Available online at www.sciencedirect.
com
Microbial metaproteomics: identifying the repertoire of proteins
that microorganisms use to compete and cooperate in complex
environmental communities
Robert L Hettich1, Ritin Sharma1,2, Karuna Chourey1 and
Richard J Giannone1
The availability of genome information for microbial consortia,
including unculturable species, from environmental samples
has enabled systems-biology interrogation by providing a
means to access genomic, transcriptomic, and proteomic
information. This provides a unique opportunity to characterize
the molecular activities and interactions of these microbial
systems at a comprehensive level never before possible. Such
information not only provides details about the organizational,
functional, and metabolic activities of such systems, but also
the untapped reserve of molecular activities that might be
invoked and exploited under certain environmental conditions.
Since bacteria naturally exist in complex ecosystems, it is
imperative to develop and utilize analytical approaches that can
provide molecular level details on systems consisting of mixed
microbial membership. This is the realm of metaproteomics —
the characterization of the complement of proteins expressed
by a microbial community in an environmental sample.
Addresses
1
Oak Ridge National Laboratory, Oak Ridge, TN 37831-6131, USA
2
Graduate School of Genome Science and Technology, University of
Tennessee, Knoxville, TN, USA
Corresponding author: Hettich, Robert L. (hettichrl@ornl.gov)
Current Opinion in Microbiology 2012, 15:373–380
This review comes from a themed issue on Microbial proteomics
Edited by Bertrand Séraphin and Robert Hettich
For a complete overview see the Issue and the Editorial
Available online 24th May 2012
1369-5274/$ – see front matter, # 2012 Elsevier Ltd. All rights
reserved.
http://dx.doi.org/10.1016/j.mib.2012.04.008
Background and rationale for metaproteomics
The explosion of high throughput DNA sequencing has
enabled an unprecedented systems-biology view of the
molecular machinery of environmental microbial samples,
as evidenced most recently by whole community sequence
information (often termed metagenomics [1]) on a variety of
important ecological habitats. Obviously the genomic com-
plexity of a microbial consortium is substantially greater
than that of a single isolate, thereby complicating the depth
and completeness of the measured metagenome infor-
mation. Notwithstanding, metagenomics provides a
www.sciencedirect.com
reasonably extensive catalog of genomic information that
can be translated into a list of all possible protein products.
Interrogation of the protein complement (termed either
whole community proteomics [2] or metaproteomics [3])
seeks to identify the functional expression of the meta-
genome and elucidate the metabolic activities employed
by a community at the moment of sampling. Early meta-
proteome research focused on low complexity microbial
consortia from acid mine drainage biofilms [4], sludge
water bioreactors [5], or synthetic communities in gnoto-
biotic mice [6]. These systems provided excellent starting
points to develop, evaluate, and optimize advanced pro-
teomic methods (and associated informatics) for more
complex systems.
The experimental requirements for proteome character-
izations include high throughput measurements, sensitive
protein/peptide detection, large dynamic range, ability to
deal with very complex mixtures, accurate mass measure-
ments, and ability to characterize and resolve peptide
sequences. In this regard, mass spectrometry (MS) has
emerged as the unchallenged technological platform. Early
work in proteomics was conducted with two-dimensional
(2D) gel electrophoresis [7,8], often accompanied by MS
detection; this general approach has been supplemented
by the use of multiple dyes in a single gel electrophoresis
experiment (DIGE) [9]. More recently, the ability to inter-
face multidimensional chromatographic separations
(either offline or online) with MS (termed LC–MS) has
enabled an unprecedented glimpse into very complex
samples containing thousands of proteolytic peptides
[10]. Figure 1 illustrates how this experimental approach
is combined with metagenomic information for peptide/
protein identifications. The advent of high performance
MS platforms, such as hybrid Q-TOFs, LTQ-FTICR-MS,
and LTQ-Orbitrap-MS [11], has provided much improved
capabilities for the rapid scanning and ultra-high perform-
ance (mass accuracy and mass resolution) that are essential
for more advanced and discriminatory proteomic measure-
ments. A key component that has also advanced dramatic-
ally over the past five years is computational informatics (as
enhanced by the availability of massively parallel comput-
ing architectures), which provides the necessary tools to
mine and analyze the resulting complex raw datasets.
The emergence of metaproteome information for a
variety of environmental samples is beginning to reveal
Current Opinion in Microbiology 2012, 15:373–380
374 Microbial proteomics

