, UK) and QuantiPro® (SigmaAldrich)) suffered significant interference from humic substances
in solution. This study partially agrees with results by Whiffen
et al . (2007), who indicated that polyphenolic compounds interfere with the quantification of protein in soil extracts when the
Bradford method is used. These findings suggest that the measurement of protein concentrations in soil extracts is not necessarily
reliable and, indeed, may hinder the steps required for possible protein identification. Roberts & Jones (2008) suggested that
protein quantification should be performed using acid hydrolysis
followed by determination of the amino acids released.
© 2009 The Authors
Journal compilation © 2009 British Society of Soil Science, European Journal of Soil Science, 60, 845–859
848 F. Bastida et al.
Extraction and purification of soil proteins
Any method for extracting proteins should fulfil the following
requirements: (i) achieve the quantitative extraction of proteins
from the soil matrix, (ii) include a step for purifying the proteins
obtained, in order to eliminate molecules that might interfere in
the subsequent analysis and (iii) the extraction conditions should
not alter the protein structure, which would hamper MS analysis.
However, such a method is difficult to achieve for soil. Proteins
can unfold and denature so that their three-dimensional (3-D)
structure is altered but their primary structure remains intact.
Many of the interactions that stabilize the 3-D conformation
of the protein are relatively weak and are sensitive to various
environmental factors, including high temperature, acid or alkaline
pH and a large ionic strength. Proteins vary greatly in their degree
of sensitivity to these factors. The composition of the extraction
buffer is therefore important for maintaining the structure and
function of the target protein. To prevent denaturation, the buffer
pH is based on the pH stability range of the protein. The preferred
working range is from pH 3.0 to 9.5, outside which severe damage
to the protein should be expected because of hydrolysis of the
peptide bond, protein unfolding, cleavage of disulphide bonds
or racemization. It is important to note that an alkaline pH is
commonly used for extraction of humic substances, thus inevitably
causing the enrichment of compounds interfering with protein
analysis. On the other hand, precipitation with acid-pH solutions
may lead to a precipitation of humic acids that could be linked to
proteins. Moreover, the different chemicals may release organic
matter and proteins from different sites in the soil matrix. For
example, extraction with salt solutions will release exchangeablybound proteins while alkaline pyrophosphate or NaOH will also
desorb proteins bound covalently to soil minerals. There is no
doubt that all these factors influence protein extraction.
Unfortunately, there are no published studies that compare the
efficiency, in terms of yield and quality, of different extraction
protocols for soil proteins, taking into account factors such as
pH, buffer or temperature. However, we should consider that pH
is not only important because it influences the protein structure
per se; modification may affect the adsorption of proteins to
minerals, thus affecting the yield of the extraction method.
Protein adsorption to minerals and organic matter is dependent
on pH because proton activity controls both the physicochemical
properties of the organic and inorganic adsorbents and, therefore,
the extent of interaction (Quiquampoix et al ., 1993). As a model
of protein adsorption on clays, Revault et al . (2005) showed that,
at any pH above 4, a conformational change takes place within
10 minutes of contact between recombinant prion protein and
montmorillonite particle surfaces.
Other factors, such as ionic strength, divalent cations (Ca2+
and Mg2+ ) or reducing agents (dithiothreitol, DTT or βmercaptoethanol), may be needed to maintain activity or structure.
In addition, proteins can also be hydrolysed by proteases. Methods used to minimize this enzymatic proteolysis include working
at colder temperatures (4◦ C) and adding chemicals that inhibit
protease activity, such as phenylmethylsulfonyl fluoride (PMSF)
(Jehmlich et al ., 2008). All these aspects should be considered in
a protocol for protein extraction because they may affect the MS
analysis and thus the protein identification.
In the literature, several techniques for protein extraction
are described, although the results showed different extraction
efficiencies and the techniques have not been shown to be suitable
for all soil types (Table 1). Below, we describe the techniques
published for protein extraction from soil. One of the simplest
techniques consists of suspending the soil in a buffered solution.
In this case, 1–100 g of fresh soil is suspended in 1.5 volumes
of a cold solution of 0.3 m K2 HPO4 plus 0.5 volumes of 0.3 m
EDTA solution (Ogunseitan & LeBlanc, 2005) (Table 1). The
suspension is then sonicated in an ice bath to break the soil
aggregates and to lyse the cells, facilitating the release of protein
molecules. The samples are then stirred for 4 hours at 0–4◦ C
before being centrifuged to eliminate soil particles. The proteins
are precipitated by adding a solution of 60% (w/v) (NH4 )2 SO4 ,
adjusting the pH to 7 by adding 1 m K2 HPO4 , before incubating
at 0–4◦ C for 2–6 hours. The precipitated proteins are recovered
by centrifugation at 25 000 g for 25 minutes, at 4◦ C, and resuspended in an appropriate volume of 0.2 m K2 HPO4 at pH 7.
The protein extract can then be dialysed for further purification.
Murase et al . (2003) studied the extracellular proteins extracted
from a glasshouse soil using the following procedure: a soil
sample (100 g) was mixed with 300 ml of 67 mM phosphate
buffer (pH 6) and shaken for 1 hour at 25◦ C. The supernatant was
filtered through a 0.2-μm cellulose acetate membrane to eliminate
bacterial cells. After adding trichloroacetic acid (TCA) to a final
concentration of 5%, the extract was kept at 4◦ C for 12 hours.
Then, the solution was centrifuged at 2100 g for 30 minutes
to remove TCA-soluble compounds. The pellet was submitted
to two cycles of suspension in ethanol and diethylether, with a
centrifugation, in order to purify proteins. This method uses a
phosphate buffer at pH 6. More alkaline pH values extract a large
amount of non-proteinaceous organic matter and thus the extracted
protein may be difficult to separate by SDS-PAGE. Proteins were
collected from glasshouses with total carbon contents ranging
from 1 to 13%. It was possible to identify some of the proteins
extracted by N-terminal amino acid sequencing. One of the most
abundant extracellular proteins was a homologue of thermo-stable
cellulose, produced by a thermophilic fungus. Other soil samples
collected from forests, paddy fields and tea cultures were used to
extract proteins, but no clear bands were found on the SDS-PAGE,
even with silver staining.
