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Exoproteomics: Exploring the world around biological systems

October 2012
Expert Review of Proteomics 9(5):561-75

October 2012
9(5):561-75

DOI:10.1586/epr.12.52

Source
PubMed

Authors:

Jean Armengaud

Atomic Energy and Alternative Energies Commission

Joseph Alexander Christie-Oleza

The University of Warwick

Geremy Clair

Pacific Northwest National Laboratory

Vero Malard

Atomic Energy and Alternative Energies Commission

Show all 5 authorsHide

The term 'exoproteome' describes the protein content that can be found in the extracellular proximity of a given biological system. These proteins arise from cellular secretion, other protein export mechanisms or cell lysis, but only the most stable proteins in this environment will remain in abundance. It has been shown that these proteins reflect the physiological state of the cells in a given condition and are indicators of how living systems interact with their environments. High-throughput proteomic approaches based on a shotgun strategy, and high-resolution mass spectrometers, have modified the authors' view of exoproteomes. In the present review, the authors describe how these new approaches should be exploited to obtain the maximum useful information from a sample, whatever its origin. The methodologies used for studying secretion from model cell lines derived from eukaryotic, multicellular organisms, virulence determinants of pathogens and environmental bacteria and their relationships with their habitats are illustrated with several examples. The implication of such data, in terms of proteogenomics and the discovery of novel protein functions, is discussed.

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10.1586/EPR.12.52

561

ISSN 1478-9450

www.expert-reviews.com

Review

Secreted proteins represent a speciﬁ c fraction

of the proteome in the sense that they have to

be transported across membranes to ensure for

the biogenesis of membranes and cell walls,

nutrient uptake, motility and interactions. A

signiﬁ cant part of them are active outside the

cell. Proteolysis in the extracellular environment

may be important in some cases, and only the

most stable proteins will remain abundant in

the extracellular environment. Most secreted

proteins are synthesized as precursors with a

cleavable N-terminal signal sequence, but a

signiﬁ cant fraction is secreted by nonclassical

pathways, that is, without signaling peptides.

This signal peptide is required for targeting of

the protein to the membrane-embedded export

machinery [1] . Upon translocation across the

membrane, the signal peptide is either cleaved

from the precursor via a membrane-bound

signal peptidase or served as a signal anchor

into the membrane. The most predominant

signal peptide-dependent pathways are the

Sec- and Tat-secretory pathways [2–4] . The

Sec pathway is ubiquitous and essential for

viability in bacteria, archaea and eukaryotes [5] .

This export system translocates unfolded

proteins across the endoplasmic reticulum (ER)

membrane in eukaryotes and the cytoplasmic

membrane of bacteria and archaea, through a

protein-conducting channel. The twin-arginine

translocation (Tat) machinery exports folded

proteins across the cytoplasmic membrane

in bacteria and archaea, as well as across the

chloroplast thylakoid membrane in plants [4] .

Tat recognizes longer and less hydrophobic,

signal peptides than those of the Sec system,

comprising two contiguous arginine residues

[4] . In Gram-positive bacteria, protein substrates

of sortases are translocated via Sec-dependent

secretory pathway but are covalently anchored

to the cell wall. Lipoproteins are also secreted (in

Gram-positive bacteria) or exported (in Gram-

negative bacteria) via the Sec pathway (or Tat-

dependent pathways for some of them) but are

anchored to the cytoplasmic membrane (in both

types of bacteria) or the outer membrane (on

the periplasmic side in Gram-negative bacteria).

Integral membrane proteins are inserted via

YidC in a Sec-dependent or independent

manner. The Sec- and Tat-dependent secretion

Jean Armengaud*

Joseph A

Christie-Oleza

Gérémy Clair

Véronique Malard

and

Catherine Duport

3 ,

CEA , DSV, IBEB, Lab Biochim System

Perturb, Bagnols-sur-Cèze, F-30207,

France

School of Life Sciences, University of

Warwick, Coventry CV4 7AL, UK

Université d’Avignon et des Pays de

Vaucluse, UMR408, Sécurité et Qualité

des Produits d’Origine Végétale,

F-84000 Avignon, France

INRA, UMR408, Sécurité et Qualité des

Produits d’Origine Végétale, F-84914

Avignon, France

*Author for correspondence:

Tel.: +33 046 679 6802

Fax: +33 046 679 1905

jean.armengaud@cea.fr

The term ‘exoproteome’ describes the protein content that can be found in the extracellular

proximity of a given biological system. These proteins arise from cellular secretion, other protein

export mechanisms or cell lysis, but only the most stable proteins in this environment will remain

in abundance. It has been shown that these proteins reﬂ ect the physiological state of the cells

in a given condition and are indicators of how living systems interact with their environments.

