metaproteomic characterization of dissolved organic matter in coastal seawater
TRANSCRIPT
SHORT CONTRIBUTION
Metaproteomic characterization of dissolved organic matterin coastal seawater
Mitsuhiro Yoshida • Keitaro Yamamoto •
Satoru Suzuki
Received: 13 June 2013 / Revised: 19 November 2013 / Accepted: 10 December 2013 / Published online: 29 December 2013
� The Oceanographic Society of Japan and Springer Japan 2013
Abstract We performed a comprehensive metaproteomic
analysis of the dissolved organic matter (DOM) in Japanese
coastal waters using liquid chromatography–tandem mass
spectrometry and demonstrated that these proteomes were
characterized by proteins with various functions, including
metabolic enzymes, membranes, and photosynthetic pro-
teins. The protein sources included cyanobacteria, hetero-
trophic bacteria, and eukaryotic phytoplankton. Most of the
components were similar among samples and also similar
to pelagic components. We also observed differences in the
compositions of the microbial communities of origin
among the different dissolved protein samples and differ-
ences in the relative abundance of specific dissolved pro-
tein types (e.g., cytoskeletal proteins), possibly indicating
potential dynamics in the coastal DOM pool.
Keywords Dissolved organic matter � Dissolved
proteins � Proteomics � LC–MS/MS � Microbial loop �Coastal area
1 Introduction
Dissolved organic matter (DOM) in seawater is the largest
pool of organic matter in the oceanic carbon and nitrogen
cycles (Siegenthaler and Sarmiento 1993), and it plays
important roles in utilization and production by heterotro-
phic bacteria and the subsequent food-web processes in the
marine microbial loop (e.g., Ducklow 2002). Among the
high-molecular-weight DOM ([1 kDa) in seawater, dis-
solved proteins are essential sources of nitrogen and energy
acquisition for heterotrophic bacterial production through
the microbial loop. These dissolved proteins are derived
from proteins that contain 85 % of the organic nitrogen in
marine organisms (Tanoue 1995). Thus, the proteins from
marine organisms are eventually transferred to the dis-
solved pool via biogeochemical processes; however, the
processes involved in the production and fate of marine
dissolved proteins and the source organisms for these
proteins are not yet well understood.
Regarding the protein species dissolved in seawater,
Tanoue et al. (1995) first reported that specific bacterial
porin proteins are ubiquitously distributed in the ocean. His
coworkers then expanded this research to include other
protein species (Yamada and Tanoue 2003) and to identify
the source bacterial groups for dissolved proteins (Suzuki
et al. 2000). Throughout the 1990s, gel electrophoresis,
western blotting, and N-terminal sequencing were the main
approaches used to study dissolved proteins (Suzuki et al.
1997; Tanoue et al. 1995; Yamada and Tanoue 2003).
Following recent advances in comprehensive ‘omics’
studies, the metaproteomic approach of using the liquid
chromatography–tandem mass spectrometry (LC–MS/MS)
method allows us to detect expressed proteins and thus
verify microbial functions in situ in various environments,
including marine waters (Morris et al. 2010; Sowell et al.
Electronic supplementary material The online version of thisarticle (doi:10.1007/s10872-013-0212-6) contains supplementarymaterial, which is available to authorized users.
M. Yoshida � K. Yamamoto � S. Suzuki
Center for Marine Environmental Studies (CMES), Ehime
University, 3 Bunkyo-cho, Matsuyama, Ehime 790-8577, Japan
Present Address:
M. Yoshida (&)
Japan Agency for Marine-Earth Science and Technology
(JAMSTEC), 2-15 Natsushima-cho, Yokosuka,
Kanagawa 237-0061, Japan
e-mail: [email protected]
123
J Oceanogr (2014) 70:105–113
DOI 10.1007/s10872-013-0212-6
2009, 2011). Using this technique, the specific proteomes
of the DOM fractions in seawater were recently charac-
terized in the pelagic environment (Dong et al. 2013;
Powell et al. 2005; Wang et al. 2011). In contrast, little
effort has been devoted to the proteomic content of the
dissolved fraction in coastal areas (Dong et al. 2013).
Coastal marine ecosystems have high biological activity,
abundant biomass, and various DOM sources that are
unlikely to be observed in oceanic pelagic ecosystems.
These considerations suggest that a wide variety of marine
dissolved proteins in the coastal ecosystem may possibly
be derived not only from marine biota but also from ter-
restrial sources.