Glomalin is a glycoprotein that is produced by the hyphae of
arbuscular mycorrhizal fungi in soil (Wright & Upadhyaya, 1996).
This abundant soil protein might be involved in stabilization
of aggregates. To extract weakly-associated glomalin from soil,
Wright & Upadhyaya (1996) used 20 mm citrate, pH 7, at 121◦ C
for 30 minutes. This is a very energetic method for extraction
of glomalin. A monoclonal antibody was used to assess the
glomalin amount by immunofluorescence and by an enzymelinked immunosorbent assay (ELISA).
© 2009 The Authors
Journal compilation © 2009 British Society of Soil Science, European Journal of Soil Science, 60, 845–859
Precipitation with TCA. Protein
pellet washed with ethanol
and diethyl ether
No protein
purification/concentration
procedure
Protein concentration using
dialysing membrane and PEG
(15–20 kDa)
Protein precipitation with
acetone at 4◦ C
Separation of protein from
humic substances by gel
filtration over Sepharose 4B
20–50 mm sodium citrate (pH 7),
121◦ C, 30 minutes
Different extracting solutions: distilled
water, four salt solutions (CaCl2 ,
KCl, NaCl and Na2 SO4 ) and five
buffers (succinate, bis-TRIS, PO34 – ,
pyrophosphate, citrate) all at pH 6
50 mm TRIS-HCl, 2 mm DTT, 4 mm
EDTA, 0.1% Brij 58 (pH 7.6)
Soil solution extraction using glass
ceramic suction plates
Precipitation with 60% (w/v)
(NH4 )2 SO4 .
0.3 m K2 HPO4 , 0.3 m EDTA (pH 8)
67 mm phosphate buffer (pH 6)
Protein purification
or concentration method
Extracting solution
and conditions
Identification of the biological
origin of the protein extracted
from a complex matrices.
Assignation of the catalytic
function of these proteins.
This procedure was
compatible with a MS
analysis of protein
Optimization of protein
extraction in order to avoid
the co-extraction of humic
substances and other
compounds which can react
with Bradford reagent
A diminution of protein
expression level was detected
in a Cd-contaminated soil
Protein extraction and separation
by SDS-PAGE. The sequence
of some isolated proteins can
be obtained by means of
automatic sequencer
Specific method in order to
extract glomalin. This is an
arbuscular mycorrhizal protein
with an important role in the
formation of soil aggregates
Protein extraction from complex
microbial communities with
emphasis on the preservation
of enzymatic activities
Advantages of procedure
Few protein bands were detected in
SDS-PAGE. No changes of
SDS-PAGE protein profiles were
detected in Cd-contaminated soil
with regard to control soil
Not possible to analyse the
complete proteome of soil. Only
proteins isolated from dissolved
organic matter characterized
The protein was not analysed from
molecular point of view. Humic
substances possibly form part of
the fraction obtained using this
extracting solution. This is an
energetic method of extraction.
Other proteins were not isolated
and analysed
No protein separation by
SDS-PAGE or mass spectrometry
analysis of the proteins was
performed
Ogunseitan & LeBlanc (2005)
Possible interference of humic
substances and other substances
as a result of the poor protein
purification of this method. This
could produce an over-estimation
of protein content using the
Bradford assay. The separation of
extracted proteins by SDS-PAGE
was not tested
Only extracellular proteins of soil
were extracted and analysed in
some samples
Schulze et al . (2005)
Singleton et al . (2003)
Criquet et al . (2002)
Wright & Upadhyaya (2006)
Murase et al . (2003)
Reference
Disadvantages of procedure
Table 1 Methods of protein extraction and purification from soil and their potential application in the study of the soil metaproteome
Soil metaproteomics: an emerging science
849
© 2009 The Authors
Journal compilation © 2009 British Society of Soil Science, European Journal of Soil Science, 60, 845–859
Masciandaro et al . (2008)
Benndorf et al . (2007)
The protein recovery from soil was
small. An incubation or
inoculation of soil was necessary
to obtain a sufficient level of
proteins
Important background on the
SDS-PAGE and no identification
of protein bands by mass
spectrometer
Two-phase extraction (phenol
and water) to separate protein
from humic compounds
PVPP and membrane molecular
cut-off 10 000
0.1 m NaOH, 30 minutes
0.1 m sodium pyrophosphate pH 7.0;
67 mM phosphate buffer (pH 6.0);
0.5 m potassium sulphate (pH 6.6)
Functional analysis of soil
metaproteome was carried out.
It was possible to extract
intra- and extracellular
proteins
Study of humus-enzymes
complexes and intracellular
proteins depending on the
buffer
Protein purification
or concentration method
Extracting solution
and conditions
Table 1 (Continued )
Advantages of procedure
Disadvantages of procedure
Reference
850 F. Bastida et al.
The extraction of reactive glomalin protein from soil is considered to be harsh (involving high temperatures) and would
be expected to quickly and effectively denature albumin beyond
Bradford detection (Rosier et al ., 2006). Thus, we should consider
the effect on the structure of proteins when high temperatures are
applied. The denaturation of some proteins may be a handicap
for their detection by MS. Usually, trypsin is used for protein
hydrolysis, acting on residues of arginine and lysine. For this reason, if damage by high temperatures is occurring in these amino
acid residues, the hydrolysis may be affected and MS analysis
may be constrained. However, the results of Rosier et al . (2006)
suggest that the glomalin extraction process does not denature all
plant-derived sources of protein in soil, so other proteins can be
extracted by high-temperature methods.