High-throughput proteomic approaches based on a shotgun strategy, and high-resolution mass

spectrometers, have modiﬁ ed the authors’ view of exoproteomes. In the present review, the

authors describe how these new approaches should be exploited to obtain the maximum useful

information from a sample, whatever its origin. The methodologies used for studying secretion

from model cell lines derived from eukaryotic, multicellular organisms, virulence determinants of

pathogens and environmental bacteria and their relationships with their habitats are illustrated

with several examples. The implication of such data, in terms of proteogenomics and the

discovery of novel protein functions, is discussed.

Exoproteomics: exploring

the world around biological

systems

Expert Rev. Proteomics 9(5), 561–575 (2012)

KEYWORDS: exoproteomics • hypothetical proteins • metaproteomics • peptide signal • proteogenomics • proteomic

surfaceome • secretion systems • secretome • shotgun proteomics • subcellular localization predictor • toxins

• virulence factors

For reprint orders, please contact reprints@expert-reviews.com

Expert Rev. Proteomics 9(5), (2012)

562

Review

pathways also lead to the release of some proteins into the

extracellular medium

(FIGURE 1) . In Gram-positive bacteria, some

exported proteins share a determinant at their carboxyl termini

for covalent attachment to the cell wall. Such a signal consists of

a signature motif (such as LPXTG or PEP amino acid sequences),

a hydrophobic transmembrane α-helix, and a cluster of basic

amino acids (primarily arginine residues) and is processed by

sortases [6] . In eukaryotic cells, Sec-dependent extracellular

proteins are transported from the ER to the Golgi apparatus and

subsequently to the cell surface, where they are delivered to the

microenvironment by fusion of the Golgi-derived vesicles with the

plasma membrane [7] . This pathway of protein export is referred

to as the classical secretory pathway. Other secretory pathways

also exist in eukaryotic cells, allowing for protein secretion

independently of the presence of an identiﬁ able signal peptide

and the ER–Golgi. Diverse mechanisms have been proposed,

including lysosomal secretion, endosomal recycling and release

of membrane vesicle exosomes into the extracellular ﬂ uid [8, 9] . In

bacteria, the mechanisms responsible for nonclassical secretion are

still largely undocumented. Nevertheless, cytoplasmic proteins

found in the extracellular milieu are thought to be released

during septum formation, and furthermore, membrane vesicles

from the envelope of growing Gram-negative bacteria have been

shown to be secretory vehicles for proteins [10 –12] . Furthermore,

extracellular cytoplasmic proteins have been shown to participate

in a range of biological processes including virulence [13] and as

nonactive (enzymatically) binding substrates [14] . Such proteins,

which display two unrelated functions, have been referred to as

‘moonlighting’ proteins and could be selectively secreted [13] .

Among the proteins secreted by bacteria and archaea, S-layer

proteins are worth mentioning as they are abundantly assembled

on the surface of the cells as paracrystalline sheets and associated

with many other secreted proteins [15] .

The term ‘secretome’ refers to the pool of proteins which are

actively secreted via classical or nonclassical mechanisms or via

the release of exosomes, as well as the secretion machinery itself

[16] . The secreted proteins comprise membrane-linked proteins

as well as other translocated proteins. Proteins released into the

extracellular milieu belongs to the latter group of protein. A

proportion of the exported proteins are inserted in the membrane

via the presence of transmembrane hydrophobic segments or can

also be associated in some cases with other transmembrane-

inserted proteins, as exempliﬁ ed by some components of the

ﬂ agellum apparatus of motile bacteria [17] . The speciﬁ c fraction

of secreted proteins bound to the external surface of the cell, also

known as the ‘surface proteome’ or ‘proteomic surfaceome’, can be

evaluated by means of shaving-based approaches [18 –20] . For this,

intact cells are exposed to limited proteolysis, to cleave surface

proteins present on the outer membrane, cytoplasmic membrane

and/or cell wall. The peptides derived from shaved proteins can be

identiﬁ ed by mass spectrometric analysis but are restricted to the

exposed, nonhydrophobic regions, sometimes hindering protein

identiﬁ cation because of low sequence coverage. Numerous studies

have been reported regarding bacterial [21, 22] or human cell [23, 24]

surfaceomes and plant cell-wall proteomes

[25] . An interesting recent innovation is

the speciﬁ c detection of secreted proteins

on the bacterial cell surface directly by

MALDI-TOF mass spectrometry using

colonies of Mycobacterium marinum [26] .