To investigate the biological origin and chemical char-
acterization of these coastal dissolved proteins, we per-
formed an LC–MS/MS-based metaproteomic analysis of
the dissolved proteins in Japanese coastal waters. To our
knowledge, this study is the first report to describe the
comprehensive characteristics of dissolved proteins in
various coastal environments, including intensive sampling
locations in an inland sea where a red-tide bloom was
observed.
2 Materials and methods
2.1 Sample collection and preparation of dissolved
proteins in coastal DOM
Surface seawater samples (20 L) were collected at stations
(Stns) U1 (32�930N, 132�570E), U2 (32�950N, 132�570E),
and U3 (32�960N, 132�570E) in the Uwa Sea and at Stn H
(33�900N, 132�740E) in Horie Port, Matsuyama, Ehime
Prefecture, Japan (Fig. 1). The sampling at Stns U1-3 and
H was conducted on June 1 and October 6, 2010, respec-
tively. Stns U1 and U2 are located near fish farms. Blooms
of the red-tide dinoflagellate Gymnodinium-like cells were
observed at Stn U3 during the sampling (Fig. S1).
Each seawater sample was filtered with a plankton net
(mesh size 150 lm) immediately after sampling and
poured into a 20-L sampling bag. The concentration and
desalting of dissolved proteins were carried out according
to the procedures of Tanoue (1995). Sodium azide [NaN3
100 lM (w/v)] and sodium dodecyl sulfate [SDS 0.01 %
(w/v)] were added to the filtrates to prevent bacterial
growth and to help solubilize the proteins, respectively.
Then, the filtrates were further filtered through a 0.2-lm
cartridge filter (Advantec Toyo, Tokyo, Japan) to remove
particulates and microorganisms. Dissolved proteins in the
filtrate were concentrated using a Pellicon 2 cassette tan-
gential flow filtration system equipped with two 10-kDa
regenerated cellulose filter cassettes (Millipore, Billerica,
MA, USA). The concentrate was desalted with 0.01 %
(w/v) SDS/35 mM ammonium bicarbonate rinsing solution
(pH 7.8).
Bovine serum albumin (BSA 66.4 kD) was used as a
standard protein to evaluate the recovery of DOM by the
tangential flow filtration system. Seawater that had been
prefiltered through the 0.2-lm cartridge filter was spiked
with the BSA standard. The spiked solution was then run
through the Pellicon 2 cassette tangential flow filtration
system according to the procedure described above.
Quantification of the standard BSA added in the recovery
experiments was performed by densitometric measurement
of the band detected in SDS–PAGE analysis (see details
below). The recovery of the standard protein by the tan-
gential flow filtration system was 19–34 %.
Dissolved proteins in the concentrate from tangential
flow filtration were precipitated with ice-cold 10 % tri-
chloroacetic acid (TCA) for at least 12 h at 4 �C (Suzuki
et al. 1997; Tanoue 1995). The TCA-insoluble fraction was
centrifuged at 14,000g for 30 min at 4 �C, and then the
pellets were washed sequentially with 100 % ethanol and
100 % diethyl ether and were air-dried. Each dried pellet of
dissolved protein samples was redissolved in a sample
buffer solution of 62.5 mM Tris–HCl (pH 6.8), 2 % (w/v)
SDS, 5 % (v/v) 2-mercaptoethanol, and 10 % (w/v) glyc-
erol, heated to 95 �C, and maintained at this temperature
for 3 min. The sample was then analyzed using SDS–
polyacrylamide gel electrophoresis (SDS–PAGE).
2.2 One-dimensional SDS–PAGE of dissolved proteins
The rehydrated dissolved protein concentrate was loaded
onto a 14 % Bis–Tris gel according to the method of
Laemmli (1970). Electrophoresis was run at 20 mA in a
Tris–glycine-SDS buffer electrode solution. Low-molecu-
lar-weight markers were used for reference. After electro-
phoresis, the proteins in the gel were stained with colloidal
Coomassie Brilliant Blue-R250 (CBB-R; Bio-Rad, Hercu-
les, CA, USA).