Criquet et al . (2002) tried different solutions for protein extraction from an evergreen oak litter: these were distilled water, four
salt solutions (CaCl2 , KCl, NaCl, Na2 SO4 ) and five buffers (succinate, bis-TRIS, phosphate, pyrophosphate and citrate). All were
standardized at pH 6 and 0.1 m. Moreover, they studied several
factors that affected the extraction of proteins and their quantification including powdering of litter, the nature and molarity of the
extraction solvent, the amount of litter used for protein extraction, the concentration methods of the extracts, the addition of
polyvinylpolypyrrolidone (PVPP) and Tween 80 during the extraction, the extraction time, incubation time with the colorimetric
reagent and the colorimetric method used. The results show that
the best extraction method was the addition of 80 g of powdered
litter to 700 ml of a solution containing CaCl2 (0.1 m), Tween 80
(0.1%) and 50 g PVPP. The flask was shaken for 6 hours and,
after centrifugation and filtration, the supernatant was dialysed
against 2 mM bis-TRIS buffer at pH 6. The authors did not use
TCA, for protein precipitation, because the acidification of litter
extracts precipitates humic acids so that protein pellets are brown.
These humic acids interfere with the measurement of protein concentration by the Bradford method. A 10 000-MWCO cellulose
dialysis tube was used as the concentration method. These authors
did not evaluate the compatibility of this protein extraction method
with SDS-PAGE protein isolation or molecular analysis by MS.
It is important to stress the function of cations in protein extraction from soil. Large quantities of proteins were desorbed from
humic acids by CaCl2 , so proteins were not precipitated simultaneously (Criquet et al ., 2002). Ladd & Butler (1975) corroborated
this and indicated that humic acids bind enzymes reversibly by a
cation-exchange mechanism, and that inorganic divalent cations,
in sufficiently large concentrations (from 10−1 to 10−2 m), displace the enzymes from the complexes.
Singleton et al . (2003) used a method based on that described
by Ogunseitan (1993) to analyse the proteins of a soil spiked
with Cd. The method consists of the suspension of 1 g of soil (at
50% WHC) in 1 ml of extraction buffer plus 100 μl of a protease
inhibitor cocktail. The optimized buffer solution contained 50 mM
Tris-HCl, 10% sucrose, 2 mm DTT, 4 mm EDTA and 0.1% Brij
58, with the final solution pH adjusted to 7.58. Addition of
chelating compounds (i.e. EDTA) could avoid the linkage between
© 2009 The Authors
Journal compilation © 2009 British Society of Soil Science, European Journal of Soil Science, 60, 845–859
Soil metaproteomics: an emerging science
proteins and organic matter or minerals, by cationic exchange
(Ladd & Butler, 1975). In order to break down soil aggregates and
facilitate the cellular lysis in a suitable form, two methods were
assayed: (i) a snap-freeze method using liquid nitrogen; and (ii) a
bead-milling method using glass beads. Two methods of protein
concentration were used on the extracts: a volume-reduction
method, through heating, and an acetone-precipitation method.
The snap-freeze method followed by the heating concentration
step yielded 32% more protein when compared with the amount
of protein obtained from the bead beating. After SDS-PAGE
of protein extracts, cleaner gels and significantly more protein
bands were observed using the acetone-precipitation technique.
However, the protein profiles obtained by SDS-PAGE, for soil
protein extracted from control and Cd-contaminated soils and
concentrated by both acetone precipitation and heating, showed
no differences in protein pattern between the treatments.
Schulze et al . (2005) extracted dissolved organic matter using
suction plates installed at different depths and analysed its protein
content. The collected solution was filtered through a 0.2-μm
acetate filter membrane prior to freeze-drying, and thus the
samples were sterilized and only extracellular proteins were
analysed. Proteins associated with mineral particles were extracted
by the following method: the particulate organic matter was
removed from samples by heavy liquid flotation with sodium
polytungstate. After centrifugation, the supernatants were removed
and the pellets washed with deionized water in order to eliminate
salts. Proteins were removed from the inorganic material by
dissolving the latter in 10% HF. After neutralization, proteins
were separated from organic molecules by gel filtration and
subjected to SDS-PAGE. After silver staining, the gel was
cut into slices and proteins were digested using trypsin. The
mixtures of tryptic peptides obtained were separated by nanoflowliquid chromatography (LC) prior to analysis by tandem mass
spectrometry (MS/MS). The authors were able to identify the type,
functions and biological origin of the extracellular proteins and a
reduction in the number of identified proteins was observed with
soil depth in a deciduous forest.
More recently, Benndorf et al . (2007) proposed a new protein
extraction method for functional analysis of the soil and derivation
of subterranean-water metaproteomes. They developed an extraction method in which the separation of proteins from inorganic and
organic constituents of the soil matrix was achieved by a combination of 0.1 m NaOH treatment and phenol extraction. The NaOH
solution released humic substances and proteins from the soil
mineral fraction and lysed microorganism cells. The subsequent
phenolic extraction separated proteins from humic substances. Precipitation of proteins was performed by sequential washing steps
with 0.1 m ammonium acetate in methanol, 80% acetone and 70%
ethanol. Proteins in the extracts were separated by SDS-PAGE
and 2D-electrophoresis, and individual proteins were analysed by
LC coupled with mass spectrometry via electrospray ionization
(ESI-MS). To assess the suitability of the extraction method for
the functional soil metaproteome, it was applied to soil that had
been incubated with 2,4-dichlorophenoxy acetic acid (2,4-D) and
851
inoculated with 2,4-D-degrading bacteria. Thus, it was possible
to introduce abundant target proteins for metaproteome analysis.
Some of these proteins were identified as chlorocatechol dioxygenases, consistent with the bacterial pathways expected to be
expressed in these soil samples.
