Exploring Mixed Microbial Community Functioning Recent Advances in Metaproteomics

MINIREVIEW
Exploring mixed microbial community functioning: recent

advances in metaproteomics
Alma Siggins1, Eoin Gunnigle1 & Florence Abram2
1
Microbial Ecology Laboratory, Department of Microbiology and Ryan Institute, National University of Ireland, Galway (NUI, Galway), Galway,
Ireland; and 2Functional Environmental Microbiology, Department of Microbiology, National University of Ireland, Galway (NUI, Galway), Galway,
Ireland
Correspondence: Florence Abram, Abstract

Functional Environmental Microbiology,
Department of Microbiology, National System approaches to elucidate ecosystem functioning constitute an emerging
University of Ireland, Galway (NUI, Galway), area of research within microbial ecology. Such approaches aim at investigating
University Road, Galway, Ireland. Tel.: all levels of biological information (DNA, RNA, proteins and metabolites) to
+353 0 91 492390; fax: +353 0 91 494598; capture the functional interactions occurring in a given ecosystem and track
e-mail: florence.abram@nuigalway.ie
down characteristics that could not be accessed by the study of isolated com-
ponents. In this context, the study of the proteins collectively expressed by all
Received 16 May 2011; revised 7 October
2011; accepted 13 December 2011.
the microorganisms present within an ecosystem (metaproteomics) is not only
crucial but can also provide insights into microbial functionality. Overall, the
DOI: 10.1111/j.1574-6941.2011.01284.x success of metaproteomics is closely linked to metagenomics, and with the
exponential increase in the availability of metagenome sequences, this field of
Editor: Ian Head research is starting to experience generation of an overwhelming amount of
data, which requires systematic analysis. Metaproteomics has been employed in
Keywords very diverse environments, and this review discusses the recent advances
environmental proteomics; human gut
achieved in the context of human biology, soil, marine and freshwater environ-
MICROBIOLOGY ECOLOGY
microbiota; marine and freshwater

ments as well as natural and bioengineered systems.
environment; soil; bioengineered systems.
from its natural environment might not necessarily dis-

Introduction
play the same characteristics under laboratory conditions
Microorganisms occupy virtually every habitat on our as it does within its ecological niche. Therefore, the study
planet, and their activities largely determine the environ- of mixed microbial communities within their natural
mental conditions of todays world. Indeed, microorgan- environment is key to the investigation of the diverse
isms are heavily involved in biogeochemistry, ensuring roles played by microorganisms, and to the identification
the recycling of elements such as carbon and nitrogen of the microbial potential for biotechnological applica-
(Madsen, 2011). In addition, microorganisms are exten- tion, including but not limited to: pharmaceutical, diag-
sively used to degrade anthropogenic waste prior to nostics, waste treatment, bioremediation and renewable
release into the environment (Hussain et al., 2010; Park energy generation. An emerging field of research in
et al., 2011). In their natural habitat, microorganisms microbial ecology encompasses system approaches
coexist in mixed communities, the complexity of which is (Fig. 1), whereby all levels of biological information are
specific to each environment, for example from six esti- investigated (DNA, RNA, proteins and metabolites) to
mated individual taxa for an acid mine drainage biofilm capture the functional interactions occurring in a given
(Ram et al., 2005), up to 106 estimated taxa per gram of ecosystem and identify characteristics that could not be
soil (Wilmes & Bond, 2006). As most of the microorgan- accessed by the study of isolated components (Raes &
isms present in the environment have not been cultured, Bork, 2008; Roling et al., 2010). Recent technological
their investigation requires the use of molecular tech- advances, including the development of high-throughput
niques that bypass the traditional isolation and cultivation omics methods, make such system approaches possi-
of individual species (Amann et al., 1995). Moreover, ble, where mixed microbial communities are viewed as
even when isolation is possible, a single species removed one meta-organism. Metagenomics, metatranscriptomics,
FEMS Microbiol Ecol && (2011) 116 2011 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
2 A. Siggins et al.
metaproteomics and metametabolomics are employed to intestinal tract (Verberkmoes et al., 2009a; Rooijers et al.,
determine respectively the DNA sequences of the meta- 2011), human oral cavity (Rudney et al., 2010), animal
organism under study, the collectively transcribed RNA, guts (Toyoda et al., 2009; Burnum et al., 2011) and natu-
the translated proteins and the metabolites resulting from ral and bioengineered systems (Ram et al., 2005; Wilmes
cellular processes. All of the generated data can then be et al., 2008a; Jehmlich et al., 2010). Typically, metaprote-
used to identify the metabolic pathways and cellular pro- omic approaches involve up to seven main steps (Fig. 2),
cesses at work within an ecosystem. Yet another level of namely sample collection, recovery of the targeted frac-
information is required to access the molecular interaction, protein extraction, protein separation and/or frac-
tions occurring within the ecological niche under investi- tionation, mass spectrometry analysis, databases searches
gation, and this is achieved by the application of and finally data interpretation, whereby the expressed
metainteractomics (Fig. 1; Lievens et al., 2010; Medina & proteins and pathways identified are used to access infor-
Sachs, 2010; Janga et al., 2011). Ultimately, system mation about system functioning (for detailed descrip-
approaches aim to develop mathematical models that can tions of the methodologies involved, see Wilmes & Bond,
be used to predict the behaviour of a biological system in 2006 and Verberkmoes et al., 2009b). Each environment
response to environmental stimuli (Fig. 1; Raes & Bork, offers specific challenges and limitations within this work-
2008; Roling et al., 2010). flow. Typically, sample collection and recovery of the tar-
Metaproteomics, which is the identification of all the geted fraction (Fig. 2) can be problematic in the marine
proteins expressed at a given time within an ecosystem and freshwater context, where microorganisms can be
(as defined by Wilmes & Bond, 2004), is an indispensable recovered from hundreds of litres of water away from
element of system approaches and plays a key role in the laboratory facilities. The protein extraction step (Fig. 2)
determination of microbial functionality. Microbial meta- has proven specifically difficult when dealing with soil
proteomics has been applied in the context of diverse samples, which naturally contain interfering humic acids.
environments such as soil (Benndorf et al., 2007; Wil- Such compounds are usually co-extracted together with
liams et al., 2010; Wang et al., 2011), sediments (Benn- proteins and are known to interfere with protein quantifi-
dorf et al., 2009; Bruneel et al., 2011), marine (Morris cation, separation and identification (Bastida et al., 2009).
et al., 2010; Sowell et al., 2011), freshwater (Ng et al., The use of gel-based methods for protein separation pre-
2010; Habicht et al., 2011; Lauro et al., 2011), human sents some disadvantages regardless of the origin of the
DNA/proteins RNA/proteins Proteins/proteins

interacons interacons interacons
Metainteractomics Fig. 1. System approach for the character-

ization of microbial ecosystems. Metagenomics
DNA RNA Proteins Metabolites
(DNA sequencing of all the microorganisms
from an ecosystem), metatranscriptomics
Metagenomics Metatranscriptomics Metaproteomics Metametabolomics (analysis of RNA collectively transcribed by all
the microorganisms from an ecosystem),
metaproteomics (analysis of proteins
collectively expressed by all the microorganisms
from an ecosystem) and metametabolomics
(analysis of metabolites collectively produced
Metabolic pathways, cellular by all the microorganisms from an ecosystem)
processes and cellular networks are employed to access the metabolic
pathways and cellular processes at work in an
ecosystem. Metainteractomics (analysis of the
molecular interactions between all the
Ecosystem funconing microorganisms from an ecosystem) is used to
investigate the cellular network in an
ecosystem. All the resulting data provide
insights into ecosystem functioning and are
Ecosystem modelling used to generate a model, which in turn can
allow the prediction of the behaviour of an
ecosystem in response to environmental
In silico characterizaon of ecosystem aributes changes.
2011 Federation of European Microbiological Societies FEMS Microbiol Ecol && (2011) 116
Recent advances in metaproteomics 3
addition to being an integrative component of system