2.3 N-terminal amino acid sequence analysis
Proteins in the bands detected by SDS–PAGE were sub-
jected to N-terminal amino acid sequencing. After the
SDS–PAGE separation, the proteins in the gel were
transferred onto a polyvinylidene difluoride (PVDF)
membrane (Immobilon-PSQ transfer membrane; Millipore)
at 40 mA for 45 min with a blotting device. Protein spots
on the membrane were visualized by the CBB-R250 (Bio-
Rad) staining method. The stained spots were cut out, and
the N-terminal amino acid sequence was determined using
an automated protein sequencer (ABI Procise 491HT;
Applied Biosystems, Foster City, CA, USA). The sequence
obtained from each protein spot was subjected to a
106 M. Yoshida et al.
123
BLASTp homology search against the NCBI GenBank
nonredundant (nr) protein database (Altschul et al. 1997).
2.4 Enzymatic digestion of dissolved proteins
For the ‘‘shotgun’’ proteomics experiments (Dong et al.
2009, 2013; Wang et al. 2011), the SDS–PAGE gel was cut
into seven pieces based on molecular weight marker size
([97.2, 97.2–66.4, 66.4–45.0, 45.0–29.0, 29.0–20.1,
20.1–14.3, and \14.3 kDa) to isolate molecular weight
fractions, and the pieces were subjected to enzymatic
digestion with the endoproteinase trypsin (in-gel digestion;
Wilm et al. 1996). Briefly, the gel pieces were destained
and dehydrated. After dithiothreitol reduction and iodoa-
cetamide alkylation, the trypsin solution (modified
sequencing grade; Promega, Madison, WI, USA; 10 lg/
mL) was added, and the pieces were incubated at 37 �C
overnight. In-gel digests were extracted with 50 % (v/v)
acetonitrile/5 % (v/v) trifluoroacetic acid rinses to recover
peptides, and the extracts were dried in a SpeedVac and
stored in a freezer (-20 �C) until LC–MS/MS analysis.
2.5 Mass spectrometry analysis
A tandem mass spectrometry (MS/MS) measurement was
performed using an in-line high-performance liquid chro-
matography (LC) system composed of a Paradigm MS4
microflow LC pump (Michrom BioResources, Auburn,
CA, USA) and an HTC PAL auto-sampler (CTC Analytics,
Zwingen, Switzerland) connected to an ion trap mass
spectrometer (LCQ Advantage; Thermo Finnigan, San
Jose, CA, USA). Briefly, the peptide extracts from the gel
Fig. 1 Sampling stations in the
Uwa Sea (Stns U1–3) and Horie
Port (Stn H), Japan
Metaproteomics of dissolved organic matter in coastal water 107
123
sample were injected onto a peptide CapTrap column
(0.5 9 2 mm, Michrom BioResources). The peptides were
eluted from the trap and subsequently separated on a C18
reverse-phase column (0.2 9 50 mm, packed with 3-lm
Magic C18 AQ particles; Michrom BioResources) with a
100-min linear gradient of 3–65 % mobile phase B [90 %
(v/v) acetonitrile/0.1 % (v/v) formic acid] in mobile phase
A [2 % (v/v) acetonitrile/0.1 % (v/v) formic acid] over
120 min at a flow rate of 2 lL/min. The separated sample
was introduced into the mass spectrometer via a fused-
silica Fortis Tip emitter (AMR, Tokyo, Japan). The LTQ
mass spectrometer operated with the following parameters:
spray voltage, 2.5 kV; spray temperature, 200 �C; and full
scan m/z range 450–2,000. The LC–MS system was fully
automated and under the direct control of the Xcalibur
software system (v.1.3; Thermo Finnigan).
2.6 Bioinformatic analysis
To identify the coastal dissolved proteins and their bio-
logical origins by the MS/MS analysis, the datasets of
protein sequences from the microbial taxa that possibly
contribute to the marine DOM pool were selected from the
GenBank nr protein database, and an original protein
database for the analysis was constructed. The proteins
from bacteria were classified broadly into five subgroups:
Cyanobacteria, a-Proteobacteria, b-Proteobacteria, c-Pro-
teobacteria, and a Bacteroidetes/Chlorobi group, and the
proteins from eukaryotic algae were classified into five
subgroups: Dinophyta, Cryptophyta, Haptophyta, Bacilla-
riophyta, and Chlorophyta. Proteins from other taxonomic
groups, including Archaea, Crustacea, and Stramenopiles,
were also used in this study.