This and other recent studies have a large

implication on microbial diagnostics, a

ﬁ eld of growing interest in terms of human

health and microbial ecology [27, 28] . A major

fraction of the secreted proteins will ﬁ nally

end up outside the cell. These exported

proteins comprise soluble components of

transporters, chelators, proteases, toxins,

growth factors, cytokines, sensors and

numerous proteins, which are poorly

characterized, that is, hitherto annotated

as hypothetical proteins or assigned

with a putative function. The fraction

that corresponds to the protein content

present outside of the cells, either in the

conditioned medium for laboratory cell

cultures or in the extracellular matrix of

environmental samples, is, strictly, known

as the ‘exoproteome’. The exoproteome

includes both actively secreted proteins and

extracellular, but nonsecreted, proteins,

resulting from cell lysis, cell friction and

Periplasmic space

Outer membrane

Extracellular content

Cytoplasm

Exoproteome

Inner membrane

Surface

proteome

Secretome

Nonsecreted

exoproteins

Secreted

exoproteins

Expert Rev. Proteomics © Future Science Group (2012)

Figure 1. Secretome, surfaceome and exoproteome concepts illustrated with a

Gram-negative bacterium model. The exoproteome (black dotted line) regroups the

so-called ‘secreted exoproteins’ and ‘nonsecreted exoproteins’ (among them, proteins

from cellular lysis or fragments from proteomic surfaceome due to abrasion). The

secretome (red dotted line) regroups the so-called ‘secreted exoproteins’, most

membrane proteins here shown in orange, and the diverse secretion systems here drawn

in purple. The surface proteome (blue dotted line) regroups the accessible fraction of the

proteins inserted in the membrane in contact to the extracellular environment.

Armengaud, Christie-Oleza, Clair, Malard & Duport

563

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protein degradation [16] . Thus, ‘secretome’, ‘proteomic surfaceome’

and ‘exoproteome’ are three different concepts, which represent

different and partially overlapping protein fractions, as clariﬁ ed

FIGURE 1 . The term ‘exoproteome’ should be used in preference

to ‘secretome’ when describing the subset of proteins present in

the extracellular milieu, as previously recommended [16] .

Comparative genomics have revealed that the fraction of puta-

tive exported/secreted proteins encoded by microbial genomes is

rather large. A ﬁ rst study indicated that this ratio ranged from 8

to 37% for Methanococcus jannaschii and Mycoplasma pneumonia ,

respectively [29] . Another report estimated that between 25–35%

and 15–25% of proteins could be exported/secreted for Gram-

negative and Gram-positive bacteria, respectively [30] . In this

review, the authors highlight the importance of exoproteome

analysis in different biological systems. This is illustrated with

examples from cultured human cell lines, microbial pathogens

and environmental samples. It should be mentioned that other

studies on unicellular eukaryotes, plants, animals and archaea

would be also worth citing in such a review if more space was

available. The authors also describe the most recent exoproteomic

methodologies, with emphasis on shotgun proteomic approaches

and bioinformatic tools for predicting the presence of peptide

signal and other determinants, as well as the protein localiza-

tion. Finally, the authors draw the perspectives of exoproteomics,

a ﬁ eld with an enormous potential in terms of novel-function

discovery, as highlighted by the large amount of exoproteins pre-

dicted at the genomic level and detected at the proteomic level,

which remain poorly characterized, as well as for biomedical and

biotechnological applications.

Tools & strategies for exoproteome analysis

A new generation of mass spectrometers for

high-throughput proteomics

Over the last decade, substantial improvements in mass spectrom-

etry for analyzing proteins and peptides have led to fundamental

changes in the ﬁ eld of proteomics. Pioneering studies on exopro-

teomes have been carried out by fractionating complex samples

on 2D-PAGE gels, followed by the identiﬁ cation of isolated pro-

tein spots using peptide mass ﬁ ngerprinting with MALDI-TOF

mass spectrometry records. Today, complex peptide mixtures are

in most cases resolved by reversed-phase chromatography (nano-

HPLC) prior to being analyzed by high-throughput hybrid mass

spectrometers. Alternatively, they can be resolved ﬁ rst on strong

cation exchange chromatography in combination with reversed-

phase chromatography as in MudPIT experiments, or by OFFGEL

electrophoresis [31] . These novel shotgun approaches facilitate the

identiﬁ cation of several hundreds of proteins from a single sam-

ple. Scan rates ranging from ﬁ ve to 20 high-quality tandem mass

spectrometry (MS/MS) spectra acquired per second are achieved

by the most recent mass spectrometers, with attomolar sensitiv-

ity [32] . Bottom-up strategies, in which proteins in the sample are

proteolyzed with trypsin and the masses and sequences of the

resulting tryptic peptides are determined by MS/MS, are the most

frequently used approaches. With the advent of shotgun strate-

gies and high-throughput mass spectrometers, such as Q-TOF,

Q-trap and Orbitrap conﬁ gurations, more exhaustive catalogs of

proteins can be achieved without the need for tedious fractionation

[33] . However, exoproteome samples exist in extracellular, aqueous-

phase solutions containing large quantities of other chemical enti-

ties which may sometimes be incompatible with mass spectrometry.