Although some authors have not used any precipitation method
(Criquet et al ., 2002), Carpentier et al . (2005) showed that precipitation is absolutely necessary when dealing with recalcitrant
plant tissues. In plants, two major precipitation methods have been
assessed: one involves TCA-acetone and the other phenol extraction–methanol–ammonium acetate precipitation. Phenol extraction is most efficient in removing substances that interfere with
the extraction of proteins from plants and results in the best quality
gels. There is a small but significant difference between the protein patterns of the TCA–acetone precipitation and the phenol
extraction–methanol–ammonium acetate precipitation (Carpentier et al ., 2005). Possible explanations for the method-specific
loss are the following: differential inactivation of proteolytic and
other modifying enzymes, partitioning of proteins to the aqueous phase (in the case of phenol-based methods) and a different
precipitation and dissolving capacity.
With regard to the types of proteins precipitated by each
method, Carpentier et al . (2005) showed a tendency for large
proteins to be lost during TCA precipitation and observed an
enrichment of small proteins (below 25 kDa) compared with phenol extraction. This could be explained by a different degree
of proteolytic breakdown. However, this is unlikely because the
TCA-acetone-precipitated proteases are supposed to be inactivated
irreversibly (Wu & Wang, 1984). The major problem with TCA
precipitation is re-solubilization of the precipitated proteins (Maldonado et al ., 2008). This is particularly true for proteins of
relatively large molecular weight, thus promoting the presence
of proteins below 25 kDa rather than larger ones when dissolving
protein pellets from TCA precipitation. However, while these considerations are valid for some plant tissues, there is no information
about precipitation protocols for soil proteins and research comparing soil protein extraction and purification methods is needed.
Masciandaro et al . (2008) tested a method that used three different extractants, 0.1 m pyrophosphate buffer (pH 7), 67 mM
phosphate buffer (pH 6) and 0.5 m potassium sulphate, to extract
extracellular proteins and intracellular proteins, respectively, from
soil. This method was based on three previously-reported methods:
humic substances extraction with pyrophosphate at neutral pH,
extraction of microbial biomass C with potassium sulphate and
the Murase method for extraction of extracellular proteins from
soil using a phosphate buffer. Humic substances were removed
by addition of PVPP. Masciandaro et al . (2008) used two different forest soils with 5.4 and 3.8% TOC, respectively. After
purification of the extract with PVPP, a larger concentration
of extracellular protein was detected in one of the soils, using
potassium sulphate as extractant, while in the other soil similar concentrations of extracellular protein were extracted using
the three extractants. All soil extracts were prepared for loading in SDS-PAGE gels in order to obtain protein profiles and
© 2009 The Authors
Journal compilation © 2009 British Society of Soil Science, European Journal of Soil Science, 60, 845–859
852 F. Bastida et al.
determine the composition of the most abundant proteins in the
two soils. Only a few well-defined protein bands (around 20 kDa)
were observed after performing SDS-PAGE and silver staining.
A dark background was observed in the gels, which complicated
the identification of more well-defined protein bands. This may
have resulted from interfering compounds co-extracted with the
proteins.
Methods for separating proteins extracted from soil
Improvements in protein separation technology have played an
important role in the development of proteomics. Before the
introduction of SDS-PAGE, in both one and two dimensions,
few tools were available for separating proteins (Graves et al .,
2002). The availability of SDS-PAGE and the development of MS
have permitted the metaproteome to be studied in such complex
ecosystems as soils, in which several microbial communities
interact and have diverse functions. The objective of these
techniques is to separate proteins from complex mixtures and
acquire information on their expression profile.
One-dimensional electrophoresis
Polyacrylamide gel electrophoresis (PAGE) enables proteins in
more or less complex mixtures to be separated, purified and
analysed. One-dimensional electrophoresis in polyacrylamide and
SDS (a denaturing detergent), known as SDS-PAGE, is used
widely to separate mixtures of proteins extracted from the
environment, assigning them an approximate molecular mass
(Ogunseitan, 1993). In one-dimensional electrophoresis (1-DE),
proteins are separated according to their molecular mass. The
SDS detergent is added to denature the protein molecules,
which can then be separated more effectively. One-dimensional
electrophoresis in native or non-denaturing conditions (NDPAGE) also exists as a technique for isolating enzymes or native
proteins, using specific reagents that colour the separate bands in
the gel. In this case, denaturing agents are not added during the
separation process.
Two-dimensional electrophoresis
The limited power of 1-DE led to two-dimensional electrophoresis
(2-DE) being used because this permits more complex and less
purified protein mixtures to be separated. With this technique,
proteins are separated according to two different properties:
their iso-electric point (pI ) in the first dimension (iso-electric
focusing, IEF) and their molecular weight (SDS-PAGE) in the
second dimension (Michalski & Shiell, 1999). One of the weak
points of this separation technique is the poor resolution of
extremely hydrophobic membrane proteins in the pH gradient.
Moreover, significant portions of the proteome, especially lowabundance proteins, are rarely seen in 2-DE studies (Washburn
& Yates, 2000). However, one of the main reasons for using 2DE is its capacity to resolve proteins that have suffered some
degree of post-translational modification (Graves et al ., 2002).
However, the most widely used application of 2-DE is the study
of the expression profile of proteins, comparing the profiles of
two different samples both qualitatively and quantitatively. The
appearance or disappearance of spots provides information on
differences in the expression of proteins, while the intensity
of these spots gives quantitative information about the level
of protein expression (Wilmes & Bond, 2004, 2006). Imageanalysis software has helped in the understanding of the complex
images resulting from 2-DE. The problems associated with 2-DE
were solved by the introduction of differential gel electrophoresis
(DIGE), which provides a significantly more efficient, sensitive
and reliable way to detect differences in the levels of expression
of proteins between control and treated samples. This technique
uses mass- and charge-matched, fluorescent cyanide dyes to label
proteins prior to 2-DE (Nesatyy & Suter, 2007).
Techniques for visualising proteins
To visualize the bands (1-DE) or spots (2-DE) of proteins, it is
necessary to use a staining or marking method. Three methods are
used most commonly: staining with Coomassie reagents, silver
staining and fluorescent labelling (see a review by Patton, 2000).