The proteins related to the MS/MS spectra data obtained
by the mass spectrometry were identified by a similarity
search against the NCBI nr protein database from the
selected biological taxa mentioned above, using the SE-
QUEST algorithm (Thermo Finnigan). All SEQUEST
searches were performed using Bioworks software (v.3.1;
Thermo Finnigan), with the following parameters: fully
tryptic peptide, parent mass tolerance = 1.4, and peptide
mass tolerance = 1.5. The protein identification criteria
were applied based on a Delta CN ([0.1) and Xcorr (one
charge [1.9; two charges [2.2; three charges [3.75).
The integration of identified peptide hits into a related
protein was conducted with the in-house software Build-
Summary. Redundant proteins having the same peptide
hit(s) in a biological taxon were removed. The functional
assignments of the proteins identified were determined
according to the Kyoto encyclopedia of genes and genomes
(KEGG) classification system (http://www.genome.jp/
kegg/) (Kanehisa et al. 2008).
3 Results
3.1 SDS–PAGE
The profiles of the SDS–PAGE results for the dissolved
proteins from the four sampling sites (Stns U1, U2, U3, and
H) are shown in Fig. 2. The lanes of the U1, U2, and H
samples displayed a smeared staining pattern, and some
bands were observed with a smeared background. In con-
trast, the U3 sample displayed three distinct bands (namely
X, Y, and Z) within the 45.0–66.4 kDa molecular weight
range.
The protein sample from band X was subjected to
N-terminal amino acid sequencing, which indicated a
sequence of N-KHVAGEFTTG-C. This sequence did not
match reference sequences in the NCBI GenBank nr pro-
tein database. The samples from the other two bands (Y and
Z) did not yield enough protein for N-terminal amino acid
sequence analysis.
3.2 LC–MS/MS analysis
We used LC–MS/MS to identify proteins extracted from
the SDS–PAGE gel. For the above-mentioned bands, the
MS/MS spectra data matched the peptide sequences of
multiple proteins in the public database. Using the MS-
based ‘‘shotgun’’ method for gel slice sampling (Dong et al.
2009, 2013; Wang et al. 2011), the data of the MS/MS
Fig. 2 The SDS–PAGE electrophoretogram of the dissolved
proteins of the four coastal DOM samples. The left lane is a
molecular weight standard
108 M. Yoshida et al.
123
spectra obtained were processed, and the peptide sequences
in each fraction were identified. A total of 1,175 proteins,
each identified by one or more peptides in the samples,
were found in the four samples. The maximum number of
proteins (346 proteins) was observed in sample U3,
whereas the minimum number (192 proteins) was observed
in sample U1, which was located at an offshore site.
3.3 Biological sources of dissolved proteins
The taxonomic composition of the biological groups
derived from the identified dissolved proteins is shown in
Fig. 3. A wide variety of microbial taxa were identified,
including a-, b-, and c-Proteobacteria, Cyanobacteria,
Chlorophyta, the Bacteroidetes/Chlorobi group, Archaea,
viruses, and Stramenopiles. These taxa were abundantly
distributed among the four coastal water sites, accounting
for 85–95 % of the total number of identified proteins. In
contrast, the dissolved proteins associated with the
eukaryotic plankton from Bacillariophyta, Cryptophyta,
Dinophyceae, Crustacea, and Haptophyta were minor
constituents. The red-tide bloom sample U3 displayed a
relatively high abundance of Cyanobacteria, Chlorophyta,
and viruses, while proteobacterial proteins dominated at the
other three sites: U1, U2, and H (e.g., the a- and c-prote-
obacterial proteins in U2 and H).
3.4 Functional assignment
The dissolved proteins identified in this study were func-
tionally classified based on the KEGG taxonomic system
(Fig. 4). Consequently, 71, 139, 119, and 161 proteins in
the U1, U2, U3, and H samples, respectively, were related
to at least one functional assignment in a KEGG classifi-
cation category, representing 34–50 % of the total proteins
identified. Enzymes and enzyme-related proteins associ-
ated with metabolism, including carbohydrate, amino acid,
and energy metabolism, comprised a relatively high pro-
portion of the total identified proteins. Amino acid meta-
bolic enzymes were derived only from prokaryotes,
whereas other metabolic enzymes were derived from both
prokaryotes and eukaryotes. Cytoskeletal proteins, includ-
ing the tubulins (a- and b-tubulins) and actins, were
identified from the eukaryotic algae (Chlorophyta, Bacil-
lariophyta, Cryptophyta, Dinophyceae, and Haptophyta),
Crustacea, and Stramenopiles. These proteins were pre-
dominantly detected in the U2 and H site samples (11 and
13 % of the total identified proteins, respectively) and were
not detected in the samples from the other sites. Mem-
brane-associated and photosynthetic proteins were also
detected in the coastal DOM in this study. A large pro-
portion (50–66 %) of the proteins could not be identified.