For this reason, protein extraction and puriﬁ cation is a step that

must be addressed cautiously. The most straightforward method to

obtain a clean peptide solution for mass spectrometry is to separate

the exoproteome sample through a denaturing SDS–PAGE gel.

After a short electrophoretic migration, the puriﬁ ed exoproteins

within a single, small polyacrylamide band can be proteolyzed with

trypsin. The resulting, clean peptide mix can then be analyzed

by nano-LC-MS/MS. The majority of recent studies on exopro-

teomes have taken advantage of this high-throughput approach, but

2D-PAGE still has signiﬁ cant interest in the resolution of isoforms

that may result from posttranslational modiﬁ cation of proteins and

their quantitation [34] .

Shotgun proteomics & quantitation methods

In shotgun proteomics, a whole pool of peptides, which may com-

prise thousands of different entities, is being analyzed with the

objective of identifying most of the components. The presence

of proteins in the sample may be ascertained after tally of the

multiple peptide identiﬁ cations. As recently reviewed [35] , speciﬁ c

precautions should be taken into consideration for processing the

data. Filtering the MS/MS spectra assignments with a stringent

cutoff and evaluating false-positive identiﬁ cations by searching

the MS/MS spectra against decoy databases should be done to

evaluate the robustness of the approach. Over the last decade,

protein quantitation of a large number of the components became

an important dimension of shotgun proteomics. Workﬂ ows

developed over the past decade for protein quantitation by mass

spectrometry fall into two categories: labeling approaches, which

include a step in which different isotopes are introduced into

the samples to be compared, either by metabolic labeling or by

protein modiﬁ cation with chemical reagents, and nonlabeling

approaches [36] . Labeling approaches are appealing technically.

For example, multiplex analyses have been made possible with

the use of isobaric reagents, which give different, unique reporter

groups during the MS/MS fragmentation. Stable isotope labeling

by amino acids in cell culture, abbreviated as SILAC, consists in

an in vivo metabolic labeling of proteins aimed at introducing a

mass difference in the same polypeptides between different sam-

ples, enabling their direct comparison when mixed and analyzed

[37] . It has been used to quantify the type III secreted proteins of

enteropathogenic Escherichia coli [38] , the exoproteome of renal

cells [39] and a mouse skeletal muscle cell line [40, 41] . Nonlabeling

approaches are also quite popular, due to their simplicity and

relatively good reproducibility. High-throughput comparison of

samples has become a routine procedure with the use of multiple-

reaction monitoring. In this case, the intensities of a given set of

fragments from the collision-induced fragmentation of parent

peptides are measured precisely by tandem mass spectrometry

[42, 43] . More recently, MS/MS data-independent acquisition has

been shown to perform well, with an increase in the number of

Exoproteomics: exploring the world around biological systems

Expert Rev. Proteomics 9(5), (2012)

564

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quantiﬁ ed proteins [44] . However, spectral counting is nowadays

widely used and easy to perform in the common data dependent

MS/MS acquisition mode. In this quantitation procedure, the

number of MS/MS spectra of a given protein during different

sample runs, under strictly similar nano-LC-MS/MS conditions,

is counted

[45] . The comparison of protein quantities between

samples is direct, although tending to be restricted to the most

abundant proteins. The normalized spectral abundance factor can

be calculated for each protein in order to compare the abundance

of all the proteins. As the ionization properties of peptides are

sequence dependent, this comparison is only valid to compare

protein groups at a global scale and cannot be precise. Typically,

spectral counts assigned to each polypeptide are normalized by

the number of residues present [46] or the molecular weight [47 , 48] .

Values are then expressed as a ratio, by dividing each normalized

spectral abundance factor value by the total sum corresponding

to all the polypeptides detected with two or more nonredundant

peptides. As an alternative to spectral count quantitation, the

intensity of the corresponding parent ion can be extracted from

the chromatography record. A peptide elutes from the reverse-

phase column generally over several tens of seconds. This inten-

sity is repeatedly measured during this elution and the global

signal extracted. Summing the three most important intensities

recorded among the detected peptides corresponding to a given

protein has been shown to be highly representative. Extracted ion-

chromatogram, commonly abbreviated as XIC, has been reported

to increase the number of quantiﬁ able proteins compared with

spectral count, and also to be more rigorous [32] . Moreover, abso-

lute quantitation of proteins may be achieved by spiking known

quantities of isotope-labeled peptides or proteins-of-interest into

the sample and comparing both forms in the same analysis [49] .