Sample collecon approaches (Fig. 1), metaproteomics presents some valu-
able advantages over other omics technologies for func-
tional analyses. Primarily, metagenomic data only account
for the microbial potential of a system and do not pro-
Recovery of targeted fracon (microbial
fracon or extra cellular components)
vide any insights into microbial activity. On the other
hand, metatranscriptomics is one step closer to the iden-
tification of active metabolic pathways but does not allow
for translational regulation to be taken into consideration;
Protein extracon and quanficaon indeed, a lack of correlation between mRNA levels and
proteins levels has been previously documented (Gygi
et al., 1999; Pradet-Balade et al., 2001). Finally, metapro-
teomics provides significant insights into microbial activ-
Protein separaon and fraconaon
(gel-based/gel-free methods)
ity together with metametabolomics, which is the study
of the intermediate and end-products of cellular pro-
cesses. Metagenomic data typically include numerous
genes of unknown function (Ram et al., 2005), most
Mass spectrometry analysis likely involved in novel functional systems. Metaproteo-
(pepde sequence/pepde mass fingerprint) mics may be useful to identify the circumstances under
which these unknown functions are required and might
therefore help to elucidate which systems hold the most
potential for further investigation. Metaproteomics might
Databases searches or de novo pepde also prove to be valuable for the identification of key
sequencing for protein idenficaon microbial activities occurring in natural environments
that could be exploited in a bioengineered context. For
example, the investigation of the proteins expressed
within the wood termite gut (Burnum et al., 2011) or the
Data interpretaon: integraon of idenfied
proteins and metabolic pathways into system sheep gut (Toyoda et al., 2009) aimed at identifying natu-
funconing ral microbial processes involved in the degradation of
wood and cellulose. Such microbial processes could be
Fig. 2. Typical workflow for metaproteomics analysis. harnessed for the production of renewable energy from
wood or grass. For the purpose of this review, we discuss
the advancement of metaproteomics in the context of
sample. Such drawbacks are typically those associated human biology, soil, marine and freshwater environments
with two-dimensional gel electrophoresis (2-DGE): pro- as well as natural and bioengineered systems.
teins with extreme isoelectric points (basic or acidic) or
extreme molecular weight (very large or very small), lipo-
Protein expression in the human
philic proteins and low abundance proteins are typically
microbiome
excluded (Gygi et al., 2000; Ong & Pandey, 2001). Finally,
the limitations encountered in the last three steps of the In the context of human biology, the analysis of micro-
metaproteomic workflow (Fig. 2), namely mass spectrom- bial community function has been investigated mainly in
etry analysis, databases searches and data interpretation the intestinal tract and the oral cavity. Metaproteomic
are intrinsically linked to the success of the previous steps approaches in this field promise to be increasingly com-
with a disadvantage in fields where no metagenome prehensive because of the exponential progress in the
sequences are available. Overall, metaproteomics relies on generation of relevant genomic and metagenomic data-
the availability of relevant genome and metagenome sets. The Human Microbiome Project, which aims at
sequences when searching generated mass spectra against characterizing the microbial communities present at sev-
existing databases for protein identification. As such, this eral sites on the human body, has generated to date 178
approach cannot be viewed as an isolated method because full genome sequences from intestinal species (Nelson
it benefits from genome/metagenome sequencing for pro- et al., 2010), while gut metagenomic data were recently
tein identification. However, when no relevant sequences reported for 124 European individuals (Qin et al., 2010).
are available, de novo peptide sequencing can be used for The reason behind the high activity in this field of
protein identification (Lacerda et al., 2007; Fig. 2). In research lies in the apparent links between microbial
4 A. Siggins et al.
communities and human health and disease. Applying achieve an optimized use of the continuously growing
metaproteomic approaches in such context has the poten- genomic and metagenomic databases and has led to the
tial to lead to the identification of protein markers that identification of up to 5000 peptides from one sample
may be indicative of a healthy or a diseased state. In (Rooijers et al., 2011). In this study, proteins were
addition, the untargeted nature of metaproteomics makes extracted from the microbial fractions recovered from
it an ideal strategy to map unforeseen interactions faecal samples of two individuals. The extracted proteins
between the human microbial communities and its host. (50 lg per sample) were separated by one-dimensional
Despite the wealth of available genomic and metage- polyacrylamide gel electrophoresis (1D-PAGE) prior to
nomic data, only a small number of metaproteomic stud- LC-MS/MS analysis. Metagenomic data matched to these
ies have been reported to date. One of the first attempts two faecal samples were also collected. Metagenome
at characterizing protein expression from the gut revealed annotation has been identified as the main shortcoming
the difficulty of such a task in the absence of relevant for the utilization of such data for protein identification
sequences in the databases. Klaassens et al. (2007) con- (Keller & Hettich, 2009; Verberkmoes et al., 2009b; Rol-
ducted a metaproteomic analysis of faecal samples from ing et al., 2010). The strength of the iterative workflow
infants using two-dimensional gel electrophoresis proposed by Rooijers et al. (2011) is the use of nonanno-
(2-DGE) in combination with mass spectrometry analysis tated, unassembled metagenome sequences in conjunction
(matrix-assisted laser desorption/ionization time-of-flight with robustly annotated genomes from single microor-
mass spectrometry, MALDI-TOF-MS). From 100 lg of ganism. This iterative workflow could be systematically
protein, the authors could obtain more than 200 protein applied to further metaproteomic analyses provided that
spots on each 2D-gel. Fifty-five spots were excised and metagenome sequences representative of the system under
analysed by mass spectrometry, but no protein identifica- investigation are available.
tion could be obtained. After de novo sequencing was per- The characterization of the microbial communities
formed for four spots, 11 peptide sequences were from the human oral cavity is also gaining momentum
determined, of which only one showed a high-level of with the Human Oral Microbiome Project, which has
similarity with a known protein (bifidobacterial transla- made available an extensive 16S rRNA gene sequence
dolase). More recently, the identification of 2214 proteins database from oral microorganisms and with increasing
from human faecal samples was reported using a shotgun efforts to determine the genetic diversity of oral microbial
metaproteomic approach (Verberkmoes et al., 2009a). In communities (Lazarevic et al., 2009; Dewhirst et al., 2010;
this study, proteins were extracted from about 25 mg of Crielaard et al., 2011). The oral microbiota has been asso-
microbial cells recovered from faecal material of twins, ciated with infectious diseases such as periodontitis and
and the resulting peptide mixture was analysed using tonsillitis, but also with systemic diseases such as stroke
2D-liquid chromatography-tandem mass spectrometry and pneumonia (Joshipura et al., 2003; Awano et al.,
(2D- nano-LC-MS/MS). Mass spectra were searched 2008; Dewhirst et al., 2010). The microbial community of
against four databases, which all included two unmatched the oral cavity has also been suggested as a diagnostic
metagenome datasets (Gill et al., 2006), and were supple- marker for oral cancer, specifically targeting the abun-
mented with relevant sequences such as Bacteroides spp. dance of three bacterial species; Capnocytophaga gingivalis,
genomes (Verberkmoes et al., 2009a). One of the major Prevotella melaninogenica and Streptococcus mitis (Mager
breakthroughs from this study was the demonstration et al., 2005). However, up until now, the characterization
that unmatched metagenomes can be used to identify of the microbial communities from the human mouth