4 Discussion
Prior to the development of MS-based comprehensive
proteomics, several pioneering works on marine dissolved
proteins involved analysis by N-terminal sequencing (Ta-
noue et al. 1995) and western blotting (Suzuki et al. 1997,
2000). The shotgun proteomic approach by LC–MS/MS,
presented here, illustrated that the dissolved protein frac-
tions from the four coastal water samples were composed
of proteins with varying functions that originated from
various microbial taxa. The results confirmed that a few
bacterial membrane proteins were sources of coastal dis-
solved protein constituents (Yamada and Tanoue 2009;
Yamada et al. 2000). In addition to previously known
heterotrophic bacteria, such as Pseudomonas and Vibrio
(Suzuki et al. 1997; Tanoue et al. 1995; Yamada and Ta-
noue 2006, 2009; Yamada et al. 2000), photosynthetic
organisms (including cyanobacteria and some eukaryotic
algae), Archaea, and Stramenopiles also appear to play
important roles as potential contributors to the costal DOM
pool. In addition, we observed that virus-associated pro-
teins detected in the coastal DOM fractions represented
5–13 % of the total dissolved proteins in each DOM
sample (Fig. 3).
In this study, proteins from most eukaryotic planktons
(Bacillariophyta, Cryptophyta, Dinophyceae, Crustacea,
and Haptophyta) displayed a relatively low abundance of
the total proteins identified, compared with those of
prokaryotes, for example; and as mentioned above, the
relatively abundant enzymes involved in amino acid
Fig. 3 Distribution of the biological origin associated with the
dissolved proteins detected in the four coastal DOM samples
Metaproteomics of dissolved organic matter in coastal water 109
123
metabolism were derived only from prokaryotes. Given
that protists can represent as much as half of the biomass
in surface seawater (Caron et al. 1995), it should be
noted that there is a large set of proteins still unidentified
due to the lack of a proteomic database for these marine
eukaryotes, especially Dinophyceae, Haptophyta, and
Cryptophyta (Table 1). Accordingly, the observations for
these eukaryotic planktons must be interpreted carefully.
When examining the dissolved protein at Stn 3, where
red-tide blooms by a Dinophyceae species were observed
(Fig. S1), Dinophyceae proteins were minor components,
whereas those from Cyanobacteria, Chlorophyta, and
viruses were predominant. These findings might be due
to an insufficient eukaryotic phytoplankton database or
to the lability of proteins from this type of phytoplank-
ton bloom.
In addition, in the U3 sample, we found three intense
bands that were possible proteins associated with the red-
tide bloom phenomenon, but we could not identify the
Fig. 4 KEGG-based functional
assignments of the dissolved
proteins detected in the coastal
DOM samples. The KEGG
categories including more than
1 % of the total identified
proteins are presented
Table 1 The protein databases used in this study
Protein database No. of protein
entries
c-Proteobacteria 4,848,684
a-Proteobacteria 1,806,838
b-Proteobacteria 1,645,045
Viruses 1,204,312
Archaea 934,592
Bacteroidetes/Chlorobi group 875,673
Cyanobacteria 480,588
Chlorophyta 112,201
Stramenopiles 55,536
Crustacea 52,218
Bacillariophyta 49,322
Cryptophyta 3,587
Dinophyceae 3,530
Haptophyceae 1,074
110 M. Yoshida et al.
123
potential origin of the major band using N-terminal amino
acid sequencing. Instead, based on the LC–MS/MS data,
the peptide sequences suspected for each of the distinct
three bands matched with various proteins that have not yet
been reported in marine DOM, a result similar to the study
by Powell et al. (2005). Thus, these bands may have con-
sisted of mixed bands with similar molecular sizes. The
possible sources of these bands may have not been iden-
tified due to the lack of a protein database for marine
organisms, together with the data of N-terminal amino acid
sequencing as described above.
In all the sampling sites, enzymes involved in the
metabolism of carbohydrates, amino acids, and energy,
along with cytoskeletal proteins, membrane proteins,
transporters, and photosynthetic proteins, were commonly
detected. These categories were found in previous studies
that examined oceanic surface waters (Powell et al. 2005;
Suzuki et al. 1997; Wang et al. 2011; Yamada and Tanoue
2006, 2009). To date, alkaline phosphatase, ATP synthase,
ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBi-
sCo), and pyruvate carboxylase have been detected in
oceanic DOM samples (Powell et al. 2005; Wang et al.