Figure 1

Multidimensional LC Parent peptide MS Fragmentation (MS/MS)

100

Relative Abundance
100 100
Relative Abundance

Relative Abundance
90 90 90
80 80 80
70 70 70
60 60 60
50 50 50
40 40 40
30 30 30
20 20 20
10 10 10
0 0 0
0 10 20 30 40 50 60 70 80 90 100 110 400 500 600 700 800 900 1000 1100 1200 1300 200 400 600 800 1000 1200 1400

time m/z m/z

cellular peptides LC/LC-MS/MS measure peptides

trypsin
lysate (---Lys,---Arg) and fragmentation
digest

Crosscorrelation
scoring for
peptide/protein ID

in silico peptide
Metagenome in silico predict fragmentation
sequence proteins predict peptides
and fragmentation

Current Opinion in Microbiology

Integrated experimental/computational approach for metaproteome measurements. A multidimensional liquid chromatography–tandem mass
spectrometry approach is used to obtain high resolution parent ion and subsequent fragmentation information for proteolytic peptides in the environmental
sample. To enable high-throughput peptide identifications, the metagenome sequence is used to predict all possible protein products, as well as their
predicted proteolytic cleavage and tandem mass spectrometric fragmentation products. A crosscorrelation scoring approach is used to compare the
measured information on peptide parent masses and fragmentation with that predicted from the metagenome information. After peptides are scored,
filtered, and identified, the predicted protein database is used to assemble the measured peptides computationally into respective protein families.

detailed information about microbial community struc- environmental sample, peptide/protein fractionation
ture, dynamics, and functional activities, thereby permit- before detection, and subsequent high-throughput unam-
ting a better understanding of microbial community biguous peptide/protein identification. For example, the
development (recruiting, maturization), interspecies dynamic range of a proteome sample can be on the order
cooperation and competition for nutrients, and distri- of 104–106 for microbial isolates, and significantly larger
bution of metabolic activities across the community for consortia in environmental samples [2]. Thus, the
members (including defense systems) [12]. Such infor- organismal complexity and wide-range of protein expres-
mation will be crucial for the characterization of host/ sion challenges the very notion of complete proteomic
microbe interactions, such as bacterial/plant and bacterial/ characterization. However, coupling extensive separation
human interfaces — two research areas poised to advance (i.e. GE or LC) with sensitive, high performance detec-
significantly in the near future. tion (i.e. MS) provides the best opportunity to overcome
this obstacle.
This article will focus on a description of the current state
of metaproteomic research, and highlight various recent In terms of proteome measurement depth and confi-
metaproteomic studies. It is not intended to be a com- dence, the experimental ‘standard’ is multidimensional
prehensive review, as this has been featured recently chromatographic separation (either offline or online)
elsewhere [12,13,14,15,16], but rather to describe coupled with high performance tandem MS measure-
the integrated experimental/computational approach ment involving Q-TOFs or LTQ-Orbitrap-MS systems.
and the emerging nature of scientific information that While reversed-phase chromatographic separations have
this approach can provide. been widely used, advances in ultra-high pressure liquid
chromatography (UPLC) and/or modified stationary
Advances in experimental methodology phases (monolithic, sub-2 mm, or microparticle shell tech-
The success of a metaproteome measurement relies on nologies) provide increased chromatographic resolution,
three factors: unbiased protein extraction from a complex sensitivity, and speed of online LC–MS measurements