Label-free proteomic methods are, arguably, the most signiﬁ cant

contribution to proteomic analyses in recent times [35] . They have

been shown to be very robust and comparable with labeling meth-

ods [32] . These approaches will be further developed as can be

foreseen from recent studies [50, 51] .

Bioinformatic tools for predicting protein localization

During cell culture, some cells may die and be lysed resulting

in the release of large amounts of intracellular proteins into the

conditioning medium. The amount of cellular debris is depend-

ent on the physiological state of the cells, the age of the culture,

the amount of biomass produced and the design of the experi-

ment itself. The comparison between the exoproteome and

the whole-cell proteome may be informative in distinguishing

between secreted proteins and spilled cytoplasmic proteins. Other

experimental approaches such as western blotting are needed to

conﬁ rm MS observations. Nevertheless, bioinformatic tools may

also be helpful for such discrimination. Prediction software has-

been conceived to predict the presence of possible peptide signals

at the N-termini of proteins, discriminate transmembrane heli-

ces containing proteins that will be inserted into the membrane

and postulate amino acid composition, secondary structures and

disordered regions necessary for nonclassical secretion methods.

These software programs are also able to predict the cleavage

site of the signal peptides. The most recent software programs

have been further reﬁ ned on the basis of experimental datasets

and new knowledge of secretion pathways. Prediction tools have

been reviewed in part

[7, 52] . TABLE 1 lists the most widely used and

recent tools for predicting the presence of peptide signal and by

extension, protein subcellular localization. As noted in TABLE 1 , the

most popular tools are available via web servers. Most software are

based on machine-learning techniques, such as hidden Markov

models, Bayesian networks, artiﬁ cial neural networks or support

vector machine classiﬁ ers. Interestingly, meta-analytical methods

which have been recently proposed merge the results of several

predictors and propose a meta scoring for the consensus prediction

[53, 54] . Prediction for the whole theoretical secreted proteins of any

new organism may be achieved relatively quickly using such tools.

Moreover, specialized databases for protein localization are under

development. These databases list predicted secreted proteins for

the whole proteome of several hundreds of organisms, as well as

the localization of the cleavage position of their signal peptide.

Exoproteomes from human cell lines

The concepts and applications of proteomics on human biologi-

cal ﬂ uids have already been presented in a number of reviews

[55 –57] , and the authors will focus here on proteomic analysis of

human cell line exoproteomes. Proteins secreted by human cell

lines represent a valuable source of therapeutic targets and candi-

date biomarkers. These polypeptides include numerous hormones,

growth factors, cytokines and enzymes. The authors believe that

there are two major drawbacks encountered during exoproteome

analysis. First, exoproteins are secreted in biological ﬂ uids or cul-

ture media in low amounts, resulting in high dilution. Moreover,

in vitro culture of cell lines implies the presence of nutriments in

the medium, which may sometimes be incompatible with mass

spectrometry. This can make exoproteome puriﬁ cation and analy-

sis quite challenging. The second drawback is the elevated rate

of cell death observed in vitro . Even if cell death is minimized by

maintaining optimal cell culture conditions, this is an issue that

cannot be totally avoided, resulting in the spill of intracellular

proteins into the extracellular milieu. These difﬁ culties contribute

to a heterogeneity of published data, which could probably be

addressed, or at least minimized, by an optimization of sample

preparation. It is worth noting that the rapid growth of cell lines

may affect protein secretion when compared with cells in tissues

where growth is much slower. Shotgun proteomic approaches have

shown an enormous potential for deﬁ ning the exoproteomes from

human cell lines but separate conﬁ rmatory testing is required to

validate observations made by mass spectrometry.

Conditioning media

Fetal serum is widely used as a nutrient source for human cell

cultures, usually at 10% in a standard culture medium, result-

ing in a total protein charge of 6 mg/ml. Such a high protein

concentration masks proteins secreted by the cells, which are

usually found in the tens of nanograms per mililitre range. The

use of culture media without serum, that is, basal medium, has

been proposed to overcome this problem [58–60] . However, such

Armengaud, Christie-Oleza, Clair, Malard & Duport

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nonoptimal culture conditions may disturb cells, even with short