2011; Yamada and Tanoue 2006, 2009). In this study, we
also detected alkaline phosphatase, ATP synthase, and
RuBisCo, indicating that the DOM fractions harbored
many more types of bacterial enzymes than previously
reported in other coastal areas (Yamada and Tanoue 2009).
Most of the enzymes identified in the present study were
associated with nutrient (such as carbohydrates and amino
acids) and energy metabolism, and their numbers accoun-
ted for 41–64 % of the total dissolved proteins. Bacterial
enzymes were also major constituents of the coastal DOM
pool, indicating high bacterial activity in the coastal zone
that may be linked to the high frequency of the enzymes.
This bacterial activity may be a relevant factor modulating
the DOM components in coastal environments, rather than
the accumulation of refractory bacterial membrane proteins
(e.g., OprP and OmpA-like proteins) found in pelagic
waters. Some of the detected enzymes were exo- and
periplasmic enzymes, including esterases, peptidases, pro-
teases, lipases, phosphatases, and ATP synthases. These
enzymes are potentially exposed to the outside of the cell,
and some of these enzymes are associated with the outer
and inner membrane, which may act as refractory protein
mechanisms and thus protect the proteins from degradation
due to the association with lipid bilayers, as reported by
Nagata et al. (1998), as well as association with peptido-
glycan (Miyoshi and Suzuki 2004).
Residues of refractory dissolved proteins, such as porin
P and OmpA-like membranes, have been reported in
pelagic waters (Suzuki et al. 1997; Tanoue et al. 1995;
Yamada and Tanoue 2006), whereas this study did not
detect these membrane proteins in our coastal samples.
Instead, other membrane-associated proteins, e.g., proteins
from prokaryotic outer membranes, such as lipoproteins
and inner membrane-associated proteins, transporters
(including largely ABC transporters and TonB-dependent
transporters), and ATP synthases, were observed. The
subcellular localization of these proteins is also likely to be
associated with refractory characteristics and a subsequent
accumulation in seawater (Wang et al. 2011).
Among the protein classes, the cytoskeletal proteins
displayed higher proportions in the U2 and H site samples
(11 and 13 %, respectively). Actin is the most abundant
protein in many eukaryotic cells, and it forms actin fila-
ments, the main structural component of the filopodia and
microvilli (Mogilner and Rubinstein 2005). Tubulin is also
a major structural component of microtubules that form the
flagella or cilia of eukaryotes, but the protein is typically
easily degraded in natural waters because of the absence of
a proteolysis-resistant structure (Dong et al. 2010; Wang
et al. 2011). Therefore, the patchy distribution of such
cytoskeleton proteins among the four sites might be the
result of a rapid decomposition to different net products,
due to the differences in the microbial community com-
position at each site and their different activities among
these sites. In fact, the relative abundance of the dissolved
proteins associated with eukaryotes from sites U2 and H
(average 20 %) was higher than that from sites U1 and U3
(average 14 %), suggesting a mosaic pattern of potential
microbial sources of cytoskeletal protein in this inland sea.
Among the photosynthetic proteins, RuBisCo, ATP synthase,
phycoerythrin, chloroplast light harvesting protein, and phyco-
cyanin from cyanobacteria and eukaryotic algae accounted for
2–9 % of the total proteins (Fig. 4). These results imply that the
contribution of autotrophs to the bulk DOM pool is much higher
in the coastal environment than in oceanic water (Wang et al.
2011). The most abundant photosynthetic protein was RuBisCo,
which is involved in carbon fixation and is known as the most
abundant protein on earth (Cooper 2000; Dhingra et al. 2004).
However, recent reports by Nunn et al. (2009, 2010) indicated
that RuBisCo is not the most abundant protein in the proteome of
the marine diatom Thalassiosira pseudonana and was more
rapidly degraded than other proteins (photosynthesis membrane
proteins and a membrane transporter) during a degradation
experiment with T. pseudonana cells after growth. Thus, RuBi-
sCo may have a short half-life, despite being one of the most
abundant proteins produced by cells.