Coomassie Brilliant-Blue (CBB) has the advantage of being very
specific when reacting with proteins; this eliminates the problem
of interference, which occurs with silver staining. However, the
method is not as sensitive as silver staining because the detection
limit is approximately 8–10 ng of protein, silver staining being
100-times more sensitive (Switzer et al ., 1979). Silver staining
is a multi-step process utilizing numerous reagents, the quality of
which is critical. Silver stain is very sensitive because of the strong
autocatalytic character of silver reduction (Silva et al ., 2004).
Silva et al . (2004) reported two silver staining methods, one of
which is compatible with subsequent in-gel digestion, matrixassisted laser desorption/ionization (MALDI), and electrospray
spectrometry ionization (ESI) analysis. Depending on the reagents
used for CBB staining, the method may be compatible with MS,
permitting subsequent molecular analysis of the separated proteins. Silver staining is more laborious and requires numerous
changes of solution and different steps in which the incubation
times must be strictly respected, and so CBB is more practical
when numerous gels need to be processed. A weak point of silver
staining is that interfering compounds (polyphenols and humic
substances, among others) hinder the detection of clear bands
or spots because of the dark background (Criquet et al ., 2002).
Despite the good sensitivity of silver staining procedures (low
nanogram range), they are cumbersome, multi-step processes that
exhibit a poor linear dynamic response. In addition, compatibility
with MS requires omission of glutaraldehyde from silver stain formulations, which compromises staining performance (Shevchenko
et al ., 1996). However, fluorescence detection technologies offer
greater sensitivity (1 ng–1 μg of protein), analysis by MS and
broader linear dynamic responses compared with their colorimetric counterparts (Patton, 2000).
© 2009 The Authors
Journal compilation © 2009 British Society of Soil Science, European Journal of Soil Science, 60, 845–859
Soil metaproteomics: an emerging science
Alternatives to electrophoresis
One of the alternative methods to gel electrophoresis for separating and purifying proteins is liquid chromatography (LC) (Link
et al ., 1999; Graves & Haystead, 2002; Schulze et al ., 2005).
Generally, the mixture of proteins is converted into a mixture of
peptides of smaller molecular weight by digestion with trypsin,
prior to separation by LC. The advantage of this method is that it
avoids 2-DE and so a greater number of proteins can be separated
from complex mixtures. Benndorf et al . (2007) used nano-HPLC
coupled to ESI-MS for identification of proteins from a soil percolated with 2,4-D, with or without inoculation of a mixture of
2,4-D-degradaing bacteria (Cupriavidus necator JM134, Rhodoferax sp. P230, and Sphingomonas herbicidovorans B488).
Protein identification: mass spectrometry
MS provides information on the protein molecule, such as the
mass of the peptide obtained from the protein molecule and its
amino acid sequence. With this information, the original protein
can be identified by a database search. There are two strategies
for separating and identifying proteins from complex mixtures
(Figure 3). The proteome can be investigated either by ‘top-down’
analysis of the intact proteins or by ‘bottom-up’ analysis of the
peptides generated by the trypsin digestion of proteins (Nesatyy &
Suter, 2007). For ‘bottom-up’ proteomics, the proteins of interest
are digested and the resulting peptides are analysed by MS.
Tandem MS (MS/MS) spectra of the peptides provide information
on their amino acid sequences and possible post-translational
modifications (PTM). This approach begins with the separation
of a complex protein mixture by 1- or 2-DE: protein bands or
spots are excised from the gel, subjected to proteolytic digestion
and analysed by MS. Generally, the peptide mixture obtained
from the digestion of 1-DE bands undergoes a separation step
using reversed-phase liquid chromatography (RP). The ‘bottomup’ approach is particularly suited to protein identification, which
is achieved either from mass fingerprinting profiles of the peptides,
derived from the digestion of the protein by specific proteases,
or from sequences of these peptides obtained by MS/MS and a
subsequent database search.
In the ‘bottom-up’ approach, the alternative to gel electrophoresis separation is multi-dimensional separation by LC. After the
digestion of proteins, the resulting mixture of peptides is separated by strong cation exchange (SCX) chromatography in the
first dimension, followed by reversed-phase chromatography in the
second. However, protein identification using ‘bottom-up’ analysis generally suffers from incomplete protein sequence coverage
and the loss of information on PTMs, or degradation, as a result of
proteolytic digestion. In the ‘top-down’ method (Figure 3), intact
protein ions are detected, fragmented and analysed in the mass
spectrometer, yielding both the molecular weight of the intact protein and protein fragmentation spectra. This allows the complete
primary structure of the protein and all of its PTMs to be deduced.
Both strategies for analysing the proteome rely on bioinformatics
853
tools to match experimentally-produced MS data with protein and
genome sequence databases.
Generally, there are two steps in analysing proteins by MS as
follows.
1 Ionization of the sample. To analyse peptides by MS, it is
necessary for the molecules to be dry and charged; that is,
they should be in the form of desolvated ions. The most
common methods for ionization are ESI and MALDI. In
both methods, the peptides are converted into ions by the
addition or elimination of one or more protons. Normally, the
ESI apparatus is connected in-line with a chromatograph and
peptides are automatically separated and purified before being
injected in the form of ions into the mass spectrometer (Link
et al ., 1999). In the MALDI system, the sample is incorporated
in a matrix and then exposed to laser radiation, which leads
to the formation of molecular ions. The MALDI ionization
system can be automated and ionization can be carried out
directly, without purification, which represents an advantage
over ESI.
2 Mass analysis. After ionization of the peptide molecules, their
mass is analysed by a mass analyser, which separates the
molecular ions in a vacuum chamber according to their charge
and mass. Two types of mass analysers are common, the
quadrupole and the time of flight (TOF). In the first, the ions
are conducted through an electric field created by a system of
four parallel metal rods (quadrupoles). The quadrupole can act
as a mass filter that permits ions with a given m/z ratio to be
transmitted. In the second type, m/z ratio of an ion and the
necessary time to pass through a flight tube are determined.