protein expressed from mixed microbial consortia (Ver- has been mainly directed at determining the diversity
berkmoes et al., 2009a). This finding was quite remark- more than the functionality of such communities. Indeed,
able because each individual possesses a unique gut when metaproteomic data are generated for oral cavity
microbial communities (Zoetendal et al., 2006). Another samples, most studies so far only reported on the result-
interesting result reported by Verberkmoes et al. (2009a) ing phylogeny obtained from peptide species assignment
was the observation of a clear discrepancy in the distribu- and did not investigate the functions of the proteins iden-
tion of clusters of orthologous groups (COG) categories tified (Guo et al., 2006; Xie et al., 2008; Grant et al.,
between the metaproteome and the metagenome. This 2010). Realizing the potential of the generated data, some
emphasizes the advantages of using a metaproteomic of the authors involved in the study of Xie et al. (2008)
approach, whereby the measure of expression and transla- revisited their results and provided a thorough analysis of
tion of a gene product can be achieved, over a metage- the functions represented by the proteins expressed in the
nomic approach, which is only indicative of the potential oral cavity (Rudney et al., 2010). In that respect, this
of a gene to be expressed and translated. More recently, study constitutes the only true metaproteomic analysis
an iterative workflow has been successfully developed to conducted on the human oral microbiota. The authors
successfully developed a three-step method to effectively all the fully sequenced microbial genomes from the Inte-
characterize the proteins expressed in salivary samples grated Microbial Genomes database in addition to an
and identified over 1000 human proteins and 139 micro- unmatched metagenome dataset from agricultural soil
bial proteins from 200 lg of protein extract (Xie et al., (Tringe et al., 2005). The metaproteome was found to
2008; Rudney et al., 2010). The oral microbial mixed reflect survival strategies such as bacterial spore forma-
community was found to be metabolically active and tion, probably to overcome the extended summer drought
mainly involved in the transport of carbohydrate. One of typical of the soil under investigation (Chourey et al.,
the major hurdles however in this field of research is the 2010). Another study (Wang et al., 2011) individually
absence to date of oral metagenomic data. Once such analysed the rhizospheric soil metaproteome associated
data become available, future comparative metaproteomic with rice, sugar cane and the flowering plant Rehmanniae.
studies should aim at characterizing community function Separation of extracted proteins by 2-DGE allowed the
in response to disease to identify functional biomarkers visualization of approximately 1000 individual proteins
that could be used to develop diagnostic tests. on each gel, with 538 common to all three samples, from
which a subset of 287 spots were randomly selected for
protein identification. Using MALDI-TOF/TOF followed
Protein expression in soil
by MS/MS and the NCBI database to search the resulting
Soil is widely regarded as a difficult medium for the anal- mass spectra, 189 spots were successfully identified,
ysis of microbial community structure and function. including 107 proteins attributed to plants, 72 to bacteria
Although soil contains an abundance of microorganisms and fungi and 10 to fauna. The identified proteins were
(~109 cells g 1 soil; Rossello-Mora & Amann, 2001), the associated with a wide range of functional categories
high species diversity, particularly of uncultured organ- including, amongst others: energy metabolism, protein
isms, has hindered analytical progress. In addition, the turnover, amino acid biosynthesis and proteins involved
presence of inhibitory substances, such as humic acids, in resistance mechanisms.
has proved difficult for the extraction of nucleic acids In the soil environment, metaproteomics can be used
and proteins from soil, without compromising down- to understand complex community interactions associated
stream processing methods, or resulting in protein degra- with in situ bioremediation of contaminated soil sites.
dation (Solaiman et al., 2007). As a result of these issues, Earlier studies such as Renella et al. (2002) and Singleton
the success of soil metaproteomics has been somewhat et al. (2003) focused on metal contaminants such as cad-
limited. Initial studies focused on investigating the opti- mium, while more recent studies moved towards organic
mization of protein extraction protocols that remove contaminants such as 2,4-dichlorophenoxy acetic acid
interfering compounds without inhibition of successive (Benndorf et al., 2007), toluene (Williams et al., 2010)
analysis (for details, see review from Bastida et al., 2009; and diesel fuel (Bastida et al., 2010). Benndorf et al.
Chen et al., 2009). One of the earliest reports of (2007) conducted metaproteomic analyses on soil micro-
attempted identification of soil proteins from excised 1D- cosms to investigate the degradation of 2,4-dichlorophen-
PAGE bands was carried out by Schulze et al. (2005). oxy acetic acid (2,4-D). Soils were percolated with 2,4-D
This study investigated the extracellular protein fraction solution to promote the development of indigenous
from soil microparticles using LC-MS/MS analysis. The degrading species, and one of the microcosms was also
resulting mass spectra were searched against the NCBI augmented with a mixture of bacteria known to degrade
database, which led to the identification of 75 proteins 2,4-D. Proteins were extracted from 5 g of soil and sepa-
involved in the degradation of biological matter and rated by 1D-PAGE, prior to excision of protein bands.
included cellulases, collagenases, lignin phenoloxidases Protein identification was carried out using nano-LC-elec-
and laccases. Fifty per cent of the identified proteins were trospray ionization (ESI) source-MS/MS, and searching
assigned to bacterial species, while the remaining proteins the resulting mass spectra against the NCBI database. A
originated mainly from plants, fungi, vertebrates and total of only four proteins were identified, and the low
nematodes. An interesting observation from this study level of protein identification was attributed to the diffi-
was the redundancy of protein function as similar pro- culties encountered during soil protein extraction because
teins were expressed by a range of species. For example, of the presence of interfering humic compounds. Despite
cellulases were expressed by three different bacteria and the limited number of proteins identified, the authors
one fungus. More recently, a direct protein extraction could conclude that the autochthonous soil community
method led to the identification of 716 proteins from 5 g showed similar 2,4-D degradation capability as that of the
of soil (Chourey et al., 2010). In this study, the protein augmented soil sample based on the function of the iden-
fractions were analysed using 2D-nano-LC-MS/MS, and tified soil proteins (specifically, 2,4-dichlorophenoxyace-
the databases used for protein identification consisted of tate dioxygenase), as well as the monitoring of 2,4-D
6 A. Siggins et al.
degradation using HPLC separation combined with UV a surrogate host, typically Escherichia coli. The resulting
detection (Benndorf et al., 2007). More recently, changes clones are subsequently screened for specific activities
in the metaproteome of a soil microbial community were such as antibiotic resistance (Allen et al., 2009; Donato
investigated in response to glucose and toluene amend- et al., 2010). Functional metagenomics has proven very
ment (Williams et al., 2010). The microbial fraction was useful for the identification of novel enzymes involved in
isolated from 5 g of soil, and the corresponding extracted antibiotic resistance and can be used as a targeted strategy
proteins were separated by 1D-PAGE and analysed by to uncover specific metabolic activities. However, func-
MALDI-TOF/TOF followed by MS/MS. This study tional metagenomics does not provide insights into in situ
detected 187 proteins in total amongst which 47 were processes, but reveals the function of metagenomic DNA
identified when searching mass spectra against the NCBI in E. coli.
database. Proteins specifically associated with known tolu- Future studies in this field should aim at combining
ene degradation pathways could not be identified, but the metagenomics with metaproteomics within the soil envi-