Most of the functional categories and their potential
microbial origins for the coastal dissolved proteins identi-
fied in this study were observed in each proteome of the
four DOM fractions, and they have also been observed in
open oceanic samples (Dong et al. 2013; Wang et al. 2011);
however, there were several differences between the
coastal and oceanic surface proteomes. For the composition
of the potential DOM-producing microbial community, the
Metaproteomics of dissolved organic matter in coastal water 111
123
dissolved proteins derived from b-Proteobacteria, known
as the major phylogenetic group in freshwater environ-
ments such as estuaries (Cottrell and Kirchman 2003), were
more abundant in coastal water (here 5–11 %) than oceanic
surface waters (\1 %; Dong et al. 2013), suggesting the
presence of a coastal-specific DOM resulting from a
potential terrestrial input. The eukaryote-associated pro-
teins of the coastal DOM fractions were also detected
abundantly, ranging from 11 to 23 %, but were not
detected in oceanic waters. The origins of these proteins
are most likely linked with eukaryotic phytoplankton
blooms occurring in the coastal environment. In addition,
our data indicate that a larger proportion (50–66 %) of
functionally unidentified proteins, mostly hypothetical
proteins, was observed in this study compared to the open
ocean (32–43 %; Wang et al. 2011). This outcome may
reflect the fact that the sources of dissolved proteins in
coastal areas are more diverse and complex, although dif-
ferent databases were used in both studies. In the com-
parison of the four coastal samples, we also found
differences in the compositions of the microbial origin
communities for the different dissolved protein samples
and in the relative abundance of specific dissolved protein
types (e.g., cytoskeletons), possibly indicating some
potential dynamics in the coastal DOM pool. A more
detailed spatial and temporal analysis of dissolved proteins
in both coastal and pelagic environments is needed to
further understand the dynamics in the marine DOM pool
and to link the regional DOM dynamics to the possible
biological and geographical processes in a coastal area.
Acknowledgments This study was performed under the ‘‘Global
COE Program in Ehime University’’ of the Ministry of Education,
Culture, Sports, Science, and Technology (MEXT) of Japan and was
partially supported by the Sasagawa Scientific Research Grant (no.
23-728) from the Japan Science Society and a Grant-in-Aid for Sci-
entific Research from the Japan Society for the Promotion of Science
(JSPS).
References
Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, Miller W,
Lipman DJ (1997) Gapped BLAST and PSI-BLAST: a new
generation of protein database search programs. Nucleic Acids
Res 25:3389–3402
Caron DA, Dam HG, Kremer P, Lessard EJ, Madin LP, Malone TC,
Napp JM, Peele ER, Roman MR, Youngbluth MJ (1995) The
contribution of microorganisms to particulate carbon and
nitrogen in surface waters of the Sargasso Sea near Bermuda.
Deep Sea Res Part I 42:943–972
Cooper GM (2000) The cell: a molecular approach, 2nd edn. Sinauer,
Sunderland
Cottrell MT, Kirchman DL (2003) Contribution of major bacterial
groups to bacterial biomass production (thymidine and leucine
incorporation) in the Delaware estuary. Limnol Oceanogr
48:168–178
Dhingra A, Portis AR, Daniell H (2004) Enhanced translation of a
chloroplast-expressed RbcS gene restores small subunit levels
and photosynthesis in nuclear RbcS antisense plants. Proc Natl
Acad Sci USA 101:6315–6320
Dong H-P, Wang D-Z, Dai M-H, Chan L-L, Hong H-S (2009)
Shotgun proteomics: tools for the analysis of marine particulate
proteins. Limnol Oceanogr Methods 7:865–874
Dong H-P, Wang D-Z, Dai M-H, Hong H-S (2010) Characterization
of particulate organic matters in the water column of the South
China Sea using a shotgun proteomic approach. Limnol Ocea-
nogr 55:1565–1578
Dong H-P, Wang D-Z, Xie Z-X, Dai M-H, Hong H-S (2013)
Metaproteomic characterization of high molecular weight dis-
solved organic matter in surface seawaters in the South China
Sea. Geochim Cosmochim Acta 109:51–61
Ducklow HW (2002) Foreword. In: Hansell DA, Carlson CA (eds)
Biogeochemistry of marine dissolved organic matter. Academ-
iPress, San Diego, pp xv–xviii
Kanehisa M, Araki M, Goto S, Hattori M, Hirakawa M, Itoh M,
Katayama T, Kawashima S, Okuda S, Tokimatsu T, Yamanishi
Y (2008) KEGG for linking genomes to life and the environ-
ment. Nucleic Acids Res 36:D480–D484
Laemmli UK (1970) Cleavage of structural proteins during the
assembly of the head of bacteriophage T4. Nature 227:680–685
Miyoshi T, Suzuki S (2004) Degradation of outer membrane proteins
of Synechococcus sp. in vitro and in situ. J Oceanogr 60:825–833
Mogilner A, Rubinstein B (2005) The physics of filopodial protrusion.
