DOI: 10.1111/prd.

12351

REVIEW ARTICLE

Metaproteome and metabolome of oral microbial communities

Nagihan Bostanci1 | Melissa Grant2 | Kai Bao1 | Angelika Silbereisen1 |

Franziska Hetrodt1 | Daniel Manoil1 | Georgios N. Belibasakis1
1
Division of Oral Diseases, Department of Dental Medicine, Karolinska Institutet, Stockholm, Sweden
2
Biological Sciences, School of Dentistry, Institute of Clinical Sciences, University of Birmingham, Birmingham, UK

Correspondence
Nagihan Bostanci, Department of Dental Medicine, Division of Oral Diseases, Section of Periodontology and Dental Prevention, Karolinska Institutet, Alfred
Nobels Allé 8, Huddinge 14152, Sweden.
Email: nagihan.bostanci@ki.se

Funding information
Karolinska Institutet Strategic Funds; Swedish Research Council; Janggen-Pöhn Foundation

1 | PROTEO M E S : A N I M P O RTA NT PI EC E workflows that rely on mass spectrometry analysis of digested

O F TH E “ O M I C S ” PUZ ZLE
Protein function is representative of active biologic processes, and
the proteome describes the entire protein makeup of a cell, tissue,
or bodily fluid at a given point in time. The composition and modular
organization of the proteome governs the phenotype. The term pro-
1
teomics was coined in 1994 and over the last 25 years the field has
advanced with the rapid changes in mass spectrometry instrumen-
tation and innovations in quantitative chemistries and bioinformatic
tools. Although two-dimensional gel electrophoresis, accompanied
by mass spectrometry detection, was extensively used as the pro-
teomics method of choice in earlier days, 2,3 it is time-consuming,
labor-intensive, and limited by the number of resolved proteins.4
Currently, liquid chromatography coupled to tandem mass spec-
trometry is the most valuable tool for the systematic identification
and quantification of proteins on a large scale.5
Most of the proteomics workflows seek to identify a set of
proteins (discovery proteomics) or to quantify a relative or abso-
lute amount of identified proteins (quantitative proteomics) be-
tween conditions or to study the protein interactions within larger
protein complexes (interactomes) or in the context of broader bi-
ologic networks (systems biology). 6 The spectrum fragmentation
method includes two approaches, namely, “untargeted shotgun
proteomics”, which help achieve a deep coverage of the pro-
teome, and “targeted proteomics” for quantitatively comparing
large numbers of samples (hundreds to thousands) based on a de-
fined set of proteins (eg, selected reaction monitoring) (Figure 1).
Most high-throughput proteomic strategies utilize “bottom-up”
peptides from proteins with the help of specific enzymes (eg,
trypsin), but a “top-down” approach for analysis of intact proteins
is also possible. This allows not only identification of a protein
by name, but also of its intact molecular form, the presence of
isoforms, and potential modifications.7 The large-scale character-
ization of any given proteome is accomplished by comparing mea-
sured protein or peptide data with predicted protein or peptide
data derived from genomic information.
2 | M E TA PROTEO M I C S : A N E W R E S E A RC H
TO O L
Although the word proteomics covers the investigation of all pro-
teins from one species, metaproteomics refers to the study of
proteins from microorganism consortia. Because the oral cavity is
one of the most complex ecosystems in humans, it is imperative to
develop and utilize analytical approaches that can provide molecu-
lar level details on systems consisting of mixed microbial member-
ship. Metaproteomics is emerging as a complementary approach to
metagenomics and metatranscriptomics for deciphering the func-
tion of microbial communities or the identification of “microbial
functional networks”.8 Because proteins carry out most functions
in any given cells, protein quantities correlate quite well with micro-
bial metabolic activity.9 The term “metaproteomics” was defined in
2004 as “the large-scale characterization of the entire protein com-
plement of environmental microbiota, at a given point in time”.10
Progressively, metaproteomics has been successfully used for

© 2020 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd

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46     
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BOSTANCI et al. |
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F I G U R E 1   An outline of discovery and quantitative proteomic technologies. AQUA, absolute quantification; DIA, data independent
acquisition; ICAT, isotope-coded affinity tag; iTRAQ, isobaric tags for relative and absolute quantitation, amine-specific tag for relative and
absolute quantitation; SID, stable isotope dilution; SIL, stable isotope-labeled; SILAC, stable isotope labeling by amino acids in cell culture;
SRM, selected reaction monitoring; SWATH-MS, sequential windowed acquisition of all theoretical fragment ion mass spectra

analyzing microbial community structures on the basis of biomass
(total per species protein), instead of gene/genome copy counts.11
Although metaproteomics is a powerful tool in studying the pres-
ence and abundance of proteins in oral microbial communities, to date
only a handful of publications have mentioned “metaproteome and oral
microbiota” compared with hundreds of listings for metagenomics or
metatranscriptomics. Instead, mass spectrometry-based proteomics
profiling has been more widely used for dissection of the function of oral
microbiome and has been mainly applied to characterize in vitro-grown
oral biofilms or individually mixed bacteria grown in suspension.12
A quarter of a century on from its naming, proteomics has devel-
oped into a well-established field of research often used in the field of
dentistry, in particular periodontal diseases, for the identification of
host-derived biomarkers. The study of metaproteomes in dentistry is
steadily increasing but is more technically demanding than the study
of metagenomics or metatranscriptomics and so lags behind at pres-
ent. The specialized fields of proteomics/metaproteomics and their
application to characterize microbiota from the oral ecosystem will be
explored in the following sections. Such in-depth proteomic mapping
of oral biofluids or dental plaque may support patient stratification, in
13
line with precision oral health or the identified protein targets may
also warrant potential therapeutic exploitation, for instance by tar-
geted pharmacological inhibition approaches.
3 |  M E TA PROTEO M I C
C H A R AC TE R IZ ATI O N O F B I O FI LM S FRO M
TH E O R A L ECO S YS TE M
The healthy oral ecosystem is characterized by the presence of non-
shedding enamel hard tissues and a constant salivary flow. Survival
of the ~700 bacterial species that inhabit this environment is de-
pendent on their ability to adhere onto surfaces, grow, and form
biofilms.14,15 Proteins and glycoproteins of salivary origin readily ad-
sorb onto dental tissues to form a thin proteinaceous layer termed
the acquired enamel pellicle.16,17 The composition of this acquired
enamel pellicle modulates the selective bacterial attachment of early
colonizers, expressing receptors recognizing proteins of the salivary
pellicle.17,18 Rapidly, so-called bridging species such as Fusobacteria
will adhere to these early colonizers, in turn providing the surface
receptors for the incremental co-adhesion of late colonizers.17,19
These organized bacterial aggregates produce an extracellular poly-
meric substance mostly composed of exopolysaccharides, DNA,
and proteins. 20 Collectively, these bacterial aggregates embedded
in their extracellular polymeric substance define the oral biofilm.
This extracellular polymeric substance enhances adhesion to den-
tal surfaces, provides nutrient substrate, and creates a protective
barrier against environmental stresses. Additionally, the juxtaposi-
tion of cells within the extracellular polymeric substance facilitates
interspecies communication, signaling (quorum-sensing), and allows
mutually beneficial metabolic networking. 21-23
Under physiologic conditions, these biofilms may evolve in equi-
librium with the host.14 However, factors such as a deficient oral
hygiene, dietary habits, or host genetics may lead to ecological al-
terations within these bacterial communities that are the cause of
most common oral affections, that is, periodontal diseases and den-
tal caries, which then lead to endodontic infections and apical peri-
odontitis.15 For many years, the ecology of oral biofilms has been
studied using culture-dependent and close-ended molecular meth-
ods such as PCR and its variants, fluorescent in situ hybridization or
DNA hybridization assays. These classical approaches permitted the
identification of sets of bacterial taxa (1-50 species) that appeared to
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48       BOSTANCI et al.

TA B L E 1   Clinical studies characterizing the bacterial metaproteome of caries-related biofilms.

Proteomic Peptide Identified

Cohort definition Sampling application Approach number proteins Highlighted proteins Ref.

1 healthy patient After 14 d, 10 h 2DE, Discovery ≥4 521.5 ± 7.3 w/ Identified yet nonquantified, 43
volunteering to following the last MALDI- Quantitative sucrose including:
wear intra-oral treatment (w/ or TOF MS 512.0 ± 37.4 1. w/ sucrose: enolase
enamel blocks w/o sucrose), the w/o sucrose isoform 2, enterotoxin,
extra-orally enamel blocks 22 phosphglycerate mutase I.
treated or not (n = 8) were 2. w/o sucrose: lipase
with sucrose for removed from the precursor, thioesterase
14 d. oral cavity. The domain containing 1,
biofilm on enamel nucleoside diphosphate
blocks was then kinaseUpregulated w/
processed for the sucrose:
“extraction of Putative NADP-specific
extracellular matrix glutamate dehydrogenase,
proteins”. DnaK, translation elongation
factors (EF-Tu),
mannose-specific EIIAB (PTS
system), pyruvate kinase,
ATP synthase beta
Downregulated w/ sucrose:
GroEL, enolase isoform 1
17 patients Supra-gingival HILIC & Discovery 48% ≥2 7771 High abundance: 44
without caries biofilm from palatal RPLC-MS/ Quantitative 46% ≥2 2137 proteins involved in energy
lesions (n = 8), and lingual surfaces MS, LTQ- metabolism (glycolysis/
with ≥ 3 active was collected using Orbitrap glyconeogenesis) and
caries lesions excavators 24 h XL protein synthesis
(n = 9) after toothbrushing RPLC-MS/ Low abundance:
and pooled. 2 MS, LTQ- proteins involved in
biologic replicates Orbitrap regulatory functions and
were acquired on 2 Velos signal transduction
different days. Healthy:
LDH, glucose-1-phosphate
thymidylyltransferase,
1,4-alpha-glucan branching
enzyme, ornithine
carbamoyltransferase,
FtsZ, succinate-CoA ligase,
succinate dehydrogenase
Caries:
CPR0540 family
ABC transporters,
N-acetylglucosamine-6-
phosphate deacetylase,
FtsZ, PEP-PTS system,
copper-containing-nitrite
reductase, CspD

Abbreviations: ATP, adenosine triphosphate; HILIC, hydrophilic interaction liquid chromatography; LDH, L-lactate dehydrogenase; LTQ, linear trap
quadrupole; MALDI-TOF , matrix-assisted laser desorption/ionization-time of flight mass spectrometry; MS/MS, tandem mass spectrometry; NADP,
nicotinamide adenine dinucleotide phosphate; PTS, phosphotransferase system; RPLC, reversed-phase liquid chromatography; 2DE, two-dimensional
gel electrophoresis.

be infection-specific and somewhat distinct from those detected in
healthy sites. Later open-ended, high-throughput DNA-sequencing
methods, and mostly those relying on sequencing of 16S ribosomal
RNA gene amplicons, greatly expanded the catalog of species identi-
24,25
fied in the oral cavity to a much more diverse microbiota.
large-scale community surveys actually revealed that the same taxa
are often detected between healthy and diseased sites, yet in dif-
ferent relative abundances. 26 In other words, most oral diseases
are associated with compositional shifts in the microbiota that re-
sult in increased pathogenicity of the entire community, rather than
the presence of specific individuals. Whereas this comprehensive
knowledge of the taxa associated with different pathologies is a
These necessary step towards the understanding of pathologic processes,
these data alone do not permit full elucidation of the pathogenicity
triggers of biofilm communities. Therefore, recent focus was paid
to proteomic technologies with the aim of gaining deeper insights
BOSTANCI et al.
into the underlying bacterial effectors, that is, the metaproteome.
The following paragraphs describe recent findings in the bacterial
metaproteome from clinical biofilms associated with various oral
diseases. We specifically address the metaproteomic profiles iden-
tified in caries-related biofilms, and also those detected in infected
root canals associated with different types of apical periodontitis.
Metaproteomic data associated with periodontal diseases are ad-
dressed in the following sections that deal with subgingival plaque,
saliva, and gingival crevicular fluid.
4 |  TH E M E TA PROTEO M E O F C A R I E S -
A S S O C I ATE D B I O FI LM S
Dental caries is inarguably a highly prevalent multifactorial disease
affected by the complex reciprocities between microorganisms,
carbohydrate abundances, and host defense mechanisms. 27-29 It
was shown that the composition, function, and properties of saliva,
which are a key element in host defense, play an essential role in
the maintenance of the surface integrity of dental hard tissues.30,31
These protective components include the pellicle layer, which is
formed by proteins, carbohydrates, and lipids spontaneously adsorb-
ing onto enamel surfaces.32 In 1839, Alexander Nasmyth209 was the
first to describe this proteinaceous layer and postulate its embryo-
logic origin, which was finally disproved by Dawes et al33 discussing
the pellicle formation after tooth eruption and introducing the term
acquired enamel pellicle. Early in vitro investigations performing
electron microscopic analysis revealed the rapid adsorption of the
pellicle layer onto enamel specimen in less than 1 minute.32 These
initial stages of the protein adsorption process are modulated by
physicochemical interactions that involve van-der-Waals forces, hy-
drophobic interactions, and dipole-dipole effects promoting further
attachment of proteins and the development of a complex layer.34,35
The usage of scanning electron microscopy, transmission elec-
tron microscopy, and confocal laser scanning microscopy provided
deeper insights into the pellicle's thickness and ultrastructure pre-
senting different pellicle formation patterns conditional on time and
34
tooth anatomy. Recent advances in protein fragmentation meth-
ods and mass spectrometry technologies have made possible the
analysis of proteomic profiles from the acquired enamel pellicle and
have detected up to 363 various peptides and proteins.150 Abundant
proteinic pellicle constituents, such as sialylated mucins, statherins,
histatins, cystatins, or proline-rich proteins reveal binding motifs
for surface receptors of bacteria, thereby enabling the adhesion of
early colonizing species.36-38 Predominant early colonizers such as
Streptococcus salivarius, Streptococcus mitis, Streptococcus oralis, and
Streptococcus sanguinis allow the subsequent co-adhesion of Gram-
negative anaerobes, including Fusobacterium nucleatum, Prevotella
melaninogenica, and Veillonella spp.17 Of relevance to dental caries
is an ecological shift within the biofilm towards large abundances
of aciduric/acidophilic Gram-positive bacterial species, such as
Streptococcus mutans, Streptococcus sobrinus, Actinomyces spp., and
Lactobacillus spp., which are able to ferment dietary carbohydrates
|
      49
to organic acids.39 This acidic environment results in mineral loss of
dental surfaces and, if not reversed in its early stages, the subse-
quent widespread destruction of dental hard tissues.40,41
Because the availability of carbohydrates represents a crucial
environmental factor in the initiation and progression of dental car-
ies, the presence of different disaccharides was shown to influence
the proteinic composition of the extracellular polymeric substance
previously described by Cury et al.42 More recently, the same re-
search group performed two-dimensional gel electrophoresis and
matrix-assisted laser desorption/ionization time-of-flight mass spec-
trometry with the aim of characterizing the proteomic profiles of
the extracellular matrix from 14-day-old clinical biofilms exposed to
sucrose.43 To target extracellular proteins, the authors employed a
protein isolation method based on sodium hydroxide and ethylene-
diaminetetraacetic acid that was supposed to avoid disrupting intact
bacteria. In total, 54 proteins were identified, including 22 bacterial
proteins mainly related to stress responses, protein biosynthesis,
and energy metabolism (Table 1). Among these proteins isolated
from the extracellular matrix were several typical cytosolic proteins,
such as enolase, pyruvate kinase, DnaK, or translation elongation
factors. As discussed in the study, such high prevalence of cytosolic
proteins in the extracellular matrix of those aged biofilms may be a
result of leakage from membrane-damaged bacteria. The absence
of bacterial lysis was, however, not verified following the particu-
lar protein extraction protocol. Interestingly, the pathway analysis
showed diverse regulations of stress response proteins, indicating a
sucrose-induced upregulation of the chaperone DnaK, and concom-
itantly to a downregulation of GroEL.
Belda-Ferre et al 44 performed a study introducing a metapro-
teomic profile of eight healthy and nine caries-affected patients
using liquid chromatography/tandem mass spectrometry. The au-
thors detected 7771 bacterial and 853 human proteins presenting
the first accessible protein investigation of clinical oral biofilms.
The majority of bacterial metaproteome pathways identified were
associated with central metabolic and housekeeping processes,
for example, energy metabolism and protein synthesis. Based on
the metaproteomic profiles, no significant differences in abun-
dance of the bacterial genera were found between healthy and
caries-affected subjects. Representative genera accounting for
60%-90% of total diversity were Actinomyces, Corynebacterium,
Rothia, and Streptococcus. The quantitative approach employed
in this study allowed the identification and proposal of poten-
tial biomarkers according to the functions overrepresented in
healthy and diseased patients, respectively (Table 1). Potential
biomarkers abundantly present in disease were the glucose phos-
photransferase system, copper-containing nitrite reductase, and
stress response protein CspD, whereas L-lactate dehydrogenase,
succinate-CoA ligase, as well as succinate dehydrogenase, were
more predominant in healthy individuals. These pioneer findings
represent initial approaches to identify functions potentially pre-
venting tooth decay and more importantly detecting caries-prone
individuals prior to the formation of caries lesions. Mass spec-
trometry-based proteomics profiling of caries-associated biofilms
 |
50       BOSTANCI et al.