cell-incubation times. Consequently, secretion mechanisms, and

hence catalogs of secreted proteins, may be modiﬁ ed. Moreover,

a change of culture medium may induce a strong metabolic stress

due to the sudden deprivation of serum, which usually translates

to a drastic reduction in cell proliferation rate [61, 62] , activation

of apoptotic pathways [63] and the induction of signal transduc-

tion and expression of genes involved in stress-response mecha-

nisms [64] . Even a previous washing step performed to remove

contaminant proteins from the culture medium may also impact

the cells [65] . Most studies to date have shown a relatively high

number of intracellular proteins (around 50%) identiﬁ ed in exo-

proteome samples [48, 66–68] . A large, comparative study was con-

ducted recently, using 17 different culture media compositions for

the human bronchial epithelial BEAS-2B cell line. Cell viability,

proliferation rate, effect on the cell cycle and initial protein charge

were compared systematically. Of interest, while cell viability

was preserved in most media, proliferation rates were in some

cases drastically reduced. DMEM, a high-glucose-containing,

protein-free medium, was shown to preserve a good viability

and proliferation of cells and, thus, was recommended for such

exoproteomic studies [48] .

Analytical methods for conditioning & analysis of samples

The analysis of secretomes using current proteomic procedures

requires large volumes (from 8 to 100 ml) of conditioned medium

[65, 67] . Extraction of all the proteins from conditioned media is

required, as well as their concentration before mass spectrometry

analysis. Methods include precipitation, ultraﬁ ltration and lyo-

philization. Improved techniques based on carrier-assisted TCA

precipitation [59, 69, 70] and ﬁ ltration on low cutoff membranes with

centrifugation devices [48, 65, 71, 72] have been documented for exo-

proteome analysis. Applying separation methods or puriﬁ cation

prior to nano-LC-MS/MS has allowed an increase in the number

of identiﬁ ed proteins in secretome analyses. Resolution of pro-

tein mixtures into less complex fractions can be performed easily

Table 1. Main predictors and databases for peptide signal and protein subcellular localization.

Predictor name Organism target Short description Ref.

BPBAac Gram- Speciﬁ c type III secretion signals (SVM) [14 3]

CELLO Gram- N-terminal peptide analysis (SVMs)

[14 4]

EffectPred Gram- Meta-analytic approach to predict type III secreted proteins

[54]

ESLpred Eukaryotes Based on dipeptide composition and PSI-BLAST analysis

[145]

ExProt Bacteria Artiﬁ cial neural network

[29]

HMMTOP All Transmembrane topology prediction

[146]

LipoP Bacteria Lipoprotein, other signal peptides, membrane helices (HMM)

[147]

LocateP Gram+ Database (seven protein subcellular location for 427 genomes)

[148]

MetaLocGramN Gram- Metaserver (14 predictors at once), ﬁ ve subcellular localization

[53]

PA-SUB All Five machine-learning classiﬁ ers specialized for speciﬁ c taxons

[149]

Pheobius All Transmembrane protein topology and signal peptide (HMM)

[150]

Pred-lipo Gram+ Lipoprotein signal peptides (HMM)

[151]

PRED-SIGNAL Archaea Based on 69 proteins with signal peptide (HMM)

[152]

PRED-TAT Bacteria Sec and Tat signal peptide predictor (HMM)

[153]

PSLT Human InterPro motifs and speciﬁ c membrane domains

[15 4]

PSORTb Prokaryotes Homology, motif identiﬁ cation and machine learning methods

[155, 156]

SecretomeP Bacteria Nonclassically secreted proteins (neural networks)

[10]

SecretP All Combination of predictors

[157]

Sherloc Eukaryotes Sequence and text-based features (SVM)

[158]

SIEVE Gram- Speciﬁ c type III and IV secretion signals detection (SVM)

[159]

Signal-3L All Amino acid composition

[160]

SignalP 4.0 Bacteria Neural network-based method with composite scoring

[161]

SOSUI All Transmembrane helices, secondary and tertiary structure

[162]

SubLoc All Amino acid composition (SVM)

[163]

PrediSi All Position weight matrix

[16 4]

TMHMM All Transmembrane helices (HMM)

[165]

HMM: Hidden Markov model; Gram-: Gram-negative bacteria; Gram+: Gram-positive bacteria; SVM: Support vector machine; TAT: Twin-arginine translocation.

Exoproteomics: exploring the world around biological systems

Expert Rev. Proteomics 9(5), (2012)

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Review

by SDS–PAGE [48, 65, 73 , 74] , although liquid separation methods,

such as strong cation exchange chromatography

[67] or OFFGEL

electro phoresis [75] , have also proven to be successful. Other,

more-speciﬁ c methods have also been developed, as exempliﬁ ed

by afﬁ nity-based lectin chromatography for enriching glycopro-

teins from three human breast cell lines [74] . Such an approach

is highly relevant in order to enrich the secreted glycoprotein

fraction of the exoproteome sample.