Current Opinion in Microbiology 2012, 15:373–380 www.sciencedirect.com

 Microbial metaproteomics Hettich et al. 375

[17–20]. In addition, development of solution-based sep-
arations based on the venerable 2D-GE approach enable
fractionation which is more directly compatible with
enhanced sample recovery for digestion and MS measure-
ment. For example, two major offline methodologies,
isoelectric focusing (IEF) [21] and GELFrEE [22], sep-
arate complex protein mixtures by isoelectric point or
molecular mass, and provide a starting point for possible
3D-LC separations. Coupled together, these strategies
enable a multidimensional approach that better separates
complex environmental mixtures, thus augmenting
protein detection.
Likewise, hardware improvements to mass spectrometers
increase duty-cycle, selectivity, and sensitivity, and pro-
vide remarkable gains in peptide measurement metrics
[23]. In addition, the increased commercial availability of
high mass accuracy, and high resolution instruments [24–
26] provides the means to better discriminate co-eluting
peptides of similar mass-to-charge, a necessity as sample
complexity increases. These more accurate measure-
ments provide more extensive informative sequence
coverage (as shown in Figure 2 for electron transfer
dissociation of a targeted peptide) and permit the confi-
dent detection of subtle differences between and/or
modifications to expressed proteins, such as sequence
polymorphisms and/or post-translational modifications.
Concurrent optimization of both LC and MS serves to
broaden the measurement and identification metrics of a
proteomic experiment. Improved detection limits and
wider dynamic range measurement capabilities should
provide a more comprehensive approach for characteriz-
ing less abundant microbial members of a consortium,
thereby permitting a more thorough understanding of the
interplay between the different species of a community
and potentially unveiling the exquisite niche fulfillment
strategies of participating members.

Figure 2

#3663-3663 NL: 1.08E5

•High sequence coverage (18 out of 24 possible
Relative Abundance

100
fragments identified, 75%)
80
•Low mass error for fragments
60
40
20
0
0 100 200 300 400 500 600 700 800 900 1000 1100 1200 1300
#3663-3663 NL: 1.08E5

6
Relative Abundance

5
4
3
2
1
0
0 100 200 300 400 500 600 700 800 900 1000 1100 1200 1300
m/z

2
Delta Mass (amu)

-1
-2

Current Opinion in Microbiology

Tandem mass spectrometric fragmentation, achieved by electron transfer dissociation in an LTQ-Orbitrap-MS, of a proteolytic peptide reveals
extensive sequence information, as shown in the top panel. An expansion of the y-axis (middle panel) reveals numerous fragment ions (c-type and z-
type ions) that can be measured with mass accuracies in the parts-per-million range. This provides almost complete overlapping sequence information
for this (NKITSVGGTVEEI) peptide, as shown in the table on the bottom.