metaproteomic data overall reflected microbial adaptation ronment, which is largely dominated by uncultured
to the presence of toluene, noted by the expression of microorganisms. Taking advantage of the only soil
stress-related proteins (Williams et al., 2010). One of the metagenome available to date (Tringe et al., 2005),
major limitations of this study, however, is the lack attempts should be made to reanalyse existing mass spec-
of measurement of the level of toluene degradation, tra using databases inclusive of this metagenome. Further-
which consequently does not allow for any conclusions more, the pending availability of soil metagenomic data
regarding the toluene degrading capabilities of the soil as instigated by the TerraGenome Project (Vogel et al.,
community. 2009) should considerably improve the success of protein
Also focusing on bioremediation, Bastida et al. (2010) identification from soil environmental samples. In addi-
compared the protein content of an artificially hydrocar- tion, when investigating contaminant degradation, efforts
bon-amended soil with an unpolluted control. Proteins must be made to ensure that pollutants levels are moni-
were extracted from the microbial fraction recovered tored throughout the duration of the experiment to clar-
from 10 g of soil and either separated by 1D-PAGE fol- ify whether the nonidentification of proteins associated
lowed by LC-ESI-MS, or by direct LC-ESI-MS. Mass with contaminant degradation is because of the lack of
spectra were searched against the bacterial entries of the degradation activity or is masked by more abundant pro-
NCBI database. The majority of the 42 identified proteins cesses. Remarkably, despite the low number of identified
were related to cellular metabolism, indicating high proteins, Benndorf et al. (2007) could correlate the mea-
microbial activity both in the control and the polluted sured contaminant degradation with the presence of spe-
soil; however, no specific known hydrocarbon degrada- cific pollutant degrading proteins in their soil samples.
tion enzyme could be detected in the polluted soil This might imply that other bioremediation studies (Bast-
(Bastida et al., 2010). Once again in this study, no mea- ida et al., 2010; Williams et al., 2010), which did not
surement of the level of pollutant degradation was include the monitoring of the level of contaminants in
reported, and therefore, no information regarding the their experimental setup, might have been investigating
degrading capabilities of the soil communities was pollutants at concentrations that did not allow for the soil
provided. community to utilize its degrading capabilities.
Overall, the field of soil metaproteomics is still in its
infancy, mainly because of the technical difficulties typical
Protein expression in marine and
of this environment. Improvements in extraction proto-
freshwater
cols are reported regularly, and even though only one soil
metagenomic study has been published to date (Tringe Aquatic regions of the world harbour significant micro-
et al., 2005), it has been largely underused. Strikingly, the bial populations with an average of ~2.5 9 106 cells ml l
highest number of proteins identified by soil metaproteo- of seawater (Kan et al., 2005). These natural mixed
mics so far (716 proteins) was based on the use of data- microbial communities play crucial roles in essential pro-
bases including an unmatched soil metagenome (Chourey cesses, such as carbon and nitrogen cycling and organic
et al., 2010). This study however was still a proof of con- matter decomposition (Maron et al., 2006). Metagenomic
cept, where the authors aimed to demonstrate the techni- approaches have revealed the extensive microbial diversity
cal feasibility of soil metaproteomics and did not really and metabolic potential of marine and freshwater mixed
discuss identified protein functions. communities (Venter et al., 2004; Rusch et al., 2007; Ng
An interesting alternative strategy to metaproteomics, et al., 2010). These metagenomics datasets provide an
which has been employed in soil, is the use of functional ideal platform for metaproteomics, which is a growing
metagenomics, where metagenomic DNA is expressed in field both in marine and freshwater ecosystems.
Metaproteomics aims at capturing the protein expres- LC-MS/MS. The resulting mass spectra were searched
sion profile at the point of sampling, and thus, limiting against an extensive database from the Global Ocean
interferences with the natural microbial consortia in sam- Sampling (GOS) metagenomic project containing over
ples is of vital importance. Typically, once water samples 600 000 predicted proteins (Rusch et al., 2007; Yooseph
are collected, the microbial fraction is immediately con- et al., 2007). In total, this study identified 2273 proteins
centrated prior to storage pending protein extraction. To with 428 158 distinct membrane proteins per sample
stop further protein expression in response to sample (Morris et al., 2010), compared with 1042 proteins iden-
handling conditions, strategies such as chemical fixation tified when whole protein extracts were analysed (Sowell
(Kan et al., 2005) or microwave fixation (Mary et al., et al., 2009). To illustrate the need for relevant metage-
2010) have been employed. The majority of studies, how- nomic sequences when conducting metaproteomics analy-
ever, transfer concentrated pellets directly to storage at ses, Morris et al. (2010) searched their mass spectra
80 C or employ liquid nitrogen flash freezing (Sowell against the GenBank database and recorded a staggering
et al., 2009, 2011; Morris et al., 2010). 6.2-fold decrease in peptide identification compared with
As discussed earlier in the human biology context and searching against the extensive Global Ocean Sampling
in the soil environment, identification of the protein database. Overall, this study reported different metabolic
expressed by mixed microbial consortia has proven to be activities as indicated by protein expression along the nat-
challenging without the availability of relevant metage- ural nutrient gradient investigated. Specifically, Ton-B
nomic data. Kan et al. (2005) conducted the first meta- transport systems, known to use a proton motive force
proteomic analysis of a complex aquatic microbial for nutrient membrane translocation, were enriched in
community from Chesapeake Bay using 2-DGE in combi- the nutrient-rich coastal samples, while porins and perm-
nation with both MALDI-TOF and LC-MS/MS. From eases were preferentially expressed in the oligotrophic
100 lg of extracted proteins, the authors could detect open ocean samples. In addition, urea ABC transporters
over 200 protein spots on each 2D-gel. Forty-eight spots and photosystem proteins were more abundant in the
were excised, from which no protein identification could open ocean, when compared with the coastal area. Over-
be obtained using MALDI-TOF analyses. Seven of the 48 all, the authors concluded that the membrane metaprote-
spots were further analysed using LC-MS/MS and de novo omic data and derived species assignment reflected well
sequencing, which led to the identification of three pro- the microbial biodiversity and physicochemical character-
teins that shared homologies with proteins annotated istics of the oligotrophic open ocean and nutrient-rich
from the Sargasso Sea metagenome (Venter et al., 2004). environments investigated (Morris et al., 2010).
More recently, a metaproteomic study investigated the Another study employed metaproteomics to investigate
functionality of specific groups of microorganisms known the functionality of the marine microbial communities in
to be abundant in the oligotrophic Sargasso Sea (Sowell the nutrient-rich Oregon coastal seawater upwelling
et al., 2009). Protein extracts from microbial cells recov- region (Sowell et al., 2011). The marine microorganisms
ered from two samples of ~230 L of surface seawater were were isolated from ~100 L of surface seawater, and the
analysed using LC-MS/MS. The resulting mass spectra extracted whole protein fractions were analysed by 2D-
were searched against three synthetic metaproteomes nano-LC-MS/MS. The generated mass spectra were
comprising, respectively, the SAR11 clade, Prochlorococcus searched against a database composed of the predicted
and Synechococcus genomic fragments from the Sargasso proteins from the GOS project (Rusch et al., 2007) that
Sea metagenome (Venter et al., 2004). This approach led was edited to contain only the sequences found in similar
to the identification of 236 SAR11 proteins, 402 Prochlo- natural environments, as well as additional genome