Applications

The identiﬁ cation of speciﬁ c cancer biomarkers is the objective

of many exoproteome studies, with more than 28 reports on

cancer cell lines available to date [60] . These studies cover a wide

range of cancers, such as prostate, lung, breast, colorectal and

pancreatic [72, 76, 77] . Secreted proteins are indeed excellent sero-

logical tumor marker candidates as they can enter the circulatory

system and, thus, be detected during routine blood analysis.

In some cases, these candidate biomarkers may be measured

directly in patient samples, either from serum or tissues [78] .

Proteins secreted by cancer cells are a major component of the

tumor microenvironment. However, little is known about the

impact of single oncogenic lesions on the regulation of secreted

proteins at the early stages of tumor development. As c-Myc

overproduction is one of the most frequent alterations in cancer,

the effect of sustained c-Myc production in a nontransformed

human epithelial cell line was studied [79] . A substantial down-

regulation of 50 proteins was observed in the exoproteome.

These ﬁ ndings helped to uncover an autocrine/paracrine com-

ponent in the ability of c-Myc to stimulate proliferation, sustain

tumor maintenance and modulate cell migration. Resistance to

cancer treatment has also been studied by means of exoproteome

analysis. A total of 57 proteins were found to be secreted dif-

ferentially when comparing the exoproteomes of MCF-7 and

doxorubicin (an anticancer drug)-resistant MCF-7 cell lines.

IL-18 was further validated to contribute to doxorubicin resist-

ance and may, thus, represent a useful drug target for breast

cancer therapy [73] .

Other applications of human cell line exoproteomes relate

to mechanistic information, the treatment of other patholo-

gies and to drug development. Neuronal differentiation and

skeletal myogenesis have been addressed through the exopro-

teomic lens. The identiﬁ cation of proteins released from neu-

rons, astrocytes and neural precursor cells, using 2D-PAGE

and nano-LC-MS/MS, revealed that the secreted, extracellu-

lar pool includes proteins involved in cell–cell interactions,

energy metabolism, redox regulations and several molecular

chaperones, each one speciﬁ c to a given cell population [80] . A

panel of proteins secreted during cell differentiation has been

established to improve knowledge of regulatory pathways

[59] . New potential mediators of the stem cell microenviron-

ment, and cardiac and neuronal differentiation processes, have

been identiﬁ ed [81] . Among these, some proteins (e.g., protein

14-3-3), which were found to be secreted in atherosclerotic

lesions but not in normal vasculature, represent potential thera-

peutic targets. The exoproteome of adipose tissue may be useful

for the development of treatments for type 2 diabetes, as adi-

pose tissue has proven to be a central driver of the progression

of this condition

[82] .

The ﬁ eld of toxicology is of particular interest because many

new compounds, chemicals or nanoparticles have to be assessed

in terms of safety. The cellular response to stress when cells are

put in contact with such compounds may be evaluated through

changes in their exoproteome. For example, the effect of cobalt

on human lung cells has recently been described. Upon cobalt

exposure, protein secretion was generally reduced, the ER

amino peptidase 1 being the only signiﬁ cantly induced protein.

This implies a possible link with angiogenesis, the process by

which novel blood vessels are formed [48] . Nephrotoxicity is an

adverse event which strongly limits the use of the immuno-

suppressant, cyclosporine, in solid organ transplantation. An

exoproteome study highlighted the upregulation of the secreted

cyclophilins A and B, a macrophage inhibition factor and the

phosphatidyl-ethanolamine-binding protein 1, following expo-

sure of HEK-293 human cells to cyclosporine [39] . The pro-

teins identiﬁ ed in such studies represent potential biomarkers

of toxicity.

Revisiting microbial virulence determinants &

pathogenesis

Microbial pathogens are involved in a wide range of severe, and

sometimes fatal, human diseases, including nosocomial infec-

tions, food-borne infections and toxic shock syndrome. The

pathogenesis of the majority of bacterial diseases is a multifacto-

rial process. Five common, crucial steps may be listed: adhesion

to the host cell, invasion, damage to host tissues, resistance to

environmental stresses during infection and subversion of the host

immune response [83] . Completion of each stage is dependent on

orchestrated activities of speciﬁ c exoproteins. These are, in turn,

tightly controlled by a speciﬁ c set of regulators which, for most

pathogens, are still being studied. Exoproteins which play pivotal

role in the adaptability of the pathogen to the speciﬁ cities of the

host’s intracellular environment and promote efﬁ cient infection

are recognized as virulence factors [84] .