Perspectives for soil metaproteomics
Soil is a heterogeneous matrix in which a multitude of microorganisms are exposed to a variety of natural conditions and human
actions. The dynamic of the soil’s microbial populations and their
susceptibility to different external factors will determine the composition of the metaproteome of a community, which is both
time- and place-dependent. In other words, the composition of
the proteins that will be encountered will depend strongly on the
factors that influence microbial life and dynamics. This means
that the ‘richness’ and diversity of the ‘protein community’ are
relatively unknown. Nevertheless, we should stress that currently
soil metaproteomics is an area under development and that much
more effort needs to be devoted to protein extraction and genome
sequencing.
It is generally considered that some 40% of the total N of the
soil’s organic matter is protein-like material (proteins, peptides
and amino acids) (Kögel-Knabner, 2006). According to Nannipieri
& Smalla (2006), it is surprising that only approximately 4%
of total N in the soil is found in the microbial biomass (MBN,
microbial biomass N), while a large proportion (30–45%) is
present as amino acids after acid hydrolysis. This suggests
© 2009 The Authors
Journal compilation © 2009 British Society of Soil Science, European Journal of Soil Science, 60, 845–859
854 F. Bastida et al.
Figure 3 Basic scheme for protein identification by mass spectrometry.
that the quantity of extracellular protein-like nitrogen (proteins,
peptides or amino acids) is considerably larger than its intracellular
counterpart. Among the reasons for this, is that the extracellular
proteins result not only from root exudates or excretion by
microorganisms, but are also protected in the soil. Knicker (2004)
showed that the survival of peptide-like structures is a ubiquitous
phenomenon. In fact, the potential involvement of the fine silt and
clay fraction in protecting peptides from microbial and chemical
degradation has been shown recently (Knicker et al ., 2000). In
addition, the stability of proteins and their durability in soil can
be enhanced further by interactions with other soil molecules,
specifically polyphenolics and carbohydrates (Rillig et al ., 2007).
Knicker & Hatcher (1997) suggested that proteins might be
provided with long-term protection by their incorporation into
hydrophobic domains of soil organic matter and it has been
suggested that covalent coupling occurs as well as humic-protein
linkage (Hsu & Hatcher, 2005). In this respect, the organic
matter (more specifically, humic substances) stabilizes a large
quantity of proteins but, at the same time, interferes with their
extraction and separation. We next describe the significance of
soil metaproteomics in the future, although it must be emphasized
that the science is in its infancy and its growth will depend largely
on the development of a suitable methodology and genomic
database.
Indicators of soil degradation and biogeochemical
potential
There is no doubt that human actions influence greatly the
dynamic of microbial populations and their activity in the soil
(Gianfreda & Bollag, 1994; Cardelli et al ., 2004; Mertens et al .,
2006; Carson et al ., 2007; Bastida et al ., 2008a; Dell’Amico
et al ., 2008; Yevdokimov et al ., 2008). Direct activities such as
ploughing, adding fertilizers and contamination affect microbial
life in soil (Kandeler et al ., 2006). The study of soil metaproteomics may be related closely to the evaluation of the effect that
certain degradative processes have on microbial functionality, for
example which proteins are ‘lost’ in the soil through desertification, hydrocarbon contamination or the presence of metals and
other compounds? The answers to this will tell us more about
which proteins and microorganisms are most involved in each of
these degradative phenomena compared with the situation in a
control soil.
In recent decades, soil enzymology has become a serious subject
of study. A large number of enzymes have been shown to act as
indicators of soil quality and/or degradation, because they are sensitive to amendments, climatic conditions and contamination by
heavy metals and other chemical agents (Nannipieri et al ., 1990;
Moreno et al ., 2003; Ros et al ., 2003; Bastida et al ., 2008a).
However, the measurement of a soil’s enzymatic activity is not
© 2009 The Authors
Journal compilation © 2009 British Society of Soil Science, European Journal of Soil Science, 60, 845–859
Soil metaproteomics: an emerging science
necessarily a measure of its actual state. In the laboratory, experiments are carried out under controlled conditions, using optimal
techniques (stirring, etc.) and in the presence of substrates that are
difficult to find in natural conditions. In most cases, the measured
enzymatic activity of a soil is a potential measurement rather than
a real one reflecting the state of the microbial communities found
therein. Among the enzymes that have traditionally been used to
evaluate soil microbiological activity are ureases, proteases, glucosidases, phosphatases and xylanases, all of which are related
to soil elemental cycles. These and others represent the catalytic
potential of an ecosystem, and their identification may be useful
for establishing the nexus of the relationship between the presence
of certain species or microbial taxa and the soil’s biogeochemistry,
so helping us to understand the dynamic of the elements in the
soil.
By studying the soil metaproteome, we could identify which
enzymes are affected by changes in soil conditions, because the
variations that occur at population level and in the expression of
proteins are specific to the perturbation (Maron et al ., 2007). It has
been demonstrated that anthropogenic actions, such as heavy metal
contamination, seriously affect the concentrations of proteins in
the soil (Singleton et al ., 2003). Therefore, the identification of
proteins that show some variation in the face of such a process
may be of basic importance for studying the functionality of these
soils; in other words, it will be possible to identify proteins that
act as indicators of soil quality or degradation processes.
As more genome and environmental sequencing projects are
undertaken, the application of functional environmental ‘omic’
approaches is becoming feasible (Wilmes & Bond, 2004). At
the microbial ecology level, this is of fundamental importance
because the identification of proteins may be useful for identifying
both enzymes and microorganisms that are affected by certain
pressures, and even which populations supplant their function,
because a certain degree of redundancy of microbial populations
in complex ecosystems has been demonstrated (Benndorf et al .,
2007). The identification of proteins and microorganisms resistant
to degradation phenomena could open up new fields in these
studies. If this is possible, we could then aim at designing
‘made to measure’ soil recovery programmes, using particular
microbial populations, that would come to the aid of the affected
microorganisms, or, at a more specific level, using enzymes
involved in the degradation of products considered harmful to
the soil’s functionality.