has also been applied to characterizing in vitro-grown oral biofilms

or single bacterial species grown in suspension.45 In such studies
St. mutans was found to antagonize by Streptococcus gordonii and
St. sanguinis.46,47 Among 1209 protein spots detected in a two-di-
mensional gel electrophoresis approach, Yoshida et al3 found that
the expression level of one peroxide-resistance protein, Dpr, was
4.3-fold higher when co-cultured with St. gordonii compared with
the St. mutans mono-species biofilm. Based on this discovery, the
authors constructed a knockout strain of St. mutans and found that
its growth was inhibited by both St. gordonii and St. anguinis in du-
al-species biofilms. Considering that both streptococci use oxygen
availability and produce hydrogen peroxide against St. mutans,48
Dpr might be one of the essential proteins for the survival of St.
mutans in the presence of other oral streptococci. When mixed
with St. oralis or Actinomyces naeslundii, St. mutans is initially pres-
ent at low levels and gradually becomes elevated later on during
biofilm maturation, until saturation.49 In addition, a higher number
of St. mutans proteins was detected at 67 hours (1153) compared
with at 115 hours (1009), including many related to intracellular
polysaccharide storage, lipoteichoic acid metabolism, and biofilm F I G U R E 2   Predicted functional partners of Streptococcus
mutans glucosyltransferase-SI, depicted via string analysis.
formation. This suggests that St. mutans may display specific time
Downloaded from the Uniprot database (https://www.unipr​ot.org)
windows of higher activity during its growth. An example of a St.
mutans protein interaction network is provided in Figure 2.

a comprehensive map of the taxa associated with endodontic infec-

5 |  TH E M E TA PROTEO M E O F
E N D O D O NTI C I N FEC TI O N S A S S O C I ATE D
W ITH A PI C A L PE R I O D O NTITI S
Endodontic infections, the bacterial colonization of the pulpal space,
occur as a sequel to caries, trauma, or periodontal diseases and almost
invariably lead to pulpal necrosis.50 The necrotized pulpal space is
rapidly colonized by polymicrobial bacterial communities that adhere
51-53
to root canal walls and form organized biofilms. Bacterial commu-
nities and their by-products gain access to periapical tissues through
apical or lateral foramina. This interaction between bacteria and host
defenses induces a series of inflammatory changes within periapical
tissues causing the development of apical periodontitis. As a result
of the balance between the pathogenicity of the bacterial communi-
ties and host defenses, such apical lesions may clinically appear as
asymptomatic (chronic lesions) or symptomatic (acute or abscessed
54,55
lesions). Additionally, primary apical periodontitis lesions, caused
by bacteria that initially invaded the necrotic pulp, are further dif-
ferentiated from posttreatment apical periodontitis lesions, caused
by bacteria that persisted into the root canal or repenetrated after
55-59
an endodontic treatment was performed. Because the success
of endodontic treatment primarily relies on eradication of the infec-
tion, a thorough understanding of the ecology and pathogenicity of
intra-radicular biofilms is of prime interest. This last decade, con-
siderable efforts, mostly using high-throughput sequencing of 16S
ribosomal RNA gene amplicons, have been devoted to defining the
taxonomic composition of endodontic infections associated with dif-
ferent types of apical periodontitis. Together, these studies formulate
tions.60 Whereas nucleic acid-based taxonomic characterization
provides important insights into the ecology of these biofilms, it is
less informative regarding their metabolic functions, physiology, and
pathogenicity. Therefore, one important challenge that remains is to
identify the proteome expressed by these communities and which
ultimately modulates bacterial interactions and virulence.
Nandakumar et al61 were the first to employ proteomic ap-
proaches, that is, liquid chromatography/tandem mass spectrometry,
to characterize the bacterial metaproteome expressed in endodontic
infections. The authors used paper points to sample seven infected
root canals indiscriminately associated with primary or posttreatment
apical periodontitis (Table 2). The proteomic profiles identified were
divided into proteins belonging to either the taxon Enterococcus fae-
calis (n = 57) or to nonenterococcal species (n = 31). In both cases,
the majority of the proteins identified were outer membrane proteins
potentially contributing to virulence, pathogenicity, and antibiotic re-
sistance (ABC transporter-adenosine triphosphate-binding protein or
N-acetylmuramoyl-L-alanine amidase family 4) (Table 2). For instance,
proteins assigned to E. faecalis included the bacteriocin BacB along
with TetM, which mediates resistance to tetracycline by protecting its
ribosomal binding sites, or several vancomycin sensors and transcrip-
tion factors (VanSB, VanSG2, and VanTG2), indicating the presence of
translated vancomycin resistance operons. Among the most frequently
identified proteins from nonenterococcal species were several viru-
lence-related adhesins (fibrinogen-binding protein, fibronectin-bind-
ing A domain protein), proteases (collagenase/metalloprotease), the
stress response chaperonin GroEL, and the penicillin binding protein
PBP-2B, conferring first-level resistance to beta-lactams.
BOSTANCI et al. |
      51

TA B L E 2   Clinical studies characterizing the bacterial metaproteome of biofilms from endodontic infections.

Cohort Proteomic Peptide Identified

definition Sampling application Approach number proteins Highlighted proteins Ref.

7 patients Root canals sampled LC-MS/MS Discovery >2 89 (57 of Enterococcal origin: 61
with infected with paper points enterococcal ABC transporter-ATP-binding
root canals during the course origin, 31 of protein, N-acetylmuramoyl-
associated of an endodontic nonenterococcal L-alanine amidase family 4,
with primary re-treatment. origin) BacB, TetM, VanSB, VanSG2,
(n = 4) or VanTG2
persistent Nonenterococcal origin:
(n = 2) AP, penicillin-binding protein
plus 1 case of 2B (Lactococcus lactis,
unclassified Bacteroides vulgatus),
AP. microbial collagenase
metalloprotease (Bacillus
spp.), fibrinogen-binding
protein (Streptococcus
agalactiae), fibronectin-
binding A domain protein
(Exiguobacterium spp.)
12 root canals Cases of nanoLC- Discovery NR 261 (173 in Pathogenicity related: 62
associated asymptomatic AP LTQ Velos abscess cases Glycosyltransferase 1, tight
with (12) were sampled Orbitrap & and 88 in adherence protein G,
asymptomatic by syringe aspiration LC-QTOF asymptomatic coagulation factor 5/8 type
AP and 2 from the root canal. root canals) domain
cases of Sampling was Resistance/survival related:
acute apical repeated after the DnaK, GroEL, HslU, UvrABC,
abscess. chemo-mechanical beta-lactamase, TetR
preparation. Acute
abscesses (2) were
sampled by pus
aspiration through
the mucosa.
10 patients Apical sections of nanoLC- Discovery NR 733 (460 in root Related to antibiotic 63
displaying root canals (3-5 mm) LTQ Velos sections and 360 resistance & stress response:
post- resected during Orbitrap in the associated beta-lactamase, TetR, MarR,
treatment apical surgery prior apical lesions) DnaK
apical to cryo-pulverization. The taxon with the highest
periodontitis The associated number of proteins identified
(8:M, 2:W). apical lesion was also was genus Enterococcus.
collected.
20 patients Root canals sampled 2D-LC- Discovery NR 720 Most commonly identified 64
displaying with paper points ESI-MS/ protein:
dental during an endodontic MS LPXTG-motif cell wall anchor
root canals re-treatment. protein (taxonomically
associated assigned to Streptococcus
with post- anginosus)
treatment AP OMPs including:
(n = 20) Chitinase 3, aspartate kinase,
kinase-domain protein.
Proteolysis and DNA repair
including:
IgA-specific serine
endopeptidase, collagenase,
unfoldase HslU
Antibiotic resistance
including:
MdtB, beta-lactamase, TFs
including AsnC-type, LacL-
type and QseB-type

Abbreviations: AP, apical periodontitis; LC-MS/MS, liquid chromatography with tandem mass spectrometry; nanoLC, nanoflow liquid
chromatography; LTQ, linear trap quadropole; NR, not reported; OMPs, olfactory marker proteins; QTOF, quadrupole time-of-flight; 2D-LC-ESI-MS/
MS, two-dimensional liquid chromatography electrospray ionization with tandem mass spectrometry.
 |
52       BOSTANCI et al.

TA B L E 3   In vitro studies characterizing the bacterial metaproteomes associated with caries or periodontal diseases

Proteomic
Author Year Bacteria in the biofilm model Identified proteinsa  application Ref.

Mono-species biofilms
Llama-Palacios 2017 Aggregatibacter actinomycetemcomitans 87 2DE, MALDI TOF 2
et al MS/MS
Sadeghinejad 2016 Streptococcus mutans 314 LC-ESI-MS/MS 195
Bandara 2013 Candida albicans 7 quantified 2DE, MALDI TOF 196
MS/MS
Kishi et al 2012 Porphyromonas gingivalis 165 2DE, MALDI TOF 197
MS/MS
Chew et al 2012 Fusobacterium nucleatum alkaline-induced 421 2DE, MALDI-MS 77
biofilms & LC-ESI ion trap
MS/MS
Seneviratne et al 2010 Candida glabrata 24 2DE, MALDI TOF 76
MS/MS
Pham et al 2010 Tannerella forsythia 348 iTRAQ-labele, 198
ESI-qQ-TOF-MS/
MS
Ange et al 2008 Porphyromonas gingivalis 116 1DE, LC-MALDI 78
TOF/TOF-MS
Seneviratne et al 2008 Candida albicans 25 2DE, MALDI TOF 75
MS/MS
Rathsam et al 2005 Streptococcus mutans biofilms 242 protein spots 2-DGE MALDI 45
TOF MS
Multispecies biofilms
Bao et al 2018 Aggregatibacter actinomycetemcomitans + 6 5469 Orbitrap Fusion 90
species biofilmsc 
Bao et al 2017 Anaeroglobus geminatus + 10 species biofilmsb  3213 Orbitrap Fusion 88
Mohammed et al 2017 Fusobacterium nucleatum and Porphyromonas 542 LFQ, 82
gingivalis LTQ-Orbitrap
Kuboniwa et al 2017 Porphyromonas gingivalis and S gordonii 1153 Orbitrap Fusion 181
Bao et al 2015 Aggregatibacter actinomycetemcomitans + 10 3352 Q-Exactive MS 211
species biofilmsb 
Chavez de Paz 2015 Enterococcus faecalis and Lactobacillus salivarius, 3proteins from 5 2DE, high 199
et al Actinomyces naeslundii or Streptococcus gordonii protein spts capacity ion trap
ultra MS
Yoshida et al 2015 Streptococcus mutans, Streptococcus gordonii, 1 protein from 1209 ion-trap mass 3
Streptococcus sanguinis detected protein spectrometer
spots (Q-Tof2)
Maeda et al 2015 Porphyromonas gingivalis and Streptococcus oralis 593 Q-ExactiveHybrid 79
Quadrupole-
Orbitrap MS
Hendrickson et al 2014 Fusobacterium nucleatum, Streptococcus gordonii, 1210 LTQ MS 200
and Porphyromonas gingivalis
Hendrickson et al 2012 Fusobacterium nucleatum, Streptococcus gordonii, 1122 LTQ MS 81
and Porphyromonas gingivalis
Klein et al 2012 Streptococcus mutans, alone or mixed with 1966 MudPIT LTQ- 49
Actinomyces naeslundii and Streptococcus oralis Orbitrap mass
spectrometer
Zainal-Abidin 2012 Porphyromonas gingivalis, Treponema denticola, 1485 2DE, LC-MALDI 86
et al and Tannerella forsythia TOF/TOF MS
Kuboniwa et al 2009 Fusobacterium nucleatum, Streptococcus gordonii, 1156 LTQ MS 83
and Porphyromonas gingivalis

(Continues)
BOSTANCI et al. |
      53

TA B L E 3   (Continued)

Proteomic
Author Year Bacteria in the biofilm model Identified proteinsa  application Ref.

Host-biofilm interaction models

Bao et al 2015 10-species biofilm modela  vs 3D cultureb  3363 Q-Exactive MS 91
Custodio et al 2015 Candida albicans 15 nanoUPLC 201
flight mass
spectrometry
Cavalcanti et al 2014 Streptococcus oralis and Fusobacterium nucleatum 11 Q-T of 202
Ultima mass
spectrometer

Abbreviations: ETD, electron transfer dissociation; FDR, false-discovery rate; IT, ion trap; iTRAQ, isobaric tags for relative and absolute quantitation;
LCQ, liquid chromatography quadrupole; LFQ, label-free quantitative; LTQ, linear trap quadrupole; MALDI, matrix-assisted laser desorption
ionization; MALDI-TOF/TOF, matrix-assisted laser desorption/ionization-time of flight mass spectrometry; MS, mass spectrometer; Q-TOF MS,
quadrupole time-of-flight mass spectrometry.
a
Maximum identified/quantified proteins were reported based on the following rules: 1: only the maximum identified protein number was reported if
the experiment was performed under different conditions; 2: total protein numbers were reported if the experiment has replicates; 3: the number of
identified and quantified proteins were reported if only regulated protein were reported.
b
10-species biofilm model (consisting of Campylobacter rectus, Fusobacterium nucleatum, Porphyromonas gingivalis, Prevotella intermedia, Tannerella
forsythia, Treponema denticola, Veillonella dispar, Actinomyces oris, Streptococcus anginosous, Streptococcus oralis).
c
6-species biofilm model (consisting of A. oris, C. albicans, F. nucleatum, St. oralis, St. mutans, and V. dispar).