www.sciencedirect.com Current Opinion in Microbiology 2012, 15:373–380

376 Microbial proteomics
Bioinformatic considerations
In the context of environmental proteomics, the pre-
dicted protein database is constructed from metagenomic
datasets. Therefore, its quality is inextricably linked to
the depth, quality, and relevance of the metagenome.
Certainly, the transition in DNA sequencing from Sanger
approaches to 454-methodology and now Illumina has
had dramatic subsequent impacts on metaproteome
measurements. In particular, the shorter reads accessible
with the newer sequencing approaches initially con-
founded metaproteome measurements, but have become
addressable with improved informatic assembly methods.
These factors, along with biological complexities intrinsic
to environmentally derived samples (i.e. homologous
proteins/domains, horizontal gene transfer, and/or strain
variation) require additional proteomic measurement
constraints to control the false discovery rate (FDR) while
confidently identifying unique peptides and proteins,
including those that differ only slightly. One practical
solution involves the use of high mass accuracy, high
resolution mass spectrometers, such as FTICR or Orbi-
trap instrumentation, that have the discriminatory power
(<5 parts-per-million mass accuracy) to enable distinction
between very similar peptide masses. In fact, the appli-
cation of high mass accuracy allows one to achieve extra-
ordinarily low FDR levels (<0.1%). With this
unprecedented level of confidence, peptide scoring filters
may be relaxed without consequence, resulting in an
increase in true positives without affecting the FDR.
The field of proteome bioinformatics encompasses a
range of computational operations, including protein
database searching and filtering of raw mass spectra,
peptide-spectrum matching followed by peptide-to-
protein assembly, data mining, graphical representation,
and data dissemination. Within the past three to five
years, there has been a tremendous increase in the range
and availability of software architectures to accomplish
these functions [27]. While the field is too vast to ade-
quately detail in this limited review, it is worth noting that
some of the basic database searching and data extraction
methodologies have become quite standardized and fairly
routine, leaving more attention to be paid to data mining
and analyses. This has led to a noticeable increase in the
number of interdisciplinary interactions between exper-
imental and computational researchers, often focused on
design of an integrated approach for specific problems,
such as characterizing proteomes across microbial species
in the absence of specific genomic data [28], or evaluating
new approaches to integrate metagenomic and metapro-
teomics information [29,30].
Recent exponential growth in metaproteomics
research
One of the first large-scale whole community proteome
measurements involved an uncultured microbial consor-
tium from acid mine drainage [4]. Extension of this
Current Opinion in Microbiology 2012, 15:373–380
approach was used for the strain-resolved characterization
of the dominant microbial species [31], and revealed
genome recombination as a crucial component for adap-
tation to specific ecological niches [32]. Further studies
uncovered the ecological distribution and population
succession information [33,34]. More recent work has
been directed at quantification approaches (both label-
free and stable isotope labeling) for metaproteomics,
including the demonstration of extensive isotopic label-
ing of an environmental biofilm community in the lab
[35,36]. Additionally, the use of stable isotope probing
(SIP) has been demonstrated for metaproteomics, allow-
ing for the characterization of microbial and protein turn-
over in a microbial community [37,38,39].
Recent extensions of metaproteomics have tended to
focus on three major types of ecosystems: (1) aqueous
(lakes and oceans), (2) terrestrial (soils and sediments),
and (3) eukaryotic host microbiomes (termites, mice,
plants, and humans). For aqueous ecosystems, metapro-
teomics has been used to decipher biological information
about microbial populations in highly productive or nutri-
ent-limited ocean ecosystems. For example, investigation
of a highly productive coastal upwelling system revealed
abundant microbial proteins involved in the prevention of
oxidative damage and protein refolding [40]. Related
work revealed how nutrient transport functions dominate
the SAR11 metaproteome at nutrient-limited locations in
the Sargasso Sea [41]; this general approach has been
extended to investigate ocean-scale shifts in microbial
nutrient utilization and energy transduction [42]. In a
more industrial context, metaproteomics has become a
critical research component for investigating and optimiz-
ing sewage sludge treatment by biological agents
[5,43,44].
For terrestrial ecosystem research, most work is focused
on characterization of microbes in soil in an effort to
better understand carbon/nitrogen flow, contaminant
remediation, and permafrost metabolic potentials
[45,46]. For example, metaproteomics has helped charac-
terize microbial metabolic activities relevant for biore-
mediation at nutrient-stimulated [47–49], xenobiotic [50],
hydrocarbon [51], and heavy metal-contaminated sites
[52,53]. Recent advances in in situ proteome extraction
techniques from soils have opened the door to a much
deeper level of metaproteome measurement [54,55]. Soil
metaproteomics has become a high interest research
target area, although significant challenges with regard
to microbial diversity, environmental matrices, and lim-
ited metagenomic information complicate this issue at
present.
In perhaps the most complex level of microbial metapro-
teomics, there is substantial interest in understanding the