rococcus proteins and 404 Synechococcus proteins, which sequences from two Oregon coastal isolates (Sowell et al.,
were found to largely reflect cellular adaptations to strin- 2011). Overall, this study identified 481 unique protein
gent environmental conditions under which the microor- families, with 29% of the total detected spectra related to
ganisms are competing for nutrients (Sowell et al., 2009). transport functions. The types of transport proteins iden-
A comparative metaproteomic study investigated the tified in this nutrient-rich environment (e.g. specific for
membrane proteins expressed by the mixed microbial nitrogen, carbon and sulphur containing compounds) dif-
communities from surface waters in the South Atlantic fered from those detected in the oligotrophic Sargasso Sea
(Morris et al., 2010). This study compared metaproteo- (e.g. specific for phosphate and phosphonate, known to
mic profiles over a large geospatial natural gradient be abundant during phosphorus starvation; Sowell et al.,
extending from an oligotrophic gyre to a nutrient-rich 2009). Differential protein expression as a response to
coastal upwelling region. Microorganisms were recovered natural nutrient gradients within the marine environment
from 10 samples of 100200 L each, and the enriched was also observed in the South Atlantic ocean (Morris
extracted membrane protein fractions were analysed by et al., 2010).
8 A. Siggins et al.
In freshwater environments, two recent studies investi- communities in Ace Lake were further investigated
gating microbial community functions in Ace Lake throughout the water column by sampling from six
Antarctica directly combined metagenomics with meta- depths with the aim of capturing the interactions between
proteomics (Ng et al., 2010; Lauro et al., 2011). Ng et al. microbial populations that defined nutrient cycling (Lauro
(2010) targeted a dominant green sulphur bacterium et al., 2011). The metagenomic analysis could assign
(Chlorobioaceae) known to be prevalent at specific depths ~28% of open reading frames to COG categories, while
(1214 m) in the water column, while Lauro et al. (2011) the metaproteomic study identified 1824 proteins. Inter-
investigated the microbial communities at work through- estingly, phylogenetic diversity was found to increase with
out the water column (523 m). In both studies, micro- depth and was associated with an increased amount of
bial fractions were recovered from 1- and 10-L samples hypothetical proteins, which accounted for 67% of the
obtained after drilling the ice cover of the lake. Whole identified proteins at 23 m depth. In addition, most of
protein extracts were analysed using 1D-PAGE followed these proteins did not match any orthologues from
by LC-MS/MS. Mass spectra were directly searched known microorganisms and could belong to novel func-
against the assembled metagenome from the same sample tional pathways (Lauro et al., 2011). Combining the
(Ng et al., 2010; Lauro et al., 2011). physicochemical data available for Ace Lake with match-
Metagenomic analysis of samples from 12.7 m depth of ing metagenomic and metaproteomic data, Lauro et al.
Ace Lake assigned 76% of the predicted open reading (2011) could describe the carbon, nitrogen and sulphur
frames (ORF) to a single species of green sulphur bacte- cycles throughout the water column. Briefly, for the car-
rium, (C-Ace; Ng et al., 2010), highlighting the predomi- bon cycling, cyanobacteria were found to carry out aero-
nance of this organism in this environment. The resulting bic carbon fixation in the upper stratum of the lake,
composite genome was then directly used to construct a anaerobic carbon fixation occurred further down the
database for searching metaproteomic mass spectra to water column and was performed by green sulphur bacte-
inform on the functional activities of C-Ace at the time ria conjointly with sulphate-reducing bacteria, while fer-
of sampling. This approach led to the identification of mentation processes were thought to take place at the
504 proteins corresponding to ~31% of the total pre- bottom of the lake. In addition, remineralization of par-
dicted proteome of C-Ace (Ng et al., 2010). As observed ticulate organic carbon to dissolved organic carbon was
in the context of the gut microbiome (Verberkmoes et al., found to occur at the surface of the lake with further het-
2009a), the comparison of the distribution of COG cate- erotrophic conversion carried out by Actinobacteria and
gories between the metaproteome and the metagenome members of SAR11 clade. In the lower stratum of the
revealed that some functional categories were misrepre- lake, remineralization of particulate organic carbon was
sented in the metaproteome. Specifically, proteins thought to result from the joint activities of fermentative,
involved in translation and energy production and con- sulphate-reducing and methanogenic microorganisms
version were statistically overrepresented, while amongst ultimately leading to the production of CO2 and CH4.
others, proteins involved in defence mechanisms and Carbon monoxide oxidation was also thought to be an
inorganic ion transport and metabolism were underrepre- important energy generation pathway throughout the
sented. In addition, the analysis of genes and correspond- water column as indicated by the detection of CO dehy-
ing proteins that did not have any orthologues in known drogenase genes (Lauro et al., 2011). The nitrogen cycle
green sulphur bacteria gave some insights about potential was found to involve nitrogen assimilation throughout
adaptive mechanisms in C-Ace. Evidence of a require- the lake, as indicated by the detection of glutamine and
ment for specific polysaccharides structures was attributed glutamate synthetases in the metaproteome, with reminer-
to cold adaptation, while the presence of genes encoding alization localized at the lower stratum. Interestingly, no
DNA restriction and modification system could present a evidence of nitrification occurring throughout the water
protective advantage against bacteriophages. Also worth column could be detected. Indeed, the metagenome did
noting was the construction of a pathway for sulphide not include any ammonia oxidation genes or any signa-
oxidation for C-Ace, and the lack of evidence for assimi- tures of known nitrifying bacteria or archaea. As Ace Lake
latory sulphate reduction suggesting the strict dependence is associated with continuous low level of nitrate, the
of C-Ace on sulphate-reducing bacteria, located at 14 m authors suggested that the absence of nitrification might
depth in Ace Lake (Ng et al., 2010). To conclude, the be a strategy to conserve bioavailable nitrogen (Lauro
authors could access the proteins necessary for the success et al., 2011). Finally for the sulphur cycle, green sulphur
of C-Ace in Ace Lake under cold, oligotrophic, oxygen bacteria were found to consume the sulphide produced
limited and extreme light conditions and therefore access by the sulphate-reducing bacteria, to generate sulphate
the biology of this organism in its natural habitat (Ng and thus providing the replenishment of the sulphate-
et al., 2010). The structure and functions of the microbial reducing bacteria substrate and ensuring the continuous
turnover of sulphur compounds in the lake. Even though detected in the lake. A search against the entire UniProt
genes for assimilatory sulphate reduction were detected in database led to the identification of a further 120 proteins
the metagenome, no evidence of their expression could from various species. Such a study demonstrates that, if
be found in the metaproteome. However, dissimilatory the microbial communities investigated are largely domi-
sulphide reduction was found to be an active pathway as nated by known species, LC-MS/MS analysis can lead to
indicated by the identification of proteins from sulphide extensive results in the absence of relevant metagenomic
reductase complex from green sulphur bacteria (Lauro data. However, even if such results are valid, they are
et al., 2011). Because of the crucial role seemingly played nonetheless biased towards dominant species and might
by C-Ace in the lake, as outlined by metagenomics and not be suitable for integration in system approaches
metaproteomics, and the low number of viral particles (Fig. 1).
where this bacterium is located (12.7 m depth), the
authors suspected that C-Ace was not following the previ-
Protein expression in natural and
ously developed model of viral/population dynamics in
bioengineered systems