A subsequent study by Provenzano et al62 investigated the
metaproteome of endodontic infection associated with asymp-
tomatic apical periodontitis lesions and acute abscesses by means
of liquid chromatography/tandem mass spectrometry. The authors
reported a significantly larger number of proteins identified from
abscess samples (n = 173) compared with asymptomatic apical peri-
odontitis lesions (n = 88) (Table 2). It is, however, important to note
that the direct comparison between these two clinical presentations
of endodontic infection may be somewhat irrelevant as they were
sampled from largely different ecosystems. Indeed, whereas sam-
ples from asymptomatic apical periodontitis lesions were collected
from root canals, pus aspiration was used as a proxy for abscess
cases. Irrespective of the type of endodontic infection, the results
demonstrate that the majority of bacterial proteins identified were
related to housekeeping functions such as translation, energy me-
tabolism, or DNA processing. In addition, the authors also reported
several proteins involved in pathogenic processes, such as stress re-
sponse (DnaK, GroEL), antibiotic resistance (beta-lactamase, TetR),
and biofilm formation (tight adherence protein G). Three years later,
the same group extended their investigations to infected root canals
63
associated with posttreatment apical periodontitis. In this study,
metaproteomic analyses were performed on cryo-pulverized apical
root fragments and their associated lesions (soft tissue) were col-
lected by apical surgery (Table 2). Among the 460 bacterial proteins
identified in root sections and the 360 identified within the associ-
ated lesions, most bacterial proteins were involved in housekeeping
functions, such as transcription/translation and energy metabolism.
As observed in their previous study, several other identified proteins
contribute to pathogenicity (extracellular matrix binding protein
ebh, accessory colonization factor), antibiotic resistance (beta-lac-
tamase, TetR, MarR, cation/multidrug efflux), and stress response
(DnaK, chaperonin HscA homolog). More recently, a study by
Francisco et al64 also investigated the bacterial metaproteome of
teeth associated with posttreatment apical periodontitis. Following
sampling of root canals with paper points, metaproteomic analysis
was performed using two-dimensional liquid chromatography/tan-
dem mass spectrometry (Table 2). A total of 720 bacterial proteins
were identified, among which the majority were related to house-
keeping functions. In addition, the authors reported the presence
of several proteins implicated in bacterial adhesion (LPXTG-motif
anchor protein), proteolysis, or DNA repair, all of which contribute
to pathogenicity. Equally important, these analyses also identified
beta-lactam–degrading enzymes (beta-lactamase), multidrug efflux
pumps (MdtB), and transcriptional regulators of antibiotic-resistance
genes (AsnC-type, LacL-type, and QseB-type).
Collectively, these studies reported rather similar metaproteomic
profiles among the various types of endodontic infections and api-
cal periodontitis lesions studied (Table 2). Among the functional
categories obtained from gene ontology annotations, housekeeping
processes were among the most abundant.62-64 This indicates the
presence of metabolically active communities within the root canal
system, and further suggests that active bacteria may invade periapi-
cal tissues, as evidenced by samples collected from apical lesions.63 In
addition to proteins related to housekeeping functions, many others
appeared to be involved in pathogenic processes. Typically, detected
proteins such as fibrinogen-binding protein, coagulation factor 5/8
type domain, fibronectin binding A domain, polysaccharide lyases,
or the extracellular polymeric substance producing enzyme glyco-
syltransferase I, are crucial for adhesion and biofilm formation.65-67
Equally important for pathogenicity are the numerous proteolytic
enzymes detected including collagenases, metalloproteases, serine
proteases, and extracellular peptidases.61-64 Bacterial proteolytic
activities are indeed involved in acquisition of amino acids, degra-
dation of host connective tissue, and immune evasion through the
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54       BOSTANCI et al.

TA B L E 4   Clinical application of metaproteomics in saliva

Cohort definition Sample Proteomic application Approach Peptide number

Oral health adopted from92

Healthy (n = 4+1 + 1+N/A) Supernatant from unstimulated IEF, LC-MS/MS (LTQ or QSTAR) Discovery ≥2
(consortium of 4 research saliva (individual and pooled)
groups)
Healthy (n = 1) Supernatant from unstimulated 2D SCX-LC-MS/MS, LCQ-IT Discovery ≥2
saliva (individual)
Healthy (n = 2) Supernatant from unstimulated 2DE, nanoLC-MS/MS, QSTAR Discovery ≥2
saliva (pooled) XL
Healthy (n = 1) Supernatant from unstimulated Free flow electrophoresis, LC- Discovery ≥1
saliva (individual) ESI MS/MS, LTQ
Healthy (n = 1) Supernatant from unstimulated CIEF-nanoRPLC-MS/MS, LC- Discovery N/A
saliva (individual) ESI MS/MS

Healthy (n = 3+10 + 10)
(NIDCR-funded effort of 3
research groups)
Healthy (n = 6)
Healthy (n = 10)
Healthy (n = 2)
Healthy (n = 5)
Stimulated and unstimulated 2DE/ IEF, LC-MS/MS (LCQ Discovery ≥1
parotid and submandibular/ Deca, LCQ DecaXP, LTQ 2D,
sublingual saliva (pooled) LTQ-Orbitrap, QSTAR Pulsar
X), LC-MALDI TOF/TOF MS,
LC-QqTOF MS (QSTAR Pulsar
X)
Supernatant from unstimulated Proteominer beads, IEF, Discovery ≥1
saliva (pooled) SCX-LC-MS/MS, capillary
RPLC-MS/MS, LTQ-Orbitrap
XL
Supernatant from stimulated LC-ESI-MS/MS Discovery ≥2
saliva from minor salivary glands
(individual and pooled)
Supernatant from stimulated Lectin-affinity chromatography Discovery ≥2
submandibular/ sublingual (glycoprotein enrichment),
(noncannulated) saliva LC-MS/MS, LCQ Deca XP,
LTQ linear IT or hybrid LTQ
Orbitrap, SDS-PAGE, Western
blot
Supernatant from whole saliva IEF, glycoprotein enrichment, Discovery N/A
and stimulated submandibular, 1DE and 2DE LC-QqTOF MS/
sublingual and parotid saliva MS, QSTAR XL
(pooled)
BOSTANCI et al. |
      55

Maximum identified/ Nonhuman Ref

quantified proteins proteins Highlighted nonhuman proteins no.

1456 Bacterial: 12 N/A 98

102 N/A N/A 99

174 N/A N/A 100

437 N/A N/A 101

1381 31 bacterial Acinetobacter sp. ADP1, Archaeoglobus fulgidus DSM 4304, Bacillus subtilis subsp subtilis str.68 2, 102

species Bdellovibrio bacteriovorus HD100, Bordetella pertussis Tohama I, Burkholderia mallei ATCC 23 344,
Clostridium perfringens str.3 2, Clostridium tetani E88, Enterococcus faecalis V583,
Fusobacterium nucleatum subsp nucleatum ATCC 25 586, Haemophilus ducreyi 35000HP,
Helicobacter pylori 26 695, Lactobacillus plantarum WCFS1, Mesorhizobium loti MAFF303099,
Methanococcus maripaludis S2, Methanosarcina acetivorans C2A 2, Methylococcus
capsulatus str. Bath, Mycoplasma penetrans HF-2, Mycoplasma pulmonis UAB CTIP,
Parachlamydia sp UWE25, Photobacterium profundum SS9, Porphyromonas gingivalis W83,
Pyrobaculum aerophilum str. IM2, Pyrococcus horikoshii OT3, Streptococcus agalactiae NEM316,
Streptococcus mutans UA159, Streptococcus pneumoniae TIGR4, Streptomyces avermitilis MA-
4680, Thermoanaerobacter tengcongensis MB4, Vibrio vulnificus YJ016, Wigglesworthia glossinidia
endosymbiont of Glossina brevipalpis
1166 (parotid: 914, N/A N/A 103
submandibular/
sublingual: 917)

2340 N/A N/A 104

56 N/A N/A 106

277 N/A N/A 107

193 (whole saliva: N/A N/A 108

62, parotid: 34,
submandibular: 44,
sublingual: 53)

(Continues)
 |
56       BOSTANCI et al.

TA B L E 4   (Continued)

Cohort definition Sample Proteomic application Approach Peptide number

Healthy (n = 3) Supernatant from unstimulated Glycoprotein enrichment Discovery N/A

saliva (pooled) (MNP@lectins), MALDI-TOF/
TOF
Healthy (n = 14) Supernatant from unstimulated Glycoprotein enrichment Discovery N/A
saliva (pooled) (Proteominer or Library-2
beads), SDS-PAGE, SCX-
LC-MS/MS, LTQ-Orbitrap,
mTRAQ
Healthy (n = 1) Whole saliva MALDI-MS, LC-MS/MS Discovery N/A
Healthy (n = N/A) Supernatant from unstimulated Proteominer beads, SDS-PAGE, Discovery ≥2
saliva (pooled) SCX-capillary RP-HPLC,
Healthy (n = 5) Supernatant from whole saliva nanoLC–ESI–MS/MS, LTQ Discovery ≥1
(pooled) linear IT
Healthy (n = 3) Supernatant from unstimulated 2DE, PMF, MALDI QqTOF MS/ Discovery N/A
parotid saliva MS, MALDI TOF MS, MALDI
TOF/TOF MS/MS
Health HAA group (n = 18), Supernatant from unstimulated IEF, SDS-PAGE, AOD, PLS Discovery N/A
healthy LAA groups (n = 23) saliva (pooled) analysis, MALDI-TOF MS/MS,
QStar XL
Healthy (n = N/A) Supernatant from unstimulated Amylase and plasmatic protein Discovery ≥1
saliva (pooled) depletion, SDS-PAGE, SCX
chromatography, LC-MS/MS,
FT LTQ-Orbitrap Velos
Healthy (n = 6) Supernatant from saliva (pooled) protein DRC, multidimensional Discovery N/A
peptide fractionation,
high-mass accuracy MS/MS,
LTQ-Orbitrap
Healthy (n = 5) Supernatant and pellet from nano LC-MS/MS, Ultimate Discovery ≥2
stimulated (Salivette or paraffin 3000 Nano HPLC
gum) saliva

Healthy (n = 24)
Healthy (n = 8)
Periodontal diseases adopted from131,132
Healthy (n = 4), diseased
(n = 4)
Healthy (n = 5), generalized
aggressive periodontitis
(n = 5)
Generalized periodontitis
(n = 9)
Pellet from stimulated saliva nano LC-MS/MS, Ultimate Discovery ≥1
(individually), tongue samples 3000 Nano HPLC
Whole saliva from cotton LFQ LC-MS/MS, MALDI-TOF Quantitative N/A
swabs, collection after waking MS
and postprandial an whole
unstimulated saliva after waking
(individual)
Unstimulated whole saliva 2DE, MALDI-TOF, Q-TOF 2 Discovery ≥2
(individual) MS/MS, N-terminal amino
acid sequencing, Western blot
Supernatant from unstimulated 2DE, ESI-MS/MS, ELISA Discovery >2
saliva (individual and pooled)
Supernatant from stimulated 2DE, LC-ESI-MS/MS, Discovery ≥3
saliva (individual) MALDI-TOF
BOSTANCI et al. |
      57

Maximum identified/ Nonhuman Ref

quantified proteins proteins Highlighted nonhuman proteins no.

68 N/A N/A 109

Without enrichment: N/A N/A 105

93, with
enrichment: 183

45 glycoproteins N/A N/A 203

85 phosphoproteins N/A N/A 110

65 phosphoproteins N/A N/A 111

27 N/A N/A 112

65 protein bands N/A N/A 113

1256 N/A N/A 114

N/A Bacterial: Streptococcus, Rothia, Actinomyces, Prevotella, Neisseria, Veilonella, Lactobacillus, Selenomonas, 128
2176, 65 Pseudomonas, Staphylococcus, Campylobacter
genera from
12 phyla
N/A Bacterial Actinomyces, Prevotella, Streptococcus, Rothia 127
pellet: 1005
(paraffin),
313
(Salivette)
38 genera,
90 species
4280 Bacterial: Rothia, Prevotella, Streptococcus, Veillonella, Fusobacterium, Neisseria, Haemophilus, 129
2633 Actinobacteria, Proteobacteria, Firmicutes, Bacteriodetes, Fusobacteria,
5500, single-run Bacterial: F1WNZ3 (homolog of chaperone protein HscA) (Moraxella catarrhalis), A0A096BHY1 97
analysis: 3835 2234 from (glyceraldehyde-3-phosphate dehydrogenase), E0Q9Q6 (subunit of DNA polymerase III)
identified, 3700 50 genera
quantified

202 N/A N/A 115

21 (1) (Elongation factor 2) 116

126 N/A N/A 117

(Continues)
 |
58       BOSTANCI et al.

TA B L E 4   (Continued)

Cohort definition Sample Proteomic application Approach Peptide number

Chronic periodontitis in Supernatant of unstimulated IEF, 2DE, LC/MS/MS, Discovery >2

maintenance phase (n = 5), saliva (pooled) nanoLC-Q-Tof
healthy (n = 5)
Healthy (n = 5), gingivitis Supernatant from unstimulated 2DE, MALDI-Tof/Tof, Discovery >2
(n = 5) saliva (pooled) nanoLC-Q-Tof
Diabetes with (n = 15) Supernatant from unstimulated 2DE, MALDI-TOF/TOF Discovery N/A
/wo periodontitis (n = 10) saliva (individual)
Obese with (n = 13) Supernatant of stimulated saliva SELDI-TOF-MS, MALDI-TOF/ Discovery N/A
/wo periodontitis (n = 25), (individual) TOF
healthy control (n = 19)
Periodontally healthy Supernatant from stimulated Orbitrap Velos-MS (LC-MS/MS) Quantitative N/A
(n = 20), periodontally saliva (individual)
diseased (n = 20)
Healthy (n = 12), aggressive Supernatant from unstimulated LC-MRM Quantitative 1
periodontitis (n = 10), saliva (individual)
chronic periodontitis
(n = 10)
Generalized chronic Supernatant from unstimulated LTQ Orbitrap Velos (LC-MS/ Quantitative ≥1
periodontitis (n = 17), saliva (individual) MS), LC-SRM-MS
generalized aggressive
periodontitis (n = 17),
gingivitis (n = 17), healthy
(n = 16)
Periodontitis (n = 10), caries Stimulated whole saliva LC-MS/MS Quantitative N/A
(n = 10), healthy (n = 10) (individual)

Caries
Healthy elderly (n = 20),
elderly with root caries
(n = 21), healthy young
(n = 10)
Supernatant from stimulated SDS-PAGE, LC-MS/MS, 6330 Discovery ≥2
parotid saliva (pooled) LC/MSD Trap XCT Ultra IT
MS

Abbreviations: AOD, average optical density; CIEF, capillary isoelectric focusing; DRC, dynamic range compression;
FT-ICR, Fourier Transform ion cyclotron resonance; HAA, high aggregation-adherence; HPLC, high performance liquid chromatography;
ICAT, isotope-coded affinity tag; IT, ion trap; iTRAQ, isobaric tags for relative and absolute quantitation; LAA, low aggregation-adherence;
LC-ESI-MS, liquid chromatography electrospray ionization mass spectrometry; LC-MS/MS, liquid chromatography/tandem mass spectrometry;
LC-MSE, liquid chromatography mass spectrometry in data-independent analysis mode; LCQ, liquid chromatography quadrupole;
LFQ, label-free quantitative; LTQ, linear trap quadropole; MS, mass spectrometer; MS/MS, tandem mass spectrometry;
MALDI, matrix-assisted laser desorption/ionization; mTRAQ, amine-specific tag for relative and absolute quantitation;
N/A, not applicable; PLS, partial least squares; PMF, peptide mass fingerprinting; Q-TOF, quadrupole time-of-flight;
RPLC, reversed phase liquid chromatography; SCX, strong cation exchange; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis;
SELDI, surface enhanced laser desorption/ionization; TMT, tandem mass tag; TOF, time of flight; 2DE, two-dimensional gel electrophoresis;
UPLC, ultra performance liquid chromatography; NIDCR, .

proteolysis of antibodies and complement molecules.68-70 Numerous
stress response proteins such as the chaperonins GroEL, DnaK, or
HslU were also frequently detected. In addition to contributing to
peptide refolding and assembly, these stress response proteins may
also exhibit cytotoxicity and promote the excretion of pro-inflamma-
tory cytokines, thereby potentially contributing to the pathogenesis
of apical periodontitis.71,72 Finally, antibiotic-resistance factors have
been recurrently detected in endodontic infections. These findings
are clinically relevant because systemic antibiotics often become nec-
essary in cases of aggravated endodontic abscesses or exacerbations
leading to more severe septic complications.73,74 Taken together,
these metaproteomic studies indicate that bacteria survive within the
environment of endodontic infections by expressing stress response
proteins, enhancing virulence traits and antibiotic-resistance factors. 
6 | TH E M E TA PROTEO M E O F B I O FI LM S
A S S O C I ATE D W ITH PE R I O D O NTA L
DISEASES
One of the classic examples of biofilm-associated infections
in humans is periodontitis, which involves the inflammatory
BOSTANCI et al. |
      59

Maximum identified/ Nonhuman Ref

quantified proteins proteins Highlighted nonhuman proteins no.