In recent years, soil metaproteomics has become a tool for
the functional study of soil microbial communities, although
much attention to the methodological aspects is needed to
improve this area. Benndorf et al . (2007) used metaproteomics
to attribute metabolic functions to members of the edaphic
microbial community, establishing a structure-function link in
soils contaminated with 2,4-D. Schulze et al . (2005) detected
a large number of proteins in forest soils, which permitted
conjecture on the structure of the microbial communities of these
soils and a study of the seasonal variation of their proteins.
Cellulases and laccases were detected by Schulze et al . (2005) in
855
soil particles, indicating that these enzymes, once produced, were
adsorbed to mineral surfaces and thus immobilized. In addition,
the contribution of different types of organisms to the protein
pool varies between ecosystems and with soil depth and season.
Schulze et al . (2005) observed a wide variety of extracelullar
enzymes related to organic matter dynamics. However, we should
consider that the differing extraction of humic substances in
different soils and even at different depths may affect protein
extraction, as stated earlier in this work.
If we bear in mind that 1 g of soil may contain up to
10 billion bacteria, made up of 4000–7000 species (Dubey et al .,
2006), it is logical to assume that there will be an even larger
number of proteins. Until now, however, one of the problems
of soil metaproteomics has been the scant number of proteins
detected, which can be attributed to extraction problems or
to the problems associated with their identification by mass
spectrometry, especially with the limited genome database relative
to the extent of microbial diversity in soil. Quince et al . (2008)
suggested that the rates of recovery of new microbial taxa in large
samples indicate that many more taxa remain to be discovered in
soils. Benndorf et al . (2007) detected a small number of proteins
using gel electrophoresis (SDS-PAGE) in a soil contaminated by
2,4-D and all were of an intracellular nature. The heterogeneous
nature of extracellular proteins (Nannipieri, 2006) may hinder
their identification, and only those present in large amounts may
have detectable concentrations. Benndorf et al . (2007) designed
a target experiment by adding 2, 4-D to a soil and incubating it,
the result of which was that enzymes related to this compound’s
metabolism were induced. However, Schulze et al . (2005) found
proteins in natural soils, some of which were extracellular. Such
results point to several possibilities: first of all, the soil type may
play a crucial role in the quantity and type of enzymes present;
secondly, the extraction, separation and spectrometric methods
used may influence extraction and identification; and thirdly, the
metaproteomics of a soil may be governed by specific pressures
(for example from a contaminant, heavy metal, etc.).
Is metaproteomics capable of resolving the overall map of
proteins in a soil? The problem is that, unlike DNA and RNA, no
methods exist for ‘amplifying’ proteins. The proteins may occur
in a wide variety of concentrations, depending on the number and
type of organisms. In addition, dominant proteins, such as those
involved in DNA and RNA processes, would camouflage those
present at smaller concentrations (Nesatyy & Suter, 2007).
However, the identification of extracellular proteins, besides
being of fundamental importance because many of them are
responsible for the cycling of elements (Schloter et al ., 2003)
or for decontamination, may provide misleading information as
regards the microbial ecology of a soil at a given moment. Many
enzymes will be associated with organic matter and minerals
(Benı́tez et al ., 2005; Bastida et al ., 2008b), so that they may
remain active in a soil even in the absence of the producing
microorganism. So, although we may be able to assign a taxon
to a given protein, this may not necessarily mean that this
microorganism is alive in the soil at a given moment. Therefore,
© 2009 The Authors
Journal compilation © 2009 British Society of Soil Science, European Journal of Soil Science, 60, 845–859
856 F. Bastida et al.
in the identification of microorganisms by proteomics, it may be
more useful to identify intracellular proteins.
Soil remediation and restoration: opening the ‘black
box’ of added-waste proteomics
The development of metaproteomics for a heterogeneous matrix
such as soil leads us to consider the application of this technique to
other substrates such as organic wastes. The application of such
wastes to soil, as organic amendments, has become a common
practice for improving the biological quality of degraded soils in
areas with little organic matter input (Ros et al ., 2003; Bastida
et al ., 2008a), for improving the fertility of agricultural soils and
for remediating heavy metal contamination (Moreno et al ., 1999;
Renella et al ., 2005). The application of organic matter in the form
of sewage sludge, compost, municipal waste, plant materials, etc.,
is well documented, although the agents responsible for change
(enzymes) have been studied less. The characterization of the
metaproteome of these materials may be an important step for
using specific amendments to remedy the deficiency of specific
proteins in degraded soils. Identification of the proteins that are
lacking in these soils, compared with natural soils, may become a
basic tool for improving their biogeochemical status. However, we
have discussed previously the possibility that some soil proteins
exist in non-detectable amounts; the non-identification of a protein
does not necessarily show its absence. The question would then
arise as to whether organic materials exist that could provide these
specific proteins in sufficient amounts?
The main handicap in identifying the metaproteomes of these
wastes is their large organic matter content sbecause humic substances and other organic materials hinder electrophoresis and the
subsequent identification and purification of proteins (Roberts &
Jones, 2008). However, there are some studies in which the metaproteome of such organic materials has been studied (Wilmes & Bond,
2006), which encourages the study of these materials with a view to
the controlled improvement of soils or even for other purposes.