Biophys J 89:782–795
Morris RM, Nunn BL, Frazar C, Goodlett DR, Ting YS, Rocap G
(2010) Comparative metaproteomics reveals ocean-scale shifts
in microbial nutrient utilization and energy transduction. ISME J
4:673–685
Nagata T, Fukuda R, Koike I, Kogure K, Kirchman DL (1998)
Degradation by bacteria of membrane and soluble protein in
seawater. Aquat Microb Ecol 14:29–37
Nunn BL, Aker JR, Shaffer SA, Tsai YH, Strzepek RF, Boyd PW,
Freeman TL, Brittnacher M, Malmstrom L, Goodlett DR (2009)
Deciphering diatom biochemical pathways via whole cell
proteomics. Aquat Microb Ecol 55:241–253
Nunn BL, Ting YS, Malmstroem L, Tsai YS, Squier A, Goodlett DR,
Harvey HR (2010) The path to preservation: using proteomics to
decipher the fate of diatom proteins during microbial degrada-
tion. Limnol Oceanogr 55:1790–1804
Powell MJ, Sutton JN, Castillo CED, Timperman AT (2005) Marine
proteomics: generation of sequence tags for dissolved proteins in
seawater using tandem mass spectrometry. Mar Chem
95:183–198
Siegenthaler U, Sarmiento J (1993) Atmospheric carbon dioxide and
the ocean. Nature 365:119–125
Sowell SM, Wilhelm LJ, Norbeck AD, Lipton MS, Nicora CD,
Barofsky DF, Carlson CA, Smith RD, Giovanonni SJ (2009)
Transport functions dominate the SAR11 metaproteome at low-
nutrient extremes in the Sargasso Sea. ISME J 3:93–105
Sowell SM, Abraham PE, Shah M, Verberkmoes NC, Smith DP,
Barofsky DF, Giovannoni SJ (2011) Environmental proteomics
of microbial plankton in a highly productive coastal upwelling
system. ISME J 5:856–865
Suzuki S, Kogure K, Tanoue E (1997) Immunochemical detection of
dissolved proteins and their source organism. Mar Ecol Prog Ser
158:1–9
Suzuki S, Fujita N, Kimata N, Kogure K, Tanoue E (2000) Isolation
of bacteria with membrane proteins homologous to Vibrio
anguillarum porin Omp35La. Microbes Environ 15:189–195
112 M. Yoshida et al.
123
Tanoue E (1995) Detection of dissolved protein molecules in oceanic
waters. Mar Chem 51:239–252
Tanoue E, Nishiyama S, Kamo M, Tsugita A (1995) Bacterial
membranes: possible source of a major dissolved protein in
seawater. Geochim Cosmochim Acta 59:2643–2648
Wang D-Z, Dong H-P, Xie Z-X, Dai M-H, Hong H-S (2011)
Metaproteomic characterization of dissolved organic matter in
the water column of the South China Sea. Limnol Oceanogr
56:1641–1652
Wilm M, Shevchenko A, Houthaeve T, Breit S, Schweigerer L, Fotsis
T, Mann M (1996) Femtomole sequencing of proteins from
polyacrylamide gels by nanoelectrospray mass spectrometry.
Nature 379:466–469
Yamada N, Tanoue E (2003) Detection and partial characterization of
dissolved glycoproteins in oceanic waters. Limnol Oceanogr
48:1037–1048
Yamada N, Tanoue E (2006) The inventory and chemical character-
ization of dissolved proteins in oceanic waters. Prog Oceanogr
69:1–18
Yamada N, Tanoue E (2009) Similarity of electrophoretic dissolved
protein spectra from coastal to pelagic seawaters. J Oceanogr
65:223–233
Yamada N, Suzuki S, Tanoue E (2000) Detection of Vibrio
(Listonella) anguillarum porin homologue proteins and their
source bacteria from coastal water. J Oceanogr 56:583–590
Metaproteomics of dissolved organic matter in coastal water 113
123