Healthy: 236 ± 10, N/A N/A 118

diseased: 322 ± 12

Healthy: 236 ± 10, N/A N/A 119

gingivitis: 256 ± 16
42 N/A N/A 120

Ob + versus Ob-: N/A N/A 121

8, Ob + P+ versus
Ob + P-: 3
344 N/A N/A 122

28 N/A N/A 123

LTQ: 486, SRM: 60 LTQ: 24 Metallo-beta-lactamase of Treponema denticola, glucose-6-phosphate isomerase of Prevotella 124
bacterial, 8 oralis, glyceraldehyde-3-phosphate dehydrogenase of Streptococcus oralis
fungal, SRM:
N/A

4161 bacterial: Streptococcus, Prevotella, Veillonella, Rothia, Neisseria, Haemophilus, Fusobacterium, Leptotrichia, 126
1946, 20 Selenomonas,
genera, 81 Rhodotorula mucilaginosa, Veillonella atypica, Prevotella histicola,
species Prevotella melaninogenica, Streptococcus salivarius

Healthy elderly: 226, N/A N/A 125

elderly with root
caries: 219, healthy
young: 139

destruction of the periodontal tissues as a result of subgingival
biofilm colonization. Subgingival biofilm communities are com-
posed of multiple different species, mutually dependent upon
each other for survival, and exhibiting antagonistic or synergistic
interactions. The functional characterization of subgingival bio-
film communities by metaproteomic profiling can lead to valuable
information about the critical mechanisms driving community de-
velopment and structure in healthy and diseased states. Table 3
provides a summary of the proteomic approaches investigating in
vitro biofilm models.
6.1 | Insights from experimental in vitro biofilm
models
Earlier proteomic workflows mainly employed suspensions of peri-
odontal pathogens in a planktonic state with the main anim of under-
standing protein composition. Metaproteomics can be particularly
applicable to the case of in vitro polymicrobial biofilm communities
that resemble dental plaque in vivo. In the instance of Porphyromonas
gingivalis, several virulence factors that support its survival, regulate
its communication with other species, or modulate the inflammatory
 |
60       BOSTANCI et al.
response of the colonized host tissue, are expressed. Reportedly,
overexpression of outer membrane proteins (eg, adhesins) and
C-terminal domain family proteins (eg, gingipains) has been observed
in Po. gingivalis biofilms, compared with its planktonic form. 2,75-78
In multispecies biofilms, the interaction between Po. gingivalis and
early colonizing streptococci has been highlighted for warranting
further investigations. It was recently demonstrated that omission of
streptococci from the biofilm results in numeric changes of Po. gingi-
valis. In dual-species biofilms with St. oralis, the abundance of the cell
wall and membrane transport protein FimA, an important virulence
factor for Po. gingivalis, showed increased abundance compared
with when grown as a single species.79 Fusobacterium nucleatum has
the ability to interact with many other supragingival or subgingival
80
microorganisms, and has therefore been studied extensively in
biofilms models. In a dual-species biofilm, the presence of F. nuclea-
tum altered more than 300 proteins expressed in St. gordonii.81 Co-
culturing with St. gordonii promoted the pentose phosphate pathway
81
and malate oxidoreductase of F. nucleatum. When co-cultured with
Po. gingivalis, many F. nucleatum proteins related to the ethanola-
mine81 and butyrate production pathways82 were downregulated. In
a triple species biofilm model consisting of St. gordonii, F. nucleatum,
and Po. gingivalis, the expression of F. nucleatum glycolysis protein
was reduced, concomitant to a reduction in stress-related proteins
of Po. gingivalis.83
Porphyromonas gingivalis, Treponema denticola, and Tannerella for-
sythia are common members of the microbiota of subgingival bio-
films84,85 and they are frequently adapted as part of multispecies
biofilms models.84 Interestingly, Ta. forsythia was almost undetect-
able when these three species were co-cultured in a biofilm model
system for 90 hours. Further metaproteomic analysis of three spe-
cies biofilms indicated that while iron acquisition of Po. gingivalis
was altered, upregulation of glycine catabolism-related protein in Tr.
denticola was also observed, suggesting a symbiotic relationship of
Po. gingivalis with Tr. denticola, and an antagonistic one with Ta. for-
sythia.86 Another interaction between Po. gingivalis and Ta. forsythia
was observed, when the numbers of the latter were significantly re-
duced in the presence of a lysine-gingipain mutant of the former in
a biofilm. In the presence of a Po. gingivalis Arg-gingipain mutant,
a spatial rearrangement of Tr. denticola was observed, collectively
indicating that the gingipains of Po. gingivalis may qualitatively and
quantitatively affect the composition of polymicrobial biofilms.87
Aggregatibacter actinomycetemcomitans has also been shown to
successfully integrate and grow in multispecies biofilms. 88-90 The
metaproteomic analysis showed that 483 out of 728 quantified
proteins in the biofilm were differently regulated after introducing
Ag. actinomycetemcomitans, with a stark upregulation of Prevotella
intermedia proteins, and a downregulation of Campylobacter
rectus, Streptococcus anginosus, and Po. gingivalis proteins.91
Metaproteomic analysis confirmed that Ag. actinomycetemcomitans
collectively increased the metabolic rate, ferric iron-binding, and
5S RNA binding capacities of the biofilm, but did not affect the
91
numeric composition of the constituent species. Further stud-
ies using Ag. actinomycetemcomitans knockout mutants indicated
that its histone-like (H-NS) family of DNA-binding, nucleoid-struc-
turing proteins may impose regulatory effects on the pattern of
protein expression in the other species in the biofilm, such as
streptococci,  F. nucleatum, and Veillonella dispar.90 These molecu-
lar shifts within the biofilm warrant further investigation of their
potential impact on its virulence properties, and association with
periodontal pathogenesis. When adopting Anaeroglobus geminatus,
a more recently discovered putative periodontal pathogen, many
ribosomal proteins were elevated in the biofilm, including ones in-
volved in “pyruvate metabolic processes”, whereas ones belonging
to the “glycerol metabolic process” group were downregulated.
Anaeroglobus geminatus also upregulated many St. oralis, Po. gin-
givalis, and V. dispar proteins without significantly changing their
bacterial abundances in this system. 88
The metaproteomic characterization of laboratory-grown bio-
films has led to valuable information about the critical mechanisms
driving interactions between species and their host.91 Nevertheless,
the complexity of such experimental models is still far from “real life”
situations, and thus caution should be applied when extrapolating
conclusions about health and disease.
7 | I N S I G HT S FRO M C LI N I C A L S T U D I E S
Prior successful proteomic work on laboratory-grown bacterial spe-
cies has spawned an interest in extension of the metaproteomic
applications to more complex samples, such as consortia found in
subgingival plaques or in saliva. In this case, the level of organismal
diversity is substantially greater than that of a laboratory-grown
planktonic oral bacteria or laboratory-grown multispecies oral bio-
film. The application of qualitative and quantitative proteomics to
dissect protein composition of subgingival plaque has been limited
compared to saliva or gingival crevicular fluid.
7.1 | Metaproteomic profiling of saliva
For many decades, saliva was a well-studied fluid to monitor in-
flammatory mediators in various systemic and oral diseases such
as caries, gingivitis, and periodontitis.92,93 Together with its simpler,
noninvasive, and painless way of collection, saliva is a favorable ma-
trix to investigate markers for oral health and disease and under-
lying biological pathways.94,95 However, the salivary composition is
known to vary with age, gender, daily rhythm, and dietary habits,
etc., differs between unstimulated and stimulated saliva, and is tem-
perature-sensitive.92 These limitations have to be kept in mind when
collecting and analyzing saliva for proteomic applications.
Proteomic applications have to date identified and analyzed
up to 5500 proteins in saliva in humans, and the majority of these
proteins originate from the parotid, submandibular, and sublingual
glands, the three major salivary glands.93,96,97 The remaining account
for proteins from minor glands, gingival crevicular fluid, mucosal
exudates, and the oral microflora.96 While human proteins in saliva
BOSTANCI et al. |
      61

F I G U R E 3   (A) Comparative mass-spectrometric proteome analysis based on the peptide spectrum intensity of two different saliva
samples. Quantified mass-spectrometry peak patterns of the peptide were highlighted in red squares using the “Review Peak Picking”
screen in Progenesis QI. Depicted peak areas were given from peptide NNLQAEENMLK of protein fncp_C_1_554 Fusobacterium nucleatum
subsp polymorphum ATCC 10 953. (B) Comparative mass-spectrometric proteome analysis based on the peptide spectrum intensity of
two different gingival crevicular fluid samples. Depicted peak areas were given from peptide NAEEVSGAGK protein bot274_c_4_852
Bacteroidetes bacterium oral taxon 274 F0058

have been investigated extensively in health (oral and systemic)97-114
and different oral diseases, for example, periodontal disease115-124
and caries,125 studies on the salivary metaproteome (both in pellet
and supernatant) of oral microbiota have so far been underrepre-
sented, with only a few studies in periodontitis and health being
reported.97,98,102,116,124,126-129 Table 4 summarizes the proteomic ap-
proaches investigating saliva in different oral diseases and health.
In the following, biological insights into the metaproteome of saliva,
gained from different proteomic methods, will be discussed.
Guo et al102 applied a proteomic approach to characterize the
salivary supernatant metaproteome of only one healthy individ-
ual. Overall, 1381 proteins were identified in supernatant from
unstimulated saliva analyzed by liquid chromatography/tandem
mass spectrometry. Even although not further specified, the iden-
tified microbial proteins belonged to 31 different bacterial spe-
cies. Streptococcus mutans, and to a lesser extent Po. gingivalis and
Helicobacter pylori, were the species predominantly represented by
29, four, and two proteins, respectively.
Yan et al,98 supported by a consortium of four different research
groups, also characterized the salivary metaproteome of healthy
individuals. The authors collected unstimulated saliva and the re-
spective supernatant, analyzed by liquid chromatography/tandem
mass spectrometry, revealed 1456 individual proteins (≥2 peptides),
where 12 were potentially of bacterial origin. However, these pro-
teins were not investigated any further.
Wu et al116 used a two-dimensional gel electrophoresis and liq-
uid chromatography/tandem mass spectrometry approach to inves-
tigate the salivary proteome of patients with generalized aggressive
periodontitis and healthy control subjects using the supernatant of
unstimulated saliva. Overall, 21 individual proteins were identified
(>2 peptides). One protein, elongation factor 2, could not clearly
be identified as a human or nonhuman protein because of its highly
conserved protein sequence among humans and microbes.
A few years later, Jagtap et al128 used a multidimensional pep-
tide fractionation method and a combined database of human
proteins and proteins translated from oral microbe genomes to in-
vestigate the salivary supernatant of healthy individuals. This study
showed that Streptococcus, Rothia, Actinomyces, Prevotella, Neisseria,
Veilonella, Lactobacillus, Selenomonas, Pseudomonas, Staphylococcus,
and Campylobacter were highly abundant among the 65 genera
TA B L E 5   Clinical application of metaproteomics in gingival crevicular fluid
|

Maximum
62      

identified/
Proteomic Peptide quantified Nonhuman Ref
Cohort definition Sample application Approach number proteins proteins Highlighted nonhuman proteins no.

Oral health

Healthy (n = 9) Pooled nanoLC-ESI-MS/ Discovery >2 119 N/A N/A 204
MS

Healthy children Pooled iTRAQ, LC- Quantitative N/A 165 N/A N/A 205
with mixed ESI-MS/MS,
dentition (split ELISA, Western
mouth model) blot
(n = 40)

Periodontal diseases adopted from131

Chronic Pooled SDS-PAGE, Discovery >2 MALDI-TOF/ N/A N/A 206

periodontitis nanoLC- TOF: 23,
in maintenance ESI-MS/MS, LC-ESI-MS/
phase (n = 12) MALDI-TOF/ MS: 66
TOF

Healthy (n = 12), Individual LTQ IT-Orbitrap Discovery ≥1 462 Comprehensive Comprehensive bacterial database: 33 kDa chaperonin, iron uptake protein A2, 151
chronic (LC-MS/MS) bacterial phosphoenolpyruvate carboxylase, DNA mismatch repair protein mutL, DNA mismatch
periodontitis database: repair protein mutS, uncharacterized protein pXO2-25/BXB0023/GBAA_pXO2_0023,
(n = 12) 30, targeted uncharacterized protein PM1101, undecaprenyl-diphosphatase, uncharacterized fimbrial
bacterial chaperone ybgP, ankyrin repeat protein A, uncharacterized protein MG268 homolog,
database: 20 chaperone protein dnaJ, elongation factor G, DNA-directed RNA polymerase subunit alpha,
50S ribosomal protein L7/L12, glucosamine-6-phosphate deaminase, argininosuccinate
lyase, ribulose bisphosphate carboxylase, probable succinyl-CoA:3-ketoacid-coenzyme A
transferase sub, DNA-directed RNA polymerase subunit beta, 30S ribosomal protein S16,
glyceraldehyde-3-phosphate dehydrogenase, choline dehydrogenase, glyceraldehyde-3-
phosphate dehydrogenase, 3-octaprenyl-4-hydroxybenzoate carboxy-lyase, ferrochelatase,
ribonuclease P protein component, 2-dehydro-3-deoxyphosphooctonate aldolase, elongation
factor Tu, uridylate kinase
Targeted bacterial database: Fructose-bisphosphate aldolase class 1, phosphatidylserine
decarboxylase proenzyme, chaperone protein clpB
2,3-bisphosphoglycerate-dependent phosphoglycerate mutase, tRNA 5-methylaminomethyl-
2-thiouridine biosynthesis bifunctional protein mnmC, serine hydroxymethyltransferase,
chaperone protein clpB, protein translocase subunit secA, phosphoenolpyruvate
carboxykinase [ATP], 50S ribosomal protein L5, GMP synthase [glutamine-hydrolyzing],
2’,3’-cyclic-nucleotide 2’-phosphodiesterase, phosphonates import ATP-binding protein phnC,
uncharacterized RNA methyltransferase PG_1095, L-arabinose isomerase, aspartyl/glutamyl-
tRNA(Asn/Gln) amidotransferase subunit B, selenide/ water dikinase, 30S ribosomal protein
S12, UPF0082 protein BF2564, fragilysin
BOSTANCI et al.