Recently, soil bacterial communities have been analysed for
their potential as a bioresource (Dubey et al ., 2006). Soil is a
great store of microbial biodiversity and the microorganisms that
live in it have been used in industry, environmental projects
and for agricultural and even therapeutic reasons. However, such
applications have been limited for a long time because the culture
of many microorganisms in the laboratory has not been possible
(Torsvik & Øvreås, 2002). Molecular methods were developed
to overcome such problems (Dubey et al ., 2006) and others
related to the scarcity of information that morphological analysis
provides. Analysis of the metagenome has pursued two overall
objectives: to characterize the function of microorganisms in
soil (Steele & Streit, 2005) and to discover genes or transcripts
involved in functions of biotechnological or industrial value.
For example, it has been demonstrated that the soil is a source
of microorganisms that are capable of carrying out different
bio-transformations (Nakamura et al ., 2004). Traditionally, the
potential use of bacteria with a catalytic capacity has been studied
by developing cultures in vitro, although in recent years genomics
has permitted these processes to be studied at greater intensity
by identifying microorganisms or genes (by microarray, FISH,
DGGE, etc.) and quantifying genes or transcripts (by qPCR or
RT-PCR). Such approaches have revealed the microorganisms
responsible for biogeochemical processes and have enabled the
characterization of the metabolic pathways responsible for these
processes (Zak et al ., 2006).
However, if methods are improved and developed, metaproteomics can help to directly identify enzymes in soils or wastes
without resorting to other molecular techniques. Metaproteomics
can help find the microorganisms and enzymes responsible for a
given process without the need for previous metagenomic studies,
although we should not forget that such studies have permitted
the compilation of the databases on which protein identification
is based. Indeed, such studies may be more valuable in certain
organic wastes, which practically act as microbial culture media
and in which certain microbial species predominate, while some
of these enzymes may have a biotechnological, industrial, environmental and/or pharmaceutical value (Schmeisser et al ., 2007).
The future: stable isotope probes linking proteins
(SIP-proteins)
The information that could be provided by metaproteomics is
complete in the sense that scientists can identify which proteins
and enzymes are to be found in an ideal system and their possible
origin. Theoretically, it may also be possible to know what is
or might be the function of a given enzyme, although first of all
methodological problems should be solved, as stated above. There
are more questions, for example which protein is directly involved
in the degradation of a given contaminant? Which microorganisms
and enzymes are fundamental for the degradation of the soil’s
organic matter?
To detect functional relationships within microbial communities, substrates labelled with 13 C or 15 N can be used. Microorganisms capable of developing in the presence of these compounds,
and/or of metabolizing them, incorporate this carbon and nitrogen
into their biomolecules, whether nucleic acids (DNA or RNA)
(Radajewski et al ., 2000) or proteins. This can be used to identify
the microbial populations really involved in a given process.
By means of DNA/RNA-SIP (stable isotope probes) studies,
it has been possible to detect and quantify the microorganisms
(Friedrich, 2006; Whiteley et al ., 2006) involved in the degradation and assimilation of certain compounds such as alkanes or benzenes (Nijenhuis et al ., 2007). Furthermore, studies of SIP nucleic
acids have been carried out not only in microbial cultures but also
in soils (Bernard et al ., 2007), providing important information
regarding the function of microorganisms in the soil and their
possible involvement in degradation and recovery phenomena.
However, at even greater resolution, the SIP-Protein techniques provide direct information concerning which microbes are
involved in a given function. The approach has been used for
microbial cultures (Jehmlich et al ., 2008), in which it was demonstrated that the incorporation of 13 C into proteins exceeded 94%,
© 2009 The Authors
Journal compilation © 2009 British Society of Soil Science, European Journal of Soil Science, 60, 845–859
Soil metaproteomics: an emerging science
which is greater than that observed for nucleic acids, thus helping identification of the microbial function. Therefore, SIP-Protein
techniques are a promising future alternative for firmly identifying
the enzymes or microorganisms responsible for biochemical processes in the soil community, once methodological problems of
protein extraction and purification are solved and database information is complete.
Conclusion
The development of different molecular methodologies has permitted the identification and quantification of many genes in soils
(genomics) and even of RNA transcripts derived from many of
these genes (transcriptomics). However, the identification of all
the proteins in soil (metaproteomics) is a science in its infancy,
although some workers have already proposed detailed extraction, separation and identification protocols for proteins. Thus,
soil proteomics could provide a strong support for the study of the
structure and function of microbial communities in soil, together
with DNA and RNA methodologies. However, such methods are
not standardized and there are no commercial protein extraction
kits as there are for DNA and RNA. For this reason, with this
review we promote the use of soil proteomics, but emphasize the
need for improvement of the methods so that the great potential for explaining the structure and function of soil microbial
communities can be achieved.
There are three main drawbacks in soil metaproteomics. In the
first place, soil is a poor source of proteins, at least compared with
microbial cultures, and, unlike in the case of DNA or RNA, no
techniques such as PCR exist to ‘replicate’ proteins that do exist.
Second, many proteins are closely associated with compounds
that interfere with their identification, such as those of humic
origin. Third, the genome database for protein matches for soil
microorganisms is not complete, so more effort should be made
to develop this in genomic projects.
Using the few methods that have been established to date for
extracting proteins from soil, we should be capable of perfecting
protocols and standardizing them for different types of soils. Thus,
the separation of peptides from proteins, either free or bound to
humic material or minerals, could be a strategy to follow for
separating and quantifying these proteins. We should stress that
at present, because of methodological and database gaps and the
small amount of some proteins in soil, proteomics is not able to
give us a general and accurate picture of soil proteins.
In short, if future developments permit standardization, then
soil metaproteomics could be used very specifically, because
the presence or absence of different proteins would act as an
indicator of soil processes and provide some answers to the
following questions. Which proteins are responsible for degrading
a given soil contaminant and which microorganisms produce
them? Which protein profile seems to be most involved in soil
degradation or desertification processes? Which microorganisms
are capable of living in soil and developing a specific function?
This may be the base for identifying specific indicators of soil
857
quality, and providing information concerning which enzymes
or microorganisms may be involved in the improvement and
recuperation of a non-renewable natural resource, the soil.
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