symbiotic/pathogenic relationships between microbes and
their eukaryotic hosts. For example, to better understand
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the basis of cellulose degradation by termites, metaproteo-
mics was used to characterize the functional activities of
uncultivable symbiotic microbes in the termite hindgut
[56]. Metaproteomics has also been used to investigate
factors mediating plant–microbial interactions, in particular
focusing on the proteome differences between lab cultured
microbes versus their plant symbiont counterparts [57], or
factors influencing crop rhizosphere communities [58,59].
The last two to three years has seen an explosion of
research interest in the human microbiome, fueled prim-
arily by health-related issues. It is apparent that microbes
vastly outnumber human cells in even healthy individuals.
This necessitates a thorough understanding of both normal
(symbiotic) and diseased (dysbiotic) states, specifically
with regard to microbiome functional dynamics. In
response to the early interest in characterizing a possible
microbial basis for periodontal disease, a novel 3D peptide
fractionation method was used with tandem MS to charac-
terize human salivary microbiota [60], and proteomics was
used to study Porphyromonas gingivalis as part of a model
oral microbiome community [61].
One of the key microbe–host ecosystems is the human
gut, thus providing impetus for focused research in both
microbial metagenomics and metaproteomics. For
example, metaproteomics has functionally detailed the
microbiota in the developing human infant GI tract [62],
Figure 3
(a)
(b)
Comparison of clusters of orthologous group (COG) categories for human metagenomes and metaproteomes. (a) Average COG categories of two
metagenomes from the gut microbiota of two individuals from a previous study [65]; (b) average COG categories of the metaproteomes from the gut
microbiota of two individuals (figure reprinted with permission from Ref. [64]). Note the increased proportion of energy production, translation, and
carbohydrate metabolism observed in the metaproteomes relative to the metagenomes.
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Microbial metaproteomics Hettich et al. 377
and revealed temporal stability of a core proteome for an
established intestinal microbiome of an adult human [63].
To date, the deepest level of metaproteome characteriz-
ation has focused on the unaltered adult human gut
microbiomes for two healthy, matched human twins,
and provided a glimpse into the highly integrated
relationship between microbial and human proteins
[64]. Numerous proteins were identified from the most
dominant bacterial members, revealing a large invest-
ment into energy production and carbohydrate metab-
olism. Interestingly, the functional distribution of COGs
(clusters of orthologous groups) for the metagenome were
somewhat distinct from what was actually observed in the
metaproteome, as shown in Figure 3, highlighting the
reality of inequalities between genomic potential and func-
tional expression. Furthermore, several human proteins
were detected in these enriched bacterial samples, unco-
vering evidence of innate immunity response.
The field of environmental metaproteomics is developing
rapidly, and thus this discussion of specific research
examples will undoubtedly soon become dated. How-
ever, the work listed above has served as an ignition point
for the research area, heightening general interest and
specific research activity. As expected, numerous chal-
lenges inherent to environmental samples remain, in-
cluding formidable microbial complexity that requires
even greater enhancements to dynamic range, as well
RNA processing
Chromatin structure
Energy production
Cell Division
Amino-acid metabolism
Nucleotide metabolism
Carbohydrate metabolism
Coenzyme metabolism
Lipid metabolism
Translation
Transcription
Replication
Cell wall/membrane biogenesis
Cell motility
PTMs, protein folding and turnover
Inorganic ion metabolism
Secondary metabolite biosynthesis
General function prediction only
Function unknown
Signal-transduction mechanisms
Intracellular trafficking
Defense mechanisms
Cytoskeleton
Current Opinion in Microbiology
Current Opinion in Microbiology 2012, 15:373–380
378 Microbial proteomics
as issues with identifying proteins from genomically
similar species. These challenges provide the impetus
to continue the development of better experimental and
computational tools to address these difficult but not
intractable issues.
The ability to conduct metaproteomic measurements, in
particular for unculturable organisms, is already generat-
ing an exciting new level of systems-biology interrog-
ation, as evidenced by the almost exponential growth in
publications in this area over the past three years. The
simultaneous development, advancement, and integ-
ration of closely intertwined metagenomic/metaproteo-
mic approaches are helping provide a detailed look into
how communities assemble (even at the strain-resolved
level), distribute metabolic activities, progress with time,
and respond to environmental perturbations. It is clear
that microbes comprise the overwhelming number of
living organisms on earth, although how they influence
their environments remains largely unexplored. Though
still nascent, metaproteomics has the potential to deliver
extensive novel functional information for a range of
important environmental ecosystems, many of which
may contain unculturable members.
Acknowledgements
Financial research support was provided by the U.S. Department of Energy,
Office of Biological and Environmental Research, Genome Sciences
Program. Oak Ridge National Laboratory is managed by University of
Tennessee-Battelle LLC for the Department of Energy.
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