(Continues)
TA B L E 5   (Continued)

Maximum
identified/
Proteomic Peptide quantified Nonhuman Ref
BOSTANCI et al.

Cohort definition Sample application Approach number proteins proteins Highlighted nonhuman proteins no.

Healthy (n = 1), Pooled SDS-PAGE, Discovery ≥1 Healthy: 168, N/A N/A 207
chronic nanoLC-MS/MS, diseased:
periodontitis Q-Tof Ultima 167
(n = 8) API, Western
blot
Healthy Pooled 2DE, SDS-PAGE, Discovery ≥1 Healthy: 327 Bacterial: 53, Bacterial: Acidovorax ebreus methionyl-tRNA synthetase, Acinetobacter sp. malonyl-CoA 136
(n = 5), mild LC-MS/MS, yeast: 7, O-methyltransferase BioC, Acinetobacter sp urease subunit gamma, Agrobacterium
periodontal LTQ-XL viral: 3 rhizogenes putative replication protein C, Anaeromyxobacter dehalogenans methionyl-tRNA
disease (n = 3), formyltransferase, Blochmannia pennsylvanicus glucose-6-phosphate isomeras, Bordetella
moderate avium serine ydroxymethyltransferase, Bordetella pertussis cyclolysin secretion/processing
periodontal ATP-binding protein CyaB, Bradyrhizobium japonicum protein slyX homolog, Bradyrhizobium
disease japonicum cytochrome c-type biogenesis protein CycH, Buchnera aphidicola subsp Acyrthosiphon
(n = 3), severe pisum isoleucyl-tRNA synthetase, Buchnera aphidicola subsp Baizongia pistaciae DNA-directed
periodontal RNA polymerase subunit beta, Buchnera aphidicola subsp Cinara cedri 50S ribosomal protein
disease (n = 5) L2, Coxiella burnetii elongation factor Tu, Delftia acidovorans probable ubiquinone biosynthesis
protein UbiB, Desulfobacterium autotrophicum threonyl-tRNA synthetase, Desulfococcus
oleovorans alanine racemase, Dichelobacter nodosus chaperone protein DnaK, Enterobacter
aerogenes glyceraldehyde-3-phosphate dehydrogenase, Erwinia tasmaniensis triosephosphate
isomerase, Escherichia coli transcriptional activator perA, Geobacter bemidjiensis threonyl-
tRNA synthetase, Geobacter sp. dihydroorotase, Helicobacter pylori uncharacterized protein
jhp_0176, Lawsonia intracellularis ribonuclease Y, Magnetococcus sp. protein translocase subunit
SecF, Magnetococcus sp. DNA replication and repair protein recF, Methylobacterium nodulans
chaperone protein DnaK, Methylocella silvestrisATP synthase subunit delta, Novosphingobium
aromaticivorans N-Succinylglutamate 5-semialdehyde dehydrogenase, Pseudomonas mendocina
adenosylhomocysteinase, Psychromonas ingrahamii phospho-N-acetylmuramoyl-pentapeptide-
transferase, Rhodobacter sphaeroides phosphoglycerate kinase, Rickettsia bellii aconitate hydratase,
Salmonella agona flagellar P-ring protein, Serratia proteamaculans alanyl-tRNA synthetase,
Shewanella oneidensis chemotaxis response regulator protein-glutamate methylesterase of group
1 operon, Vibrio vulnificus 3-Octaprenyl-4-hydroxybenzoate carboxy-lyase, Wolbachia pipientis
chromosomal replication initiator protein dnaA, Xylella fastidiosa glycyl-tRNA synthetase beta
subunit, Bacillus anthracis enolase, Bacillus subtilis probable glucarate dehydratase, Clostridium
kluyveri DNA ligase, Clostridium tetani probable 2-phosphosulfolactate phosphatase, Enterococcus
faecalis ATP-dependent helicase/deoxyribonuclease subunit B, Lactobacillus acidophilus UPF0348
protein LBA1527, Listeria innocua phosphoribosylamine-glycine ligase, Oceanobacillus iheyensis
chaperone protein DnaK, Staphylococcus aureus conserved virulence factor B, Streptococcus
pyogenes serotype M1 elongation factor Tu, Streptococcus suis ribosomal RNA small subunit
methyltransferase H, Symbiobacterium thermophilum chorismate synthase, Thermoanaerobacter
tengcongensistriosephosphate isomerase
Yeast: Geobacter sp dihydroorotase, Saccharomyces cerevisiae elongation factor 1-alpha 1, Saccharomyces
cerevisiae phosphatidylglycerol phospholipase C, Saccharomyces cerevisiae malate dehydrogenase/
|

mitochondrial, Saccharomyces cerevisiae Ras-related protein SEC4, Saccharomyces cerevisiae histone H2B.1
      63

Saccharomyces cerevisiae heat shock protein SSC1/ mitochondrial

Viral: Acanthamoeba polyphaga mimivirus uncharacterized protein R292, carnation etched ring
virus virion-associated protein, infectious salmon anemia virus fusion glycoprotein F0
TA B L E 5   (Continued)
|

Maximum
64      

identified/
Proteomic Peptide quantified Nonhuman Ref
Cohort definition Sample application Approach number proteins proteins Highlighted nonhuman proteins no.

Chronic Individual SDS-PAGE, Discovery N/A Without N/A N/A 137

periodontitis TCA-acetone enrichment:
(n = 5) precipitation, 990, with
LC-ESI-MS/MS enrichment:
1307

Healthy (n = 15), Pooled 1DS-PAGE, Quantitative ≥2 121 N/A N/A 138

gingivitis LC-MS/MS,
(n = 15), LC-ESI-MS/MS
chronic
periodontitis
(n = 15)

Healthy (n = 5), Individual LFQ LC/MSE, Quantitative >2 154 (healthy: Bacterial: 27, Bacterial: Cytosol aminopeptidase, H. pylori aconitate hydratase, S. typhimurium DAHP 139
aggressive Q-TOF Premier, 88, yeast: 14, synthetase, S. typhimurium sulfite reductase, E. coli biotin synthesis protein, M. tuberculosis
periodontitis ELISA diseased: viral: 8 chaperonin, putative sensory transducer, E. coli cysB, E. coli DnaJ-like protein, M. tuberculosis
(n = 5) 115) dihydrolipoyl DH, Xylella fastidiosa dnaA, H. pylori DNA polymerase, H. pylori reductoisomerase,
S. choleraesuis flagellin, A. brasilense glutamate synthase, E. coli MCP-IV, pyruvate synthase
porB, P. gingivalis Methylmalonyl-CoA, Yersinia pseudotuberculosis pelY, E. coli phosphonates-
binding protein, protein translocase, C. pneumoniae h-tRNA synthetase, P. syringae
acetyltransferase, E. coli protein yeeL, M pneumoniae adhesin P1, E. coli KDR aldolase, B. subtilis
protein ypiB
Yeast: C. albicans acetyl coenzyme A, C. albicans mRNA-capping, S. pombe cysteine synthase, S.
cerevisiae DPM synthase, S. cerevisiae beta-oxidation protein, S. cerevisiae exoribonuclease 1, S.
cerevisiae MAD1, S. cerevisiae NPL4
S. cerevisiae NUP85, C. sphaerica OMP decarboxylase, protein PSL1, S. cerevisiae l-tRNA
synthetase, SLD2, RING finger protein
Viral: Sarcoma virus tyrosine- kinase, human adenovirus fiber protein, Enterobacteria phage C1,
Enterobacteria phage P22 C2, Polymerase basic protein 2, Herpes virus protein US2, Vaccinia
virus protein C13, Enterobacteria phage A protein

Healthy (n = 11), Pooled SDS-PAGE, LC- Quantitative ≥ 2 305 169 Dialister invisus DSM 15 470, Eikenella corrodens, Bacteroides, Porphyromonas gingivalis, 140
moderate ESI-MS/MS, LTQ Actinomyces, Fusobacterium, Staphylococcus
periodontitis linear ion trap,
(n = 12) ELISA

Healthy (n = 5), Pooled LFQ nanoLC- Quantitative >2 230 (healthy: N/A N/A 141
chronic ESI-MS/MS, LTQ 145,
periodontitis Velos, ELISA diseased:
(n = 5) 501)
BOSTANCI et al.

(Continues)
TA B L E 5   (Continued)

Maximum
identified/
Proteomic Peptide quantified Nonhuman Ref
BOSTANCI et al.

Cohort definition Sample application Approach number proteins proteins Highlighted nonhuman proteins no.

Healthy (n = 2), Pooled TMT label, Quantitative ≥1 619 N/A N/A 142
moderate LC-MS/MS,
periodontitis LTQ Orbitrap X,
(n = 2), severe Western blot,
periodontitis ELISA
(n = 2)

Experimental Pooled LC-MS/MS, Quantitative ≥2 202 Bacterial: 16 Chlorobium phaeobacteroides DSM 266 helicase domain-containing protein, unidentified 143
gingivitis model iTRAQ, FT-ICR eubacterium SCB49 type II restriction enzyme/ methylase, unidentified eubacterium
(n = 10) MS SCB49 hypothetical protein SCB49_12134, unidentified eubacterium SCB49 glycosyl
transferase group 1, unidentified eubacterium SCB49 ABC transporter ATP-binding protein,
Parabacteroides distasonis ATCC 8503 hypothetical protein BDI_1504, Parabacteroides
distasonis ATCC 8503 30S ribosomal protein S2, Parabacteroides merdae ATCC 43 184
hypothetical protein PARMER_01297, Chlorobium phaeobacteroides BS1 chromosome
segregation protein SMC, Chlorobium phaeobacteroides BS1 cysteine synthase, Candidatus
Azobacteroides pseudotrichonymphae genomovar. CFP2 hypothetical protein CFPG_190,
Parabacteroides johnsonii DSM 18 315 hypothetical protein PRABACTJOHN_02373,
Fusobacterium sp. 4_1_13 fusobacterium outer membrane protein family, Parabacteroides sp.
D13 helicase domain-containing protein, Fusobacterium sp. 3_1_36A2 fusobacterium outer
membrane protein, Leptotrichia hofstadii F0254 fusobacterium outer membrane protein

Experimental Individual LFQ LC-MS/MS, Quantitative >2 Identified: Identified: Induction phase: Aminopeptidase Aggregatibacter actinomycetemcomitans, ribosome-associated 144
gingivitis model and LTQ Orbitrap 287, bacterial: 16, GTPase EngA Fusobacterium nucleatum, 50S ribosomal protein L2 Fusobacterium nucleatum,
(n = 20) pooled quantified: fungal: 12, glucose-6-phosphate isomerase Prevotella oralis, 6-phosphogluconolactonase Prevotella oralis,
samples 101 ( yeast: 7 putative uncharacterized protein NOP4 Candida albicans, potential purine permease Candida
induction Quantified: albicans, putative uncharacterized protein HAP5 Candida albicans, imidazole glycerol phosphate
phase: 59, induction synthase Candida albicans, lysophospholipase 1 Candida albicans, importin alpha re-exporter
resolution phase: 5 Saccharomyces cerevisiae, DNA-directed RNA polymerase III subunit RPC9 Saccharomyces
phase: 73) bacterial, cerevisiae, serine/threonine-protein kinase I Saccharomyces cerevisiae
5 fungal, Resolution phase: Putative uncharacterized protein Porphyromonas gingivalis, UDP-MurNAc-
3 yeast, tripeptide synthetase Fusobacterium nucleatum, glucose-6-phosphate isomerase Prevotella
resolution oralis, FeS assembly ATPase SufC Treponema denticola, CTA2 subfamily 1 protein 7 Candida
phase: 4 albicans, likely protein kinase Candida albicans, potential mitochondrial protein Fmp29 Candida
bacterial, albicans, imidazole glycerol phosphate synthase Candida albicans, thymidylate synthase Candida
6 fungal, 1 albicans, DNA-directed RNA polymerase III subunit RPC9 Saccharomyces cerevisiae, vacuolar
yeast acid trehalase OS Saccharomyces cerevisiae

Chronic Individual nanoLC-MS/MS, Quantitative >1 Baseline: N/A N/A 145

periodontitis LTQ Orbitrap XL 112-185,
before and wk 1:123-
after treatment 204, wk
(n = 10) 5:68-194,
|

wk 9:55-
192, wk
      65

13:64-169
 TA B L E 5   (Continued)
|

Maximum
66      

identified/
Proteomic Peptide quantified Nonhuman Ref
Cohort definition Sample application Approach number proteins proteins Highlighted nonhuman proteins no.

Healthy (n = 40), Pooled SDS-PAGE, Quantitative ≥2 Identified: Quantified: Bacterial: Uncharacterized protein (Streptococcus sp.), helicase protein (Porphyromonas 146
moderate/ LC-ESI-MS/MS, 238, bacterial: 42, gingivalis), Sec-independent translocase (Propionbacterium acnes), uncharacterized protein
severe ICAT, mTRAQ, quantified: yeast: 11 (Bifidobacterium longum), oxidoreductase (Porphyromonas gingivalis), chromosmal partition
periodontal LTQ-linear IT 180 Upregulated protein Smc (Lactococcus lactis), RelA/SpoT family protein (Porphyromonas gingivalis),
disease (n = 40) MS, ELISA in disease: glycine dehydrogenase (decarboxylating) (Acetobacter pomorum), outer membrane protein
bacterial: 16, (Porphyromonas gingivalis), uncharacterized protein (Porphyromonas gingivalis), DNA
yeast: 3 topoisomerase (Lactococcus lactis), glutamate-tRNA ligase (Actinomyces cardiffensis), DNA-
directed DNA polymerase (Lactococcus lactis), GMP synthase (glutamine-hydrolyzing)
(Lactococcus lactis), uncharacterized protein (Propionibacterium acnes), uncharacterized protein
(Bifidobacterium longum), putative uncharacterized protein (Lactococcus lactis), aminopeptidase
(Streptococcus sanguinis), GTP-binding protein typeA (Porphyromonas gingivalis), serine
proteinase (peptidase) (Bdellovibrio bacteriovorus), leucine amidopeptidase PfLAP (Plasmodium
chabaudi), PTS system/ lactose/cellobiose-specific IICB (Enterococcus sp.), uncharacterized
protein (Propionibacterium acnes), glycosyl hydrolase (Corynebacterium genitalium), putative
carbohydrate-active enzyme (uncultured), PTS system, sucrose subfamily IIABC (Enterococcus
faecium), glutamate synthase large chain (Acetobacter pomorum), putative glycerate kinase
(Porphyromonas gingivalis), formamidase (Acetobacter pomorum), glutamate-ammonia-
ligase adenyltransferase (Acetobacter pomorum), bifunctional protein GlmU (Fusobacterium
nucleatum), multidrug efflux pump subunit AcrB (Escherichia coli)
Yeast: Beta-actin (Chiloscylium puntatum), poly (A) polymerase Trf5p (Saccharomyces cerevisiae),
actin (Cladosporium cladosporioides), translation initiation factor eIF2B (Saccharomyces
cerevisiae), DNA polymerase (Saccharomyces cerevisiae), oligopeptide transporter
(Saccharomyces cerevisiae), actin (Candida albicans), predicted protein Tcb1p (Saccharomyces
cerevisiae), DNA polymerase (Candida glabrata), actin (Collectotrichum truncotum), histone H3
protein (Tristoma integrum)

Endodontic/pulp diseases

Healthy children Individual 1D- and Quantitative ≥2 Control: N/A N/A 147
with mixed and 2D-nanoLC-MS/ 2789, root
dentition (split pooled MS, nano- resorption:
mouth model) ULTRA LC 2421
(n = 11) system