Pathogenesis through the lens of exoproteomes

During the last decade, proteomic approaches based on 2D-PAGE

have been widely used to explore the various components of the

pathogenicity of different bacteria [85, 86] and their capacities to

adapt to environmental conditions such as those encountered in

the human host [87] . Among numerous examples, the authors

may cite the 2D-PAGE analysis of the Pseudomonas aeruginosa

exoproteome which led to the identiﬁ cation of proteins involved

in adaptation to suboptimal temperature facilitating nosocomial

infection [88] , and the analysis of Corynebacterium diphteriae and

Mycobacterium tuberculosis exoproteomes which allowed the con-

struction of reference maps for these two human pathogens and

the identiﬁ cation of novel proteins involved in host cell adhesion

and invasion [89–92] . In order to take into account the biodiver-

sity, and to obtain a more objective overview of the exoproteome,

Dumas et al. analyzed the exoproteome of 12 representative

Armengaud, Christie-Oleza, Clair, Malard & Duport

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Listeria monocytogenes strains of different serotype origins and

virulence levels

[93] . As a result of these studies, the authors pro-

posed the ﬁ rst deﬁ nition of the core and variant exoproteome of

L. monocytogenes species. Similarly, the exoproteomes of 25 clinical

Staphylococcus aureus isolates were analyzed, highlighting the

importance of proteomic studies to complement genomic infor-

mation [94] . Proteomic approaches were more recently focused on

a deeper understanding of the molecular mechanisms responsible

for pathogenicity. Quantifying the rates of protein production and

turnover and evaluating their changes among strains differing in

terms of virulence and/or under different cellular conditions are

important [51, 85] . Regulatory processes occurring after transcrip-

tion may be assessed by integrating proteomic, transcriptomic

and genomic data [95] .

New perspectives in pathogenesis & toxinogenesis

Like other human pathogens, Bacillus cereus , a Gram-positive,

spore-forming bacterium, has been the focus of several prot-

eomic studies. An initial report noted the presence of col-

lagenase, phospholipases, proteases and enterotoxins in the

exo proteome of B. cereus at the onset of the stationary phase,

following a 2D-PAGE separation [96] . A comparative study of

the exoproteomes of three closely related species, namely, B.

cereus , Bacillus thuringiensis and Bacillus anthracis , implied the

presence of the same cytosolic proteins in the exoproteome, but

different secreted proteins [97] . This result highlighted the inter-

est of the exoproteome fraction in the speciﬁ city of infection.

A total of 22 proteins were identiﬁ ed in the culture superna-

tant of cells grown in the nutritional rich lysogeny broth (LB)

medium under aerobiosis [98] . The majority of these (14 of 22)

belonged to the well-characterized PlcR virulence regulon, and

the kinetics of their concentrations peaked at the early station-

ary growth phase, concomitantly with the culture supernatant

cytotoxicity. The authors concluded that the pathogenic poten-

tial of B. cereus is maximal during the transition state, that is,

at high cell density. A comprehensive analysis of the B. cereus

exoproteome was performed recently, taking advantage of the

modern shotgun proteomic approaches [99] . B. cereus ATCC

14579 was grown in pH-regulated batch cultures on chemi-

cally deﬁ ned medium under oxido-reduction potential (ORP)

conditions considered as mimicking those encountered in the

human intestine, such as low- and high ORP anaerobiosis. Since

B. cereus does not secrete a large amount of protein at early-

exponential growth phase, culture ﬁ ltrates were concentrated

using the deoxy cholate/trichloro acetic acid method to obtain

sufﬁ cient amounts of exoproteins. From these samples, 133

different proteins were conﬁ dently listed, resulting in a much

higher exoproteome coverage than previous studies. A total of

57 putative virulence factors (14 toxins, eight motility-related

proteins and 35 adhesins and degradative enzymes) were identi-

ﬁ ed with high conﬁ dence (false-positive evaluation of less than

1%), 31 of them being detected for the ﬁ rst time. Among these

newly detected virulence factors, the putative fourth compo-

nent of hemolysin BL (Hbl), enterotoxin FM, hemolysin II and

three new putative conserved enterotoxins were uncovered. Two

additive factors may explain why these proteins were not previ-

ously detected: the slightly different growth conditions and the

high-resolution mass spectrometer used and the shotgun strategy

that was developed, which most probably both favored the detec-

tion of poorly accumulating proteins. In addition, the exopro-

teomes from the three chemically deﬁ ned conditions (namely,

low- and high-oxidoreduction-potential anoxic conditions, and

fully-oxic conditions) were compared based on their spectral-

count quantiﬁ cation

[99] . The contributions of the detected tox-

ins to the cytotoxicity of B. cereus in response to different redox

environments were discussed and derived in further experiments

focusing on the regulatory network of the cellular proteome [100] .

More importantly, shotgun proteomic strategy used in this

study brings evidence that B. cereus , like other pathogens, can

deploy an arsenal of virulence factors at low density popula-

tion to promote infection. Comprehensive analysis based on a