aquatic environments (Rodriguez-Brito et al., 2010). The
authors attributed this unusual observation to the influ- Scientists have long known the significance of the role
ence of extreme light conditions on the microorganism played in the environment by mixed microbial communi-
biology. To test their hypothesis, a mathematical model ties. However, the exact processes carried out by these
was developed, where the growth of C-Ace was intrinsi- natural consortia are far from resolved. Even in relatively
cally linked to the light intensity in the lake (Lauro et al., simple natural biofilms, the expression of thousands of
2011). From this, the authors could predict that the per- proteins is required to enable survival and growth,
sistence of C-Ace in the lake was because of the absence including proteins involved in nutrient metabolism, stress
of phage. By incorporating this prediction with the infor- response and environmental signalling (Ram et al., 2005).
mation deduced from metagenomics and metaproteomics, One such biofilm community, originating from an acid
it was concluded that the emergence of phage predators mine drainage system within the Richmond mine at Iron
could deplete the Ace Lake of green sulphur bacterial Mountain, California, has been extensively studied (Ram
populations, which, because of their central role in the et al., 2005; Lo et al., 2007; Belnap et al., 2010; Denef
recycling of carbon, nitrogen and sulphur, would have et al., 2010; Mueller et al., 2010, 2011; Wilmes et al.,
severe consequences for the whole lake community (Lau- 2010; Pan et al., 2011). Indeed, one of the first publica-
ro et al., 2011). Overall, these two studies (Ng et al., tions investigating mixed microbial consortia from this
2010; Lauro et al., 2011) focused on the interpretation of site, Ram et al. (2005), is widely considered as a break-
the data generated using metagenomics and metaproteo- through study in environmental metaproteomics. Even
mics and are quite unique in that aspect as they did not though very significant, most of these studies however
stop at the demonstration of the feasibility of applying (Lo et al., 2007; Denef et al., 2010; Wilmes et al., 2010;
such technologies to Ace Lake but fully used their results Mueller et al., 2011) do not really fall under the umbrella
to gain insights into the biology of this ecosystem. of metaproteomics, but as in the case of Habicht et al.
In most environments, the difficulties associated with (2011), they investigate the physiology of one dominant
attempting to characterize the metaproteome without species within its natural habitat. Nonetheless, these
matched or unmatched metagenome sequences have been approaches collectively participated in important method-
demonstrated. However, this does not apply when investi- ological advances and will be briefly discussed here.
gating natural environments largely dominated by one Lo et al. (2007) demonstrated the use of proteomics to
species, where protein identification can be obtained determine genomic divergences between uncultivated
using a single sequenced relevant microbial isolate (Hab- microorganisms and closely related sequenced species
icht et al., 2011). Conversely, one could argue that these within the acid mine drainage biofilm. Denef et al. (2010)
types of study do not analyse the metaproteome but sim- employed this strategy to differentiate between two closely
ply investigate the microbial behaviour of one species related genotypic groups of Leptospirillum group II
within its natural environment. Such a study identified (99.7% similarity between 16S rRNA gene sequences).
1321 proteins from Chlorobium clathratiforme (species While one genotypic group typically prevails in early-
representing more than 50% of the bacterial population) stage biofilm, the other is dominant in the later stages of
throughout the water column in Lake Cadagno, Switzer- biofilm formation. To investigate this ecological diver-
land (Habicht et al., 2011). Interestingly, when searching gence, proteomic data previously published from 27 sam-
the mass spectra generated by LC-MS/MS against all ples (Denef et al., 2009) were analysed for the
Chlorobi genomes, an additional 350 proteins could be determination of ecologically preferential functions. The
identified, revealing the presence of species not previously authors identified a subset of proteins that could be
10 A. Siggins et al.
associated with the observed ecological divergence. Specif- tioned the use of 15N for quantification of expressed pro-
ically, proteins originating from an early-stage biofilm tein, an earlier study by Belnap et al. (2010) investigated
were found to be mainly involved in co-factor biosynthe- quantitative proteomic analysis more thoroughly to com-
sis and motility, while proteins associated with energy pare a laboratory-grown biofilm with a natural biofilm.
production and conversion emerged as biofilm maturity The aim of this study was to validate the use of labora-
progressed (Denef et al., 2010). Applying a similar strat- tory model systems for the investigation of ecological
egy, Wilmes et al. (2010) focused on the genomic diver- hypotheses. In total, the relative quantification of over
gence of two dominant species within the acid mine 2500 proteins between natural- and laboratory-grown
drainage biofilm, namely Leptospirillum groups II and III, biofilms was determined. This comparison showed that
using a combination of proteomics and metabolomics. A improvements in laboratory culturing conditions resulted
total of 765 proteins and 3740 metabolites were detected in the decrease of metabolic stress protein expression,
across 14 biofilm samples. Each of the two Leptospirillum such as proteins involved in defence mechanisms and oxi-
groups investigated could be associated with a distinct dative damage repair, and this correlated with increased
cluster of proteins and metabolites, indicating genomic community growth rates (Belnap et al., 2010). This study
divergence between the two bacteria. From their data, the highlighted the relevance of using model systems under
authors could identify a limited niche overlap and a low laboratory controlled conditions to gain some under-
level of interspecies competition, suggesting separate standing of natural microbial processes. A subsequent
resource utilization for each species within the biofilm study by Pan et al. (2011) used this laboratory culture
(Wilmes et al., 2010). model to successfully develop a high-throughput stable
Investigating further the acid mine drainage biofilm, isotope probing (SIP) method. Employing this new tech-
Mueller et al. (2010) combined metaproteomics with geo- nique, the authors could trace the incorporation of 15N
chemical and biological data to investigate the behaviour into thousands of proteins expressed by a mixed micro-
of the biofilm microbial communities along environmen- bial community. Also applying SIP technology, Bastida
tal gradients. The analysis of 28 biofilm samples led to et al. (2011) quantitatively traced the movement of
13
the identification of a total of 6296 proteins and differ- C-benzene between trophic levels, specifically from bac-
ences in protein expression was found to correlate with teria to eukaryotic organisms, such as Chironomus sp. lar-
environmental parameters (Mueller et al., 2010). For vae. SIP-proteomics methods could prove very valuable
example, temperature at the time of sampling was found to track down fluxes of 13C or 15N in mixed microbial
to impact on levels of cold-shock proteins and proteins communities. However, such setups could not be applied
involved in fatty acid biosynthesis, expressed at a lower in situ and are limited to laboratory-based models including
level and at a higher level with increasing temperature, microcosms.
respectively. In addition, a shift in the metaproteome was Although the incorporation of labelled carbon and
observed from ribosome biosynthesis and transcription in nitrogen has been investigated in the context of labora-
the early stage of biofilm formation to environmental sig- tory-grown mixed anoxic communities (Jehmlich et al.,
nalling, chemotaxis and biosynthesis of extracellular com- 2008), to the best of our knowledge, no protein-based SIP
ponents in the mature biofilm (Mueller et al., 2010). In a analysis of complex, engineered systems has been yet
subsequent study, Mueller et al. (2011) focused their undertaken, which could allow for optimization of eco-
investigations on the initial dominant species, Leptospiril- nomically significant processes. However, metaproteomic