Abbreviations: CIEF, capillary isoelectric focusing; LC-ESI-MS, liquid chromatography electrospray ionization mass spectrometry; FT-ICR, Fourier Transform ion cyclotron resonance; GCF, gingival
crevicular fluid; HPLC, high performance liquid chromatography; IT, ion trap; iTRAQ, isobaric tags for relative and absolute quantitation; ICAT, isotope-coded affinity tag; LC-MS/MS, liquid
chromatography/tandem mass spectrometry; LC-MSE, liquid chromatography mass spectrometry in data-independent analysis mode; LCQ, liquid chromatography quadrupole; LFQ, label-free
quantitative; LTQ, linear trap quadropole; MALDI, matrix-assisted laser desorption/ionization; MS, mass spectrometer; MS/MS, tandem mass spectrometry; mTRAQ, amine-specific tag for relative and
absolute quantitation; N/A, not applicable; PMF, peptide mass fingerprinting; PTS, phosphotransferase system; Q-TOF, quadrupole time-of-flight; RPLC, reversed phase liquid chromatography; SCX,
strong cation exchange; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; SELDI, surface-enhanced laser desorption/ionization; 2DE, two-dimensional gel electrophoresis; TMT,
tandem mass tag; TOF, time of flight; UPLC, ultra performance liquid chromatography.
BOSTANCI et al.
BOSTANCI et al. |
      67

F I G U R E 4   A typical experimental flow of label-free quantitative proteomic analysis in our laboratories. (A) Sample preparation and
protein digestion. The protein concentration of a given sample (ie, gingival crevicular fluid [GCF] or saliva) are measured and diluted to
same amount (typically 20 µg/reaction). Samples are then lysed using a filter aided sample preparation (FASP) or in-StageTip (iST) method.
The extracted proteins are reduced, alkylated and digested with trypsin in centrifugal unite (for FASP) or in C18 membrane (for iST). The
resultant peptide solutions are desalted with C18 column (for FASP) and dried with speed vacuum. (B) Mass spectrometry and data analysis.
The peptides are subjected to liquid chromatography (LC); raw data are derived after liquid chromatography/tandem mass spectrometry (LC-
MS/MS). Raw data are searched against human and human oral microbiome database (HOMD) database and analyzed by Progenesis QI

from 12 phyla represented, consistent with metagenomic studies
25
of saliva. The bioinformatic analysis of such data supported that
the salivary microbiome is active in glycolysis and protein synthesis.
Belstrøm et al126 used a liquid chromatography/tandem mass
spectrometry proteomic approach to characterize stimulated saliva
of healthy individuals and patients with dental caries or periodon-
titis. Overall, 4161 proteins were identified, 2090 human proteins
and 1946 bacterial proteins. When clustered based on the three
conditions, in health 2079 human and 1926 bacterial proteins were
identified, in caries 2084 human and 1861 bacterial proteins, and
in periodontitis 2084 human and 1924 bacterial proteins. The 1946
bacterial proteins were derived from 20 different genera and 81 dif-
ferent species, with Streptococcus, Prevotella, Veillonella, Rothia, and
Neisseria representing the most abundant bacterial genera (~ 70% of
the total bacterial mass) while Rhodotorula mucilaginosa, Veillonella
atypica, Prevotella histicola, P. melaninogenica, and St. salivarius were
the most abundant bacterial species. Between the different condi-
tions, no significant differences, either at a genera or species level,
could be observed. However, when compared with health, higher
levels of Veillonella and lower levels of Haemophilus were seen in
caries patients, while higher levels of Fusobacterium, Leptotrichia,
and Selenomonas and lower levels of Streptococcus, Rothia, and
Haemophilus were observed in patients with periodontitis. Similar
results were also obtained at species level.
Grassl et al97 identified and quantified up to 5500 proteins
from whole saliva collected by cotton swabs from healthy indi-
viduals (no periodontal assessments were performed). This is
currently the largest number of salivary proteins identified by pro-
teomic applications. Among the identified proteins, 2234 proteins
were of microbial origin belonging to 50 different bacterial gen-
era from nine phyla (mainly Firmicutes, Bacteroides, Actinobacteria,
and Proteobacteria). Streptococcus was the most abundant genus
in line with earlier studies performed by 16S RNA sequencing.
Interestingly, protein F1WNZ3, a homolog of chaperone protein
HscA from Moraxella catarrhalis, was the most abundant bacterial
protein identified. Moraxella catarrhalis is a major mucosal patho-
gen of the human respiratory tract, but its role in periodontal
health or disease has not yet been characterized. Metaproteomic
analysis of saliva also confirmed that the oral microbiome differs
between individuals and changes drastically upon eating and
toothbrushing.
A recent quantitative proteomic approach by Bostanci et al124 in-
vestigated the salivary supernatant proteome of patients with differ-
ent forms of periodontitis, patients with gingivitis, and periodontally
healthy control subjects. In supernatant from unstimulated saliva,
486 proteins were quantified (≥1 peptide) by a two-step proteomic
application, starting with a label-free quantitative liquid chromatog-
raphy/tandem mass spectrometry approach. Among these proteins,
 |
68       BOSTANCI et al.

F I G U R E 5   Metabolite reference pathway, representing a global and overall picture of metabolism. The central carbon pathway is seen
in dark blue at the centre of the map with the TCA cycle a prominent feature; yellow to the right represents amino acid metabolism; and
turquoise to the left fatty acid and lipid metabolism. Downloaded from the Kyoto Encyclopedia of Genes and Genomes PATHWAY database on
17 January 2020 (https://www.genome.jp/kegg/kegg2.html) where an interactive map can be accessed

454 originated from humans, 14 from bacteria (Ag. actinomycetem-
comitans, Cam. rectus, F. nucleatum, Po. gingivalis, Prevotella oralis, St.
anginosus, Tr. denticola, V. dispar, and St. oralis) and eight from fungi
(Candida albicans, Saccharomyces cerevisiae) (Figure 3A). Metallo-
beta-lactamase (Tr. denticola) and glucose-6-phosphate isomer-
ase (Pr. oralis) were significantly upregulated (1.80- and 1.88-fold,
respectively) while glyceraldehyde-3-phosphate dehydrogenase
(St. oralis) was downregulated (3.7-fold) in patients with aggressive
forms of periodontitis compared with healthy individuals.
Another proteomic approach conducted recently by Rabe et al127
investigated the proteome of saliva (pellet or pellet supernatant [by
sonication]) of healthy individuals, stimulated either by Salivette
or paraffin gum. The liquid chromatography/tandem mass spec-
trometry approach identified 1005 and 313 bacterial proteins (≥2
peptides) in the sonicated pellet for the paraffin gum and Salivette
group, respectively. Within the paraffin gum group, 710 and 1005
bacterial proteins were identified in the pellet and the sonicated pel-
let, respectively, with an overlap of 402 proteins. In the sonicated
pellet of both groups (paraffin gum and Salivette) together, 38 dif-
ferent bacterial genera and 90 different species were detected. The
most abundant genera were Actinomyces, Prevotella, Streptococcus,
and Rothia. In the sonicated pellet of the paraffin gum group, 13
genera (ie, Granulicatella) and 44 species were detected exclusively.
However, studies dissecting the entire salivary pellet metaproteome
of periodontal disease categories with well-defined clinical cohorts
are still missing. Although the proteomic coverage of bacterial gen-
era is considerably higher, supporting metaproteomics could provide
a valuable complement to sequencing-based measurements of the
oral microbiome. Nevertheless, this number represents less than
half of the named genera identified by next-generation sequencing,
which shows that adequate quantification of bacterial genera by
their proteomes is still challenging.
The most recent proteomic study, also conducted by Rabe
et al,129 characterized the proteome of the stimulated saliva pellet
and tongue samples of healthy individuals. In saliva, the liquid chro-
matography/tandem mass spectrometry approach identified 4280
proteins, 1647 of human and 2633 of bacterial origin. Analysis of
the tongue samples identified 4644 proteins (human: 1337, bacteria:
3307). Overall, 107 bacterial genera of seven phyla (Actinobacteria,
Bacteriodetes, Firmicutes, Fusobacteria, and Proteobacteria) were
found both in saliva and tongue samples (an overlap of 89 genera)
and those seven phyla were also the most abundant in saliva. In sa-
liva, compared with tongue samples, Fusobacterium, Selenomonas,
Bifidobacterium, and Treponema were also significantly increased.
BOSTANCI et al.
F I G U R E 6   The impact of sample preparation protocols and
sample-specific databases for quantitative proteome coverage
of saliva, gingival crevicular fluid (GCF), and biofilm samples. A)
Proteome coverage in saliva by filter-aided sample preparation
(FASP) versus in-solution digestion (InSol) B) Proteome coverage
in saliva and GCF with a standard database at the center with a
customized one including the Human Oral Microbiome Database
(eHOMD) C) Proteome coverage in in-vitro biofilm pellets/
supernatants by FASP versus in-StageTip digestion (iST)
Regarding the function of bacterial proteins in saliva, the highest
abundances were observed for proteins involved in translation and
ribosomal structure, carbohydrate metabolism, and energy produc-
tion. In particular, the high abundance of ribosomal proteins and
|
      69
proteins involved in translation could mean that these proteins are
crucial for bacterial growth.
7.2 | Metaproteomic profiling of gingival
crevicular fluid
Gingival crevicular fluid is an important transudate or exudate
that provides host-derived substances that shape subgingival
biofilms.131 Gingival crevicular fluid harbors a rich collection of
various host- and microbial-derived components (cellular and im-
munological, ie, cells, bacteria, proteins) which, since the early
1960s, has led to various studies investigating gingival crevicu-
lar fluid to gain insights into the local inflammatory status of the
gingival tissues and associated oral diseases.131,132 Although the
host-derived proteome of gingival crevicular fluid has been exten-
sively studied, the microbiota composition of gingival crevicular
fluid is less well defined.133,134 Gingival crevicular fluid or crevicu-
lar epithelial cells reportedly contain subgingival bacteria that re-
semble the microbial composition identified in subgingival plaque
samples obtained from the same periodontal sites.135 Therefore,
metaproteome characterization of gingival crevicular fluid may
provide new opportunities for studying the dynamic properties of
subgingival biofilms. However, metaproteomic approaches of oral
microbiota in gingival crevicular fluid, similar to what is seen in
saliva, are rare. While a vast number of studies have already inves-
tigated the gingival crevicular fluid proteome in health (oral and
systemic)136,137 and different oral diseases, for example, periodon-
tal disease30,34,138-149 or endodontic/pulp diseases,150 to date only
a few studies have investigated the metaproteome of oral micro-
biota in gingival crevicular fluid.136,139,140,143,144,146,151 A summary
of the findings is provided in Table 5.
The application of two-dimensional polyacrylamide gel elec-
trophoresis for global protein profiling of gingival crevicular fluid in
the context of periodontal disease can be traced back to the 1980s.
These studies indicated that gingival crevicular fluid from periodon-
titis sites contained more proteins than healthy ones, and the molec-
ular weight of many proteins was between 64 and 16 kDa.152 Since
then, new technologies have emerged and the application of quali-
tative and quantitative proteomics for dissecting microbial compo-
nents of gingival crevicular fluid is expanding (for further reading,
refer to 131,143).
In 2010, an application of liquid chromatography/tandem mass
spectrometry combined with a label-free approach by Bostanci
et al139 enabled an in-depth analysis of both microbial and host pro-
teomes in gingival crevicular fluid and led to the identification and
quantification of 154 proteins (≥2 peptides) in health and in aggres-
sive forms of periodontitis, of which 27, 14, and eight proteins were
assigned to bacteria, yeast, and viruses, respectively. Viral proteins,
for example, herpes virus protein 2, were of specific importance,
because of their higher presence in patients with periodontitis
compared with healthy subjects. Furthermore, the Po. gingivalis
 |
70       BOSTANCI et al.
methylmalonyl-CoA mutase was the only protein derived from pu-
tative periodontal pathogens identified in diseased individuals.
In the same year, Grant et al143 applied a 21-day experimen-
tal gingivitis model to characterize gingival crevicular fluid from
10 individuals using a quantitative liquid chromatography/tandem
mass spectrometry approach. This resulted in a total of 202 pro-
teins (≥2 peptides) being discovered, of which 186 were human
proteins, 16 derived from bacteria (Chlorobium phaeobacteroides
DSM 266, Eubacterium SCB49, Parabacteroides distasonis ATCC
8503, Parabacteroides merdae ATCC 43 184, Ch. phaeobacteroi-
des BS1, Candidatus Azobacteroides pseudotrichonymphae genom-
ovar CFP2, Parabacteroides johnsonii DSM 18 315, Fusobacterium
sp 4_1_13, Parabacteroides sp D13, Fusobacterium sp 3_1_36A2,
and Leptotrichia hofstadii F0254). Interestingly, during the course
of the study (day 0 to day 35), bacterial proteins did not vary
substantially.
Choi et al140 investigated the proteome of gingival crevicular
fluid in healthy individuals and in patients with moderate peri-
odontitis. In total, the gel-based liquid chromatography/tandem
mass spectrometry approach identified 305 proteins (≥2 pep-
tides), 169 of which were of bacterial origin (Dialister invisus DSM
15 470, Eikenella corrodens, Bacteroides, Po. gingivalis, Actinomyces,
Fusobacterium, and Staphylococcus). However, details regarding
their presence in health and in disease were not specified any
further.
151
Baliban et al characterized the gingival crevicular fluid pro-
teome in healthy individuals and patients with chronic periodonti-
tis by liquid chromatography/tandem mass spectrometry using two
different databases (comprehensive and targeted) for the search.
The approach identified a total of 462 proteins (≥1 peptides, 432
human). Among the identified 30 bacterial proteins (comprehen-
sive database), 17 proteins were found in health, and 13 in disease;
however, none were common in health and disease. The top three
proteins in health (33 kDa chaperonin, iron uptake protein A2, and
phosphoenolpyruvate carboxylase) and in disease (ribulose biphos-
phate carboxylase, probable succinyl-CoA:3-ketoacid-coenzyme A
transferase, and DNA-directed RNA polymerase subunit beta) were
suggested as potential bacterial biomarkers. In addition, the targeted
approach identified 20 bacterial proteins (health [3 proteins], disease
[15], both [2]) from putative periodontal pathogens, for example,
Treponema, Fusobacterium, and Campylobacter species.
In the same year, Tsuchida et al136 used both a gel-based and gel-
free liquid chromatography/tandem mass spectrometry approach to
investigate the gingival crevicular fluid proteome in healthy individ-
uals and in patients with periodontitis (mild, moderate, and severe).
Among the healthy group, 327 proteins were identified (≥1 pep-
tides), which were derived from humans (264), bacteria (53), yeast
(seven), and viruses (three). No proteins from putative periodontal
pathogens (e.g., Po. gingivalis or A. actinomycetemcomitans) were
identified. Proteomic data regarding the periodontitis patients were
not provided.
Bostanci et al144 applied a label-free quantitative approach using
liquid chromatography/tandem mass spectrometry to characterize
the proteome of gingival crevicular fluid in 20 individuals participat-
ing in a 21-day experimental gingivitis model. Overall, 287 proteins
were identified, of which 254, 16, 12, and seven proteins were de-
rived from humans, bacteria, fungi, and yeast, respectively. Microbial
proteins were derived from St. oralis, Pr. oralis, Po. gingivalis, Tr. den-
ticola, Ag. actinomycetemcomitans, Can. albicans, and Sa. cerevisiae
(Figure 3B). The Pr. oralis glucose-6-phosphate isomerase, the Can.
albicans imidazole glycerol phosphate synthase, and the Sa. cerevi-
siae DNA-directed RNA polymerase III subunit RPC9 were present
both during induction and resolution.
The quantitative approach by Carneiro et al146 characterized the
gingival crevicular fluid proteome of healthy individuals and patients
with moderate to severe periodontitis. In total, 238 proteins (≥2 pep-
tides) were identified and subsequently quantified resulting in 180
individual proteins, both in health and in disease. Among the quan-
tified proteins, 42 were bacterial proteins from 14 different species
and 11 were yeast proteins. Sixteen bacterial proteins (Po. gingi-
valis [3 proteins]), Lactococcus lactis [2], Acetobacter pomorum [2],
Streptococcus species [1], Bifidobacterium longum [1], Enterococcus sp.
[1], F. nucleatum [1], Plasmodium chabaudi [1], and Actinomyces cardif-
fensis [1]) were significantly elevated in periodontitis compared with
health. Here, the three proteins from Po. gingivalis (oxidoreductase,
Rel/SpoT protein, and outer membrane protein 85) were of special
importance because they demonstrated 2- to 3- fold higher levels
in disease. In yeast, three proteins (Tristoma integrum [histone H3
protein], Collectotrichum truncotum [actin], and Sa. cerevisiae [trans-
lational initiating factor eIF2B]) were significantly elevated in peri-
odontitis compared with health.
The circulating gingival crevicular fluid metaproteome holds
great promise as a reservoir of the state of microbial communities in
the gingival or periodontal pocket. However, for most gingival crevic-
ular fluid proteomic studies, gingival crevicular fluid is processed by
centrifugation to remove cellular components including gingival ep-
ithelial cells, and neutrophils as well as bacteria. Therefore, low pro-
teomic coverage of bacterial genomes may not be surprising. Future
studies for metaproteome analysis should take into consideration
the gingival crevicular fluid pellet fraction.
8 | W H AT W E H AV E LE A R NT: G E T TO
K N OW YO U R SA M PLE
There are several available mass spectrometry technologies for
qualitative/quantitative proteomics (Figure 1), each with their
own advantages and disadvantages. The selection of quantifi-
cation strategy should be dictated by the question under con-
sideration rather than simply trying to catalog large numbers of
proteins in a given sample. A typical workflow of label-free quan-
titative proteomics in our laboratories is presented in Figure 4.
Many factors can affect sensitivity, such as sample preparation,
the type of mass spectrometry used, the sample itself, and the
type of database search employed. The sample preparation prior
to mass spectrometry analysis is a critical part of any proteomic
TA B L E 6   Clinical studies characterizing the metabolomes of biofilms
Ref no. Target organisms or samples
168 Subgingival plaque metabolomics
and following use of a glucose
rinse
169 Effect of xylitol or fluoride rinse
on supragingival plaque
171 Analysis of how erythritol alters
metabolic profiles of S. gordonii
and P. gingivalis
178 Comparison of caries active
lesion biofilm with nonactive
lesions in caries affected donors
and caries free donors with and
without a glucose rinse
180 Comparison of the ArcD
deletion mutant and wild type
Streptococcus gordonii
192 Comparison of headspace
metabolites from bacterial
cultures and human saliva
191 Examined the change in
metabolites afer a 21 d biofilm
overgrowth study
170 Amino acid composition of
supragingival plaque and the
metabolism of amino acids by
supragingival plaque
173 Development of a GC-MS
method to examine Candida
albicans and Staphylococcus
aureus biofilms
172 Comparison between
planktonic cells and biofilms of
Staphylococcus aureus
Metabolomic
approach Sample details
CE-MS Supragingival plaque collected from 5 young donors, influence
of 10% glucose rinsed for 60 s and supragingival resampled on
contralateral side after 10 min
CE-MS Supragingival plaque collected from 7 young donors, influence
of either 10 mM glucose, 10 mM xylitol or glucose and xylitol,
or fluoride (225 or 900 ppm) rinsed for 60 s and supragingival
resampled on contralateral side after 10 min
CE-MS In vitro culture of S. gordonii (strain DL1) and P. gingivalis (ATCC
33 277) with 0%, 0.8%, or 10% erythritol for 6 h
GC-MS 11 CA children and 4 CF children. Both groups aged 11-15 y
CE-MS Streptococcus gordonii DL1 Challis
SESI MS In vitro culture of Aggregatibacter actinomycetemcomitans, P.
gingivalis, Treponema denticola and Tannerella forsythia; saliva
donated by 1 severe periodontitis patient and 2 healthy controls
LC- and GC- MS 168 participants across 5 categories of periodontal status sampled
at baseline and at 21 d; 50 participants used for metabolomics
assays
CE-MS Supragingival plaque collected from 16 young donors for
compositional analysis, Supragingival plaque collected from 4
donors for amino acid metabolism studies
GC-MS Candida albicans strain SC5314 (ATCC MYA-2876) and
Staphylococcus aureus Newman strain (NCTC8178) were
incubated for 24 h
LC-MS Staphylococcus aureus strain LHSKBClinical
Metabolomic discovery
BOSTANCI et al.
All metabolites of the EMP, pentose-phosphate and TCA
cycle, except erythrose-4-phospahte, were detected.
Glucose rinsing modulated the pathways
Fluoride modulated the central carbon pathways but xylitol
had no effect.
Erythritol inhibited biofilm formation for both species via
depletion of DNA and RNA; attenuated extracellular matrix
production; and alterations in dipeptide acquisition and
amino acid metabolism
A cluster of branched alcohols and esters was present
at a higher intensity in biofilms at caries affected sites
compared with CF sites, irrespective of glucose rinse
Removal of ArcD caused intracellular accumulation of
ornithine and suppression of arginine biosynthesis
120 metabolites found from the headspace from individual
bacterial cultures; 18 of which were additionally also found
in saliva
Clustered microbial communities were correlated with the
saliva metabolome; showing Synergistes was strongly
associated with cyclodipeptides in patients with existing
severe periodontitis.
Most of the major amino acids, except cysteine, were
detected and the highest was glutamate. Additional of
7 amino acids to isolated plaque induced production of
ammonia and other metabolites.
Overlaps and differences between mono-culture and
co-culture of the two microorganisms was seen at the
metabolomics level eluding to the idea that changes in
carbon source may change interactions in inter-kingdom
biofilms.
Significant changes in arginine biosynthesis was seen
between planktonic and biofilm forms
|
      71
(Continues)
TA B L E 6   (Continued)
Ref no. Target organisms or samples
183 Dental calculus metabolome
in archaeological samples
in comparison with modern
calculus samples
182 Comparison of subgingival plaque
metabolomes before and after
treatment with 0.25% sodium
hypochlorite or water
208 Exploration of how exogenous
arginine can change the
metabolome of saliva generated
biofilms during sucrose
stimulation up to 48 h
181 Metaboloic analysis of the effect
of p-ABA (4-aminobenzoate/
para-amino benzoic acid) on P.
gingivalis
174 Explored 27 oral bacterial
isolates and a complex in vitro
community of >100 species for
peptidic small molecules for
diversity and unique production
179 Subgingival plaque following use
of toothpastes containing either
1.5% arginine or 1100 ppm
fluoride
Abbreviations: CA, caries active; CF, caries-free; EMP, Embden-Meyerhof-Parnas; GCF, gingival crevicular fluid; SESI MS, secondary electrospray ionization mass spectrometry; TCA, tricarboxylic acid.
Metabolomic
approach Sample details
LC-MS and 12 historical samples and 5 modern calculus samples
GC-MS
LC-MS 17 patients treated with 0.25% sodium hypochlorite and 17
patients treated with sham (water), samples taken at baseline,
2 wk and 3 mo, no other periodontal treatment until exit of
study. Sampling using Gracey cuvette
GC-MS Biofilms were generated from a saliva pool from 6 individuals
cultured for 16 h. Biofilms were treated with arginine (75 mM)
and/or sucrose (15 mM) and sampled for up to 54 h.
CE-MS In vitro culture of P. gingivalis (ATCC 33 277) with 1mg/ml p-ABA
for 2h
LC-MS In vitro culture of Actinomyces bovis ATCC 1368, Actinomyces
meyeri ATCC 35 568, Actinomyces naeslundii OMZ 724,
Actinomyces odontolyticus ATCC 17 929, A odontolyticus ATCC
17 982, Actinomyces viscosus ATCC 15 987, Fusobacterium
nucleatum 23 726, F nucleatum oral taxon 420, Fusobacterium
periodonticum 2b 54-D1, Streptococcus gordonii ATCC 10 558,
Streptococcus infantis HOT-638, Streptococcus mitis bv2 strain
ATCC F0392, Streptococcus mutans UA159, Streptococcus
oralis HOT-707, Streptococcus parasanguinis ATCC 15 911,
Streptococcus pneumoniae TCH8431, Streptococcus sanguinis
VMC66, Streptococcus sobrinus OMZ177, Streptococcus sp strain
C150, Streptococcus vestibularis F0396, Porphyromonas gingivalis
F0568, Veillonella sp strain 6127, Veillonella oral taxon 158 strain
F0412, Actinomyces sp strain XH001 (8), Lactobacillus fermentum
SHI-2, Veillonella parvula SHI-1, and Streptococcus salivarius SHI-
3; and a saliva derived overnight biofilm from a pool of saliva
donated by 6 individuals
LC-MS 45 CA individuals and 38 CF individuals; 29 CA and 22 CA using
arginine containing paste; 16 CA and 16 CF using fluoride paste
|
72      
Metabolomic discovery
Historical samples showed decreases in dipeptides, free
amino acids and nucleotides and in carbohydrates but mono
unsaturated lipids of medium or long chain length were
stable. Oxidation and chemical degradation were found in
the archaeological samples. Metabolites discovered were
on a par to those discovered in modern GCF and saliva
samples.
The 20 most abundant metabolites correlated significantly
with the maximum pocket depth and pockets that did not
respond to therapy, ie did not get shallower over the course
of the study had the most stable metabolic profile
Metabolomics analysis demonstrated arginine uptake and
catabolism to citrulline, ornithine and putrescine. Biofilms
pretreated with arginine before sucrose challenge were
able to increase biofilm pH (8.0) by 48h, whereas untreated
biofilms produced biofilms with low pH (4.5)
p-ABA was utilized for folate biosynthesis
There is a broad diversity of peptidic small molecules
produced by different species and greater annotation of
databases is needed for identification
Arginine paste use affected 16 metabolites and promoted
biofilm pH homeostasis; fluoride pastes affected 9
metabolites and reduced acid production by supragingival
oral biofilms.
BOSTANCI et al.
BOSTANCI et al.
workflow including metaproteomics. We assessed the efficiency
of popular sample preparation protocols for saliva, gingival cre-
vicular fluid, and biofilm samples. The protocols included in-
solution digestion, filter-aided sample preparation, in-StageTip
digestion as well as enrichment of low-abundance proteins via
ProteoMiner beads. When handling saliva samples, the number
of identified proteins drastically dropped in the case of in-so-
lution digestion compared with the filter-aided sample prepara-
tion method (Figure 6A). As the presence of very high-abundance
proteins and the enormous dynamic range of protein distribu-
tion in saliva creates a challenge for proteome coverage, we also
tested the efficiency of the ProteoMiner beads with saliva. The
ProteoMiner kit is based on the use of a combinatorial peptide-
binding library and has been shown to remove a large proportion
of the high-abundance proteins in human serum, and therefore
facilitates detection of low-abundance protein biomarkers. We
found that enrichment of low-abundance proteins via beads
in saliva led to low proteome coverage compared with unpro-
cessed samples (Figure 6A). Applying a more recently available
in-StageTip digestion method210 towards the characterization of
the secreted and cell-associated proteome of laboratory-grown
biofilm samples, we found that although both methods were ef-
ficient, the in-StageTip method enabled the quantification of up
to 20% more proteins in both samples (Figure 6C). Moreover,