lum Group II, and its evolving protein expression studies of engineered processes, such as activated sludge
throughout the development of the biofilm community. systems, are extensive (Wilmes & Bond, 2006; Park &
Using replicate samples and analysing membrane and Novak, 2007; Park et al., 2008a, b; Abram et al., 2011).
cytoplasmic protein fractions, this study aimed at identi- Activated sludge systems are principally employed for the
fying key proteins that could be used as biomarkers to treatment of wastewater in developed countries. The aggre-
estimate biofilm maturity (Mueller et al., 2011). The gation of microbial biomass to form well-settling flocs has
authors identified a total of 4107 distinct proteins, corre- been identified as a key parameter for the successful opera-
sponding to ~45% of the predicted proteome of Lepto- tion of wastewater treatment plants. The study of the
spirillum Group II. Changing protein abundance patterns metaproteome of laboratory-scale-activated sludge systems
were successfully determined as a function of biofilm has been divided into two approaches: analysis of the
maturity. For example, the metabolism of simple carbon extracellular polymeric substances (EPS) that bind the
compounds was found to dominate early growth stage microbial cells together, and analysis of the protein frac-
biofilms, while the metabolism of complex carbohydrates tion located within the microbial cells themselves.
increased in late stages of biofilm formation (Mueller Researchers from the University of Massachussetts/Vir-
et al., 2011). Although Mueller et al. (2011) briefly men- ginia Tech focused on the extraction and analysis of the
protein component of EPS. Their first study (Park & allowed the detection of an average of 665 spots per gel,
Novak, 2007) identified that EPS bound to different although spot patterns differed significantly between
cations require different extraction methods, which EBPR and nEBPR samples. Furthermore, proteins pat-
resulted in the extraction of sub-proteome components terns detected from the EBPR sludge samples were more
of the activated sludge sample. Focusing on the require- consistent between aerobic and anaerobic stages, indicat-
ment for the reduction of volatile solids to 38% prior to ing the development of a more stable microbial commu-
land spreading of digested sludge (US EPA, 2003), Park nity, which was not noted in the nEBPR samples.
et al. (2008a) demonstrated the selective degradation of Although no attempt at protein identification was under-
divalent cation-bound proteins and aluminium-bound taken, the importance of this study must not be underes-
proteins by aerobic digestion, but not by the anaerobic timated, as the demonstration of a statistically different
treatment process, while the opposite was true of organic protein expression between a functioning and a nonfunc-
matter bound with ferric iron. This is of significant tioning system justifies the investigation of the metaprote-
industrial importance, as it specifically indicates by pro- ome for identification of performance issues associated
tein analysis, that a two-step treatment process, featuring with full-scale-activated sludge processes.
both anaerobic and aerobic stages, may be required for A subsequent study by this group focused on the iden-
enhanced sludge treatment. tification of proteins separated by 2-DGE. Using a combi-
Next, Park et al. (2008b) attempted to identify proteins nation of MALDI-TOF/MS and quadrupole-TOF MS/MS,
that could serve as biomarkers to monitor facility opera- and searching against three unmatched EBPR metage-
tions in activated sludge processes. Eleven intense bands nomic databases, Wilmes et al. (2008a) identified 38 of
from 1D-PAGE were investigated using LC-MS/MS and 111 excised protein spots, corresponding to 33 unique
the whole NCBI database. Seven of these bands contained proteins. It is suggested that the low identification rate
identifiable proteins associated with bacterial defence, cell (41%) may be due to strain variation between the sample
appendages, cell surface outer membrane proteins, intra- sludge (from a UK SBR) and the three metagenomic da-
cellular materials and influent sewage proteins. Interest- tabases employed (two from the United States and one
ingly, of the bands that did not return any positive from Australia). Possibly recognizing the technical limita-
matches, identifiable amino acid fragmentation patterns tions of 2-DGE, Wilmes et al. (2008b) then carried out
were obtained, indicating that these proteins are likely to analysis of extracted proteins directly via 2D nano-LC,
originate from unsequenced microorganisms in full-scale- followed by MS/MS analysis. Peptide tandem mass spec-
activated sludges, again indicating the requirement for tra were matched in silico to the three separate sludge
metagenomic sequence data. As seen in other environ- metagenomic databases employed in Wilmes et al.
ments (human microbiome, soil, marine and freshwater), (2008a), and against a combined database of the three
the use of unmatched metagenome sequences may have metagenomic datasets. This extensive analysis resulted in
improved the success of protein identification in the the identification of a total of 5029 proteins, with 36%
above investigation. A short follow-up study analysed one associated with Accumulibacter phosphatis, a major poly-
of the samples from Park et al. (2008b) over the course phosphate accumulating organism.
of a 30-day batch digestion (Park & Helm, 2008). Three While activated sludge technology is commonly
1D-PAGE bands, additional to those studied in Park et al. employed in the field of sewage treatment, anaerobic
(2008b), were investigated by LC-MS/MS using the whole digestion technology is becoming increasingly popular for
NCBI database. All three bands (1) were associated only the treatment of recalcitrant chemical-containing waste-
with divalent cations, (2) emerged during the batch water streams. Jehmlich et al. (2010) were one of the first
anaerobic digestion of the activated sludge and (3) were reported studies to investigate the metaproteome of an
identified as different subunits of methyl-coenzyme M anaerobic community, originating from a batch sulphate-
reductase, a key enzyme for methane formation in metha- reducing enrichment culture, exposed to toluene. This
nogenic archaea, and in this case associated with Methan- study analysed 150 lg of protein by 2-DGE and identified
osarcina barkeri. 202 proteins from 236 excised gel spots using nano-
The intracellular microbial protein fraction of activated LC-ESI-MS/MS analysis and searching the resulting mass
sludge has been investigated by a separate group of spectra against the bacterial entries of the NCBI database.
researchers. Wilmes & Bond (2006) applied metaproteo- Identified proteins were found to be associated with both
mics to activated sludge originating from the end of the sulphate reduction and toluene degradation. Recently, the
anaerobic and aerobic cycles of the same sequencing first metaproteomic investigation of granular biomass
batch reactor (SBR) during stages of enhanced biological originating from a continuous anaerobic bioreactor was
phosphorus removal (EBPR) and non-EBPR (nEBPR). described (Abram et al., 2011). The extracted proteins
From 100-mL sludge samples, separation by 2-DGE were separated by 2-DGE, which resulted in the detection
of 388 reproducible protein spots, of which 70 were water column. Such an approach could feasibly be trans-
excised. Thirty-three proteins were positively identified ferred to other environmental ecosystems. To date, the
using nano-LC-ESI-MS/MS and searching the resulting application of complex system approaches is still scarce
mass spectra against both the whole NCBI database and and requires a coordinated experimental design that
the Trembl database. Proteins associated with the produc- brings together expertise from each of the many techno-
tion of methane and the degradation of glucose were logies involved.
identified. One of the major hurdles in the field of anaer- The technical limitations encountered throughout the
obic community function is the lack of metagenomic metaproteomic workflow (Fig. 2) have, for the most part,
data, which would facilitate the identification of proteins. been addressed in the ecosystems discussed in this review.
However, it should be kept in mind that an exhaustive
investigation of the entire metaproteome is unlikely due
Conclusions
to the unfeasibility of developing a universal protein anal-
Overall, the field of metaproteomics is gaining momen- ysis protocol. Furthermore, it must be considered that a
tum at an exponential rate within very diverse environ- metaproteome may include intracellular, extracellular and
ments. An overview of selected studies from the membrane-bound proteins, and ideally, the three protein
ecosystems discussed in this review is shown in Table 1. fractions should be analysed for each sample. When pos-
Advances in metaproteomics finally allow for the consid- sible, opting for gel-free protein fractionation seems to
eration of the integration of such data in system lead to a higher level of protein identification when
approaches (Fig. 1). This was partly achieved in the aqua- compared with gel-based methods. For example, when
tic environment where Lauro et al. (2011) combined analysing the metaproteome of activated sludge, the use
metagenomic, metaproteomic and physicochemical data of 2-DGE resulted in the identification of 38 proteins
to describe the interaction between the microbial popula- (Wilmes et al., 2008a), while the 2D-nano-LC method led
tions defining the biogeochemical cycles throughout a to the identification of 5029 proteins (Wilmes et al., 2008b).
Table 1. Overview of selected metaproteomics studies
Number of
peptides/proteins
Environment identified Method Databases References
Human gut NA/2214 proteins LC-MS/MS 2 unmatched human gut metagenomes, Verberkmoes
several genomes from gut inhabitants and et al. (2009a)
several nonhuman gut genome
Human gut 5010 peptides/NA 1D-PAGE, LC-MS/MS Synthetic human gut metagenome Rooijers et al.
(216 genomes from gut inhabitant) and (2011)
124 human gut unassembled
nonannotated metagenomes
Soil NA/716 proteins LC-MS/MS Unmatched soil metagenome supplemented Chourey et al.
with 1606 genomes (2010)
Soil NA/122 2D-PAGE, MALDI Complete NCBInr, bacterial entries NCBInr and Wang et al.
TOF/TOF MS/MS fungal entries NCBInr (2011)
Marine 6533 peptides/1042 LC-MS/MS SAR11 clade and specific microorganisms Sowell et al.
proteins from Sargasso Sea metagenome as (2009)
well as genomes from
sequenced isolates
Marine 5389 peptides/2273 LC-MS/MS Global Ocean Sampling combined Morris et al.
proteins metagenomes (2010)
Freshwater NA/1824 proteins 1D-PAGE, LC-MS/MS Matched metagenomes Lauro et al.
(2011)
Acid mine NA/4107 proteins LC-MS/MS Biofilm_AMD_CoreDB database Mueller et al.
drainage biofilm (2011)
Activated sludge NA/5029 proteins LC-MS/MS Three distinct unmatched activated Wilmes et al.
sludge metagenomes (2008b)
Anaerobic digestion NA/202 proteins 2D-PAGE, LC-ESI-MS/MS Bacterial entries of the NCBI Jehmlich et al.
nonredundant database (2010)
1/2D-PAGE, one/two-dimensional polyacrylamide gel electrophoresis; LC-MS/MS, liquid chromatography-tandem mass spectrometry; MALDI-TOF,
matrix-assisted laser desorption ionization-time of flight; ESI, electrospray ionization.
In addition, it is now apparent that metaproteomic functional insights into anaerobic digestion. J Appl Microbiol
approaches benefit from the availability of relevant me- 110: 15501560.
tagenomic data, either matched or unmatched. As a result Allen HK, Moe LA, Rodbumrer J, Gaarder A & Handelsman J
of this combined protocol, a new difficulty is encountered (2009) Functional metagenomics reveals diverse b-
regarding the analysis and interpretation of the vast quan- lactamases in a remote Alaskan soil. ISME J 3: 243251.
tity of data generated. A major hurdle in the utilization Amann RI, Ludwig W & Schleifer KH (1995) Phylogenetic
identification and in situ detection of individual microbial
of metagenomic data, impacting directly on metaproteo-
cells without cultivation. Microbiol Rev 59: 143169.
mics, has been recognized as the assembly and the anno-
Awano S, Ansai T, Takata Y, Soh I, Akifusa S, Hamasaki T,
tation of the collected genomic fragments. Rooijers et al.
Yoshida A, Sonoki K, Fujisawa K & Takehara T (2008) Oral
(2011) proposed an iterative workflow using nonannotat- health and mortality risk from pneumonia in the elderly.
ed, unassembled metagenome sequences, which could be J Dent Res 87: 334339.
systematically used in metaproteomic investigations when- Bastida F, Moreno JL, Nicolas C, Hernandez T & Garca C
ever relevant metagenomic data are available. (2009) Soil metaproteomics: a review of an emerging
Future metaproteomic studies should aim to progress environmental science. Significance, methodology and
from proof of concept approaches to experimental designs perspectives. J Soil Sci 60: 845859.
leading to practical applications. For example, metaproteo- Bastida F, Nicolas C, Moreno JL, Hernandez T & Garca C
mic comparisons between healthy and diseased states (2010) Tracing Changes in the microbial community of a
within the human microbiome have yet to be carried out. hydrocarbon-polluted soil by culture-dependent proteomics.
Additionally, comparative in situ bioremediation investiga- Pedoshere 20: 479485.
tions will need to be conducted to access the functional Bastida F, Jechalke S, Bombach P, Franchini AG, Seifert J, von
response of the natural mixed microbial communities to Bergen M, Vogt C & Richnow HH (2011) Assimilation of
benzene carbon through multiple trophic levels traced by
common pollutants. Furthermore, when investigating bio-
different stable isotope probing methodologies. FEMS
remediation processes, pollutants should be systematically
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monitored throughout the trials, to clarify the contaminant
Belnap CP, Pan C, VerBerkmoes NC, Power ME, Samatova
degradation status as a function of time. This in turn NF, Carver RL, Hettich RL & Banfield JF (2010) Cultivation
should allow for the proper assessment of the degrading and quantitative proteomic analyses of acidophilic microbial
abilities of the microbial communities investigated. communities. ISME J 4: 520530.
To conclude, the feasibility of metaproteomic studies has Benndorf D, Balcke GU, Harms H & Von Bergen M (2007)
been successfully demonstrated in very diverse natural and Functional metaproteome analysis of protein extracts from
engineered environments. However, only few studies to contaminated soil and groundwater. ISME J 1: 224234.
date employed this strategy to answer specific biological Benndorf D, Vogt C, Jehmlich N, Schmidt Y, Thomas H,
questions such as how complex communities define the Woffendin G, Shevchenko A, Richnow HH & von Bergen M
biology of a given ecosystem (Ng et al., 2010; Wilmes et al., (2009) Improving protein extraction and separation
2008b; Denef et al., 2010; Mueller et al., 2010; Lauro et al., methods for investigating the metaproteome of anaerobic
2011), and it is now critical to move metaproteomics for- benzene communities within sediments. Biodegradation 20:
ward in order for this technology to achieve its full poten- 737750.
Bruneel O, Volant A, Gallien S et al. (2011) Characterization
tial. To this end, future studies must be designed with an
of the active bacterial community involved in natural
aim towards gaining some understanding of ecological con-
attenuation processes in arsenic-rich creek sediments.
cepts, and the data generated must be adequately analysed.
Microb Ecol 61: 793810.
In addition, attempts should be made to integrate such data Burnum KE, Callister SJ, Nicora CD, Purvine SO, Hugenholtz
in the context of system approaches to allow for the predic- P, Warnecke F, Scheffrahn RH, Smith RD & Lipton MS
tion of functional responses to environmental stimuli. (2011) Proteome insights into the symbiotic relationship
between a captive colony of Nasutitermes corniger and its
Acknowledgements hindgut microbiome. ISME J 5: 161164.
Chen S, Rillig MC & Wang W (2009) Improving soil protein
This work was conducted with the financial support of a extraction for metaproteome analysis and glomalin-related
University Fellow grant from the National University of soil protein detection. Proteomics 9: 49704973.
Ireland, Galway. Chourey K, Jansson J, VerBerkmoes N, Shah M, Chavarria KL,
Tom EL, Brodie EL & Hettich RL (2010) Direct cellular
lysis/protein extraction protocol for soil metaproteomics.
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MINIREVIEW

Exploring mixed microbial community functioning: recent

Correspondence: Florence Abram, Abstract

microbiota; marine and freshwater

from its natural environment might not necessarily dis-

DNA/proteins RNA/proteins Proteins/proteins

Metainteractomics Fig. 1. System approach for the character-

addition to being an integrative component of system

Table 1. Overview of selected metaproteomics studies

Renella G, Chaudri AM & Brookes PC (2002) Fresh additions

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