as the selection of an appropriate protein database for peptide
identification is one of the most critical bioinformatics decisions
for accurate biological and ecological interpretation of microbial
community functions, we compared saliva or gingival crevicular
fluid proteome coverage with a standard database at the center
(1 085 825 sequences; 386 116 748 residues) with a customized
one, including the expanded human oral microbiome database
(2 299 026 sequences; 689 272 484 residues). Use of the custom-
ized, manually curated database led to detection of more proteins
that were not identified using the corresponding “standard” da-
tabase (Figure 6B). These findings indicated that sample-specific
databases can enable more comprehensive protein discovery in
quantitative proteomics/metaproteomic studies.
9 |  C H A LLE N G E S A N D FU T U R E
PROS PEC T S FO R M E TA PROTEO M I C
A PPLI C ATI O N S
Mass spectrometry-based proteomic and metaproteomic analyses of
oral biologic samples are technically demanding but surely possible.
However, a wider application to oral microbiota research has been
hindered by several challenges. Proteomics workflows can comple-
ment each other but should be adjusted for each biologic sample.
The variations in protein identifications in different studies can be
attributed to the criteria used for assignment of a protein, to inap-
propriate use of the standard target-decoy strategy, or could be af-
fected by the choice of protein databases.128,153 The selection of an
appropriate protein database for peptide identification is one of the
|
      73
most critical bioinformatics decisions for accurate biologic and eco-
logical interpretation of microbial community functions.154 Building
a reference database through metagenomic sequencing is often a
prerequisite for metaproteomics. UniProtKB/Swiss-Prot contains
561 568 sequence entries (December 2019), representing 9569
species, and more than 60% of the sequences are of bacterial ori-
gin (https://www.unipr​ot.org/stati​stics/​Swiss-Prot). Incorporation
of the expanded human oral microbiome database is essential for
deeper protein identification. Currently, a total of 1570 genomes,
representing 475 taxa, are currently available on the expanded
human oral microbiome database with annotations (http://www.
ehomd.org). It is known that oral metaproteomes consist of up to
700 different species, although not necessarily all of these have a
metagenome sequence. Protein identification may become difficult
if the taxonomic composition is unknown or if protein entries are
missing from protein databases.
10 | M E TA B O LO M I C S : A N O PE N FI E LD
FO R E N D E AVO R S
Metabolomics is the study of all of the metabolites in a biologic sam-
ple and comprises heterogeneous types of small molecules. In com-
parison to proteomics (and genomics for that matter), metabolomics
reveals what is actually happening within a cell or system as opposed
to the potential for something to happen, as the end point of reac-
tions in a single snapshot is revealed. Metabolite profiling as a phrase
has been used since the 1970s155 but the word metabolomics was
coined in 1998.156 Metabolites can be classified as either endoge-
nous or exogenous, for example, in the latter case from diet or xeno-
biotics. Endogenous metabolites can be further classified as primary
and secondary metabolites, those which are involved in primary life
processes in general across species, such as amino acids and glyco-
lytic intermediates, and those which have specific biologic and po-
tentially species-specific functions, such as alkaloids and hormones,
respectively. There is an extraordinary diversity with an estimated
1030 small molecules in the biosphere157 and 150 000 in the human
metabolome.158 The metabolite reference pathway from the Kyoto
Encyclopedia of Genes and Genomes is shown in Figure 5. Human and
microbial pathways can be identified in a species-specific manner
through the interactive interface on the Kyoto Encyclopedia of Genes
and Genomes website.159 Within the diverse chemistry of metabo-
lomics it is easy to appreciate that it is impossible to extract all me-
tabolites within one sample and this gives rise to different chemical
compartments that can be explored: for example, the explicit explo-
ration of lipids falls under the umbrella term of lipidomics.
In the preparation of samples for metabolomics investigations
it is imperative to stop any reactions that will immediately change
the makeup of the sample. An inability to quench enzyme activity,
for instance, will introduce variability and artefacts to the sample.160
Typical sample preparation will help to remove and denature pro-
teins and larger molecules to help with quenching. However, there
are many approaches to this and individual projects may benefit
 |
74       BOSTANCI et al.
from optimization of sample preparation for molecules of interest
and reproducibility.
Metabolomics can be global or targeted. In global approaches
the method is untargeted and the goal is to access as many me-
tabolites as possible for hypothesis generation; the number of me-
158
tabolites is typically ~ 1500 and these are often not quantified.
Whereas, in targeted approaches, a specifically small number, for
instance, 200-500 metabolites,158 is quantitatively measured across
the samples and the analysis is hypothesis-driven. The two primary
analytical techniques for metabolomics are nuclear magnetic reso-
nance and mass spectrometry. Nuclear magnetic resonance is highly
reproducible, uses simple sample preparation, can use a wide range
of solvent systems, and is nondestructive.161 However, is has rela-
tively low sensitivity, between micromolar and millimolar metabolite
concentrations. On the other hand, mass spectrometry has high sen-
sitivity (picomolar) but the metabolites must be able to be ionized for
detection, and it is destructive.161 Both can give rise to highly com-
plex patterns that need to be decoded to discover the identities of
the metabolites. In nuclear magnetic resonance, the structural elu-
cidation is facilitated by chemical shifts and coupling constants but
can be hampered by multiple overlaid metabolites. Use of two-di-
mensional techniques can assist in decongesting the signals but still
only the most abundant metabolites are revealed. For mass spec-
trometry, metabolites signatures from the parent ion mass or from
fragment ions masses in tandem mass spectrometry experiments are
compared with libraries of known compounds to elucidate identity.
As can be seen from the numbers above, it is not always possible
to elucidate the exact chemical detected, however, for the human
metabolome, the Human Metabolome Database currently (as of
162
January 2020) contains 114 177 metabolites, which is nearing the
estimated number for human cells. Mass spectrometers are nearly
always coupled to a pre-separation technique such as liquid or gas
chromatography or capillary electrophoresis. This helps to increase
the depth to which mass spectrometry can detect metabolites and
can help with the removal of confounding contaminants. The de-
velopment of ultra-performance liquid chromatography has further
improved liquid chromatography-mass spectrometry metabolomics
with increased signal-to-noise ratios and increased peak resolution
in systems run at very high pressures.
Once analytes have been detected, the data processing be-
comes key: as with all “omics” platforms there are a vast quantity
of data produced in each experiment. Structured analysis via pipe-
lines is highly recommended and reduction of dimensionality via
multivariate techniques is frequiently employed.161 Unsupervised
techniques, such as principal component analysis and supervised
techniques, such as partial least squares discriminant analysis are
common; further analysis with support vector machines, random
forests, and self-organizing maps are other examples that can be
used. Use of model evaluation and cross-validation is useful to
prevent overfitting, for example, from partial least squares dis-
criminant analysis, as this supervised technique uses correction
for confounding factors. These methods can be bespoke or may
be accessed in collected tools such as MetaboAnalyst163 and
MetaboSignal,164 which include modules to help with interpreta-
tion of data via metabolite enrichment analysis, metabolite path-
way analysis, or integrated pathway analysis. Downstream, the
analysis facilitates biologic interpretation, sample classification,
and biomarker discovery.
11 | M E TA B O LO M I C C H A R AC TE R IZ ATI O N
O F B I O FI LM S FRO M TH E O R A L ECOS YS TE M
In the exploration of dental biofilms, either clinical samples or from
biofilm or other model systems, mass spectrometry approaches
have been used (Table 6). This use of mass spectrometry may be
expected, particularly for clinical samples, where sample quantity
may be restricted and hence a lower quantity of metabolites may
be available. A small number of studies have examined the plaque
biofilm from either caries or periodontitis donors and revealed how
these metabolomes are altered by use of exogenous interventions.
Prior to 2010, targeted approaches were used to study the me-
tabolism of bacteria similar to oral species (Streptococcus lactis) using
techniques such as thin layer chromatography165-167 and nuclear
magnetic resonance.165 However, in 2010, Takahashi et al168 pub-
lished an article demonstrating that small quantities of supragingival
plaque, collected from young donors without remarkable oral con-
ditions, could be examined by capillary electrophoresis mass spec-
trometry. The data revealed that all the metabolites of the central
carbon pathways of the Embden-Meyerhof-Parnas, pentose-phos-
phate, and tricarboxylic acid cycle, except for erythrose-4-phos-
phate, could be detected quantitatively. Similar results were found
for isolated in vitro-cultured St. mutans, St. sanguinis, Actinomyces
oris, and Ac. naeslundii. Use of a glucose rinse by the plaque donors
or incubation of glucose with the in vitro-grown strains induced
changes in all three pathways: glycolysis was activated and metabo-
lites in the pentose-phosphate pathway were increased, while succi-
nate, fumarate, and malate were decreased in the tricarboxylic acid
cycle indicating metabolic regulation crossing these different but
linked pathways.
The following year, Takahashi et al169 went on to explore the ef-
fect of fluoride or xylitol on the metabolome of supragingival plaque
using capillary electrophoresis-mass spectrometry. Either of these in-
terventions was used as a rinse for 60 seconds and plaque samples
were collected after 10 minutes of rinsing. Fluoride, a known inhibitor
of enolase, inhibited lactate production via decreases in phosphoe-
nolpyruvate. However, xylitol, a nonfermentative sugar alcohol, had
no effect on plaque metabolism. In 2016, the same group published
an article170 exploring a different set of metabolites: the amino acids.
All major amino acids, except cysteine, were detected and their ca-
tabolism to ammonia was examined. Amino acids could be degraded
through various pathways including deamination, decarboxylation,
and transamination.
Nonfermentative sugars may also be of importance in the con-
trol of periodontopathogenic bacteria. Working with isolated strains
of Po. gingivalis and St. gordonii in an extensive study, Hashino
BOSTANCI et al.
et al171 examined the response of these bacteria grown in co-cul-
ture under the influence of erythritol for up to 48 hours. In com-
parison with other polyols, sorbitol, and xylitol, erythritol most
effectively decreased the addition of Po. gingivalis to St. gordonii bio-
films. Erythritol modulated the metabolic profiles of both bacteria:
the pentose-phosphate pathway, amino acid, and nucleotide sugar
metabolic pathways were decreased in both; and both showed de-
creases in polysaccharide production.
Further in-depth discovery analysis was published by Stipetic
172
et al. This approach examined the difference between planktonic
and biofilm preparations of Staphylococcus aureus, revealing a sig-
nificant difference in arginine metabolism between the two popula-
tions. These changes could represent the response of the bacteria to
their environment and adaptations to maintain chemical and pH ho-
meostasis. Meanwhile, Weidt et al173 used a targeted and untargeted
approach on cell extracts and the spent medium from Can. albicans
and St. aureus biofilms, grown either separately or in co-culture. Both
microorganisms demonstrated rapid consumption of exogenous
sugars (glucose and fructose) and that the pentose-phosphate path-
way was enhanced in co-culture. There was a high degree of overlap
shown by principal component analysis, as might be expected for
the core metabolism of whole microbes in monoculture, but which
demonstrate an intricate inter-kingdom interaction when the co-cul-
ture is also a factor.
In a study of the peptidic small molecules secreted by a wide
range of cultured oral microbes and a saliva-seeded multispecies
biofilm, Edlund et al174 explored changes after 24 and 72 hours of
biofilm growth. These molecules are of interest as they are often
antagonistic, containing classes such as the mutobactins175 and sali-
176
varicins, and support both commensal and pathogen competition
and resilience in colonization.177 They discovered between 400-900
peptidic small molecules per isolate and time point within a range
of 100-2000 Da and were able to match 153 of these to annota-
tions. The authors highlighted that this suggested that many of the
peptidic small molecules produced by oral bacteria are unknown,
indicating that there is a large molecular space still to be explored.
The identities revealed were of single amino acids, dipeptides, and
lactone-like compounds. Changes were found throughout the cul-
ture time demonstrating a dynamic chemotype within the oral
microbiome.
Zandona et al178 explored the difference between caries-ac-
tive and caries-free plaque biofilms from children in a largely
qualitative approach. The gas chromatography-mass spectrom-
etry analysis revealed a difference between the two origins of
the plaque samples, particularly associated with higher levels
of branched alcohols and esters in caries-active sampling sites.
Furthermore, Nascimento et al179 examined the influence of ei-
ther arginine (1.5%) or fluoride (1100 ppm) containing pastes for
12 weeks on dental plaque samples from adult donors that were
caries-active or caries-free, and in active donors which were at
dentinal or enamel-associated sites or caries-free sites. The liq-
uid chromatography-mass spectrometry approach revealed 3447
metabolite features, which mapped onto 208 known metabolites.
|
      75
There was minimal separation between the metabolic profiles of
the study groups but some differences could be detected when
examining the most prominent features. For instance, arginine
use decreased phenylalanine levels in caries-free sites, whereas
agmatine levels increased in caries-free sites but increased at car-
ies-active sites after use of fluoride. Also, exploring the effects of
arginine on the oral microbiome, Agnello et al208 used a saliva pool
from seven healthy donors to create biofilms that they challenged
with 75 mmol/L arginine and 15 mmol/L sucrose after up to 48
hours. Arginine exposure, at any time point prior to sucrose chal-
lenge, except simultaneous addition, invoked resilience to biofilm
acidification, and arginine catabolites were detected. These data
suggest active metabolism of arginine in vitro and require some
more information regarding the mechanism of this intervention.
Arginine metabolism via the arginine deiminase system converts
arginine to ammonia and carbon dioxide and generates adenosine
triphosphate. The genes involved are Arc A, B, and C located on an
operon. In St. gordonii there is the addition of an arginine-ornithine
antiporter ArcD. ArcD is a transmembrane protein that mediates up-
take of arginine and concomitant export of ornithine in an adenosine
triphosphate-independent manner. It has been revealed that ArcD
is additionally involved in the metabolic interactions of St. gordonii
periodontopathogenic bacteria. Sakanaka et al180 used an ArcD de-
letion mutant (Δ arcD) to explore the metabolic role in St. gordonii
interactions. The deletion mutant caused intracellular accumulation
of ornithine and suppression of arginine biosynthesis; and also de-
creased the accumulation of Fusobacterium nucleatum in co-culture.
Furthermore, in interactions between St. gordonii and Po. gingivalis,
Kuboniwa et al181 used a multi-omic approach to further understand
the role of streptococcal 4-aminobenzoate/para-aminobenzoic
acid on the accumulation of Po. gingivalis in dual-species biofilms.
Metabolomic analysis showed that para-aminobenzoic acid is used
for folate biosynthesis, reducing stress and increasing fimbrial adhe-
sion. These intricate studies reveal the nuances of cross-feeding in
the interaction of microbes in complex biofilms.
In periodontal treatment, the alteration of the microbiome has
been explored from multiple omics angles. For metabolomics, Califf
et al182 took a multi-omics approach to examine the impact of the
use of 0.25% sodium hypochlorite treatment for chronic periodonti-
tis. Periodontal pockets were sampled with Gracey curettes and the
plaque was extracted with organic solvent (methanol-acetonitrile-wa-
ter in a ratio of 1:2:1). The maximum pocket depth was measured and
correlated with the 20 most abundant metabolites. Improvement in
maximum pocket depth, equating to successful treatment, demon-
strated significant change in these metabolites, whereas patients
who did not improve had highly stable metabolomic profiles.
Lastly in this section on oral-derived microbes and biofilms,
Velsko et al183 examined the value of dental calculus as a source
of information from historical samples. The authors obtained den-
tal calculus samples from 12 skeletons from the Radcliffe Infirmary
Burial Ground collection, housed by Oxford Archaeology in Oxford,
UK, and then compared them with fresh dental calculus samples
from around the world. The historical samples were ~ 150-250 years
 |
76       BOSTANCI et al.
old. The authors demonstrated that there was some degradation of
particular classes of metabolites, such as the dipeptides, free amino
acids, free nucleotides, and carbohydrates, but that there was per-
sistence of medium and long chain fatty acids in the historical sam-
ples when compared with fresh samples. There was also evidence of
oxidation and chemical degradation in the historical samples, sug-
gesting that water-soluble metabolites were the least well preserved
but that there is value in metabolomics analysis of such valuable his-
torical resources.
12 |  M E TA B O LO M I C S PRO FI LI N G O F TH E
S A LI VA RY M I C RO B I O M E
There have been many explorations of the salivary metabolome ex-
amining a wide range of conditions of sampling184-186 and disease
status.187-190 However, there are fewer which have specifically ad-
dressed the contribution of the microbiome to the salivary metabo-
lome. While it may be difficult to deconvolute the host response and
the microbial contributions to the salivary metabolome, there have
been some studies that have reported changes which may contrib-
ute to our understanding of the oral microbiome-derived metabo-
lome to saliva. These are also listed in Table 6.
Marchesan et al191 explored the change in the salivary metabo-
lome before and after 21 days of experimental biofilm overgrowth
in donors in five categories of periodontal status. The authors iden-
tified 272 microbial species in the plaque samples from the biofilm
overgrowth sites and clustered these into microbial communities
with associated key phyla. Linear regression was used to discover
metabolites associated with the different microbial communities.
Strikingly, Synergistetes were highly associated with cyclodipeptides
cyclo (-leucine-proline) and cyclo (-phenylalanine-proline), and these
microbes and metabolites were correlated with microbial communi-
ties associated with more severely affected periodontal disease bio-
film donors. The finding of these cyclodipeptides was unexpected
by the authors and they speculated as to the functionality of these
molecules in quorum-sensing, control of virulence factors, and inhi-
bition of the growth of commensal organisms to the benefit of the
emergence of pathobionts.
Bregy et al192 compared volatile metabolites found in the head
space above bacterial cultures of Ag. actinomycetemcomitans, Po.
gingivalis, Ta. forsythia, and Tr. denticola with those that could be
found in saliva from either a patient with severe periodontitis or
two healthy control donors. The authors found 120 metabolites in
the bacterial cultures and these formed distinct clusters depending
on the originating species. Of these 120, 18 were found in saliva
with increased intensity in the periodontitis donor compared with
controls; and additionally, Po. gingivalis, Ta. forsythia, and Tr. denticola
were detected in the patient with periodontitis but not in the control
donors. The mass spectrometry technique used, secondary electro-
spray ionization, is often used to derive volatile metabolic finger-
prints of individual species,193,194 and so may be able to pinpoint the
contributions of bacteria to the complex mixture of saliva. Further
in-depth analysis of a much wider range of oral microbes would help
in further elucidation.
13 | CO N C LU S I O N S A N D FU T U R E
D I R EC TI O N S
Metaproteomics and metabolomics are two contemporary fields
that strongly complement the foundations laid by metagenomics
and metatranscriptomics in understanding microbial communities in
the oral ecosystem, as they help us to better understand their func-
tions under divergent environmental challenges and health states.
However, considerable efforts are still required to develop analy-
sis pipelines optimized and standardized for their characterization.
Future developments and in-depth understanding of how oral com-
munities change functionally and taxonomically in health and in dis-
ease and in changing environmental conditions will enlighten clinical
interventions and have the potential to alleviate oral health problems.
AC K N OW L E D G M E N T S
This work was supported by Karolinska Institutet Strategic Funds
(NB and GB), the Swedish Research Council (NB) and the Janggen-
Pöhn Foundation (DM).
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How to cite this article: Bostanci N, Grant M, Bao K, et al.
Metaproteome and metabolome of oral microbial
communities. Periodontol 2000. 2021;85:46–81. https://doi.
org/10.1111/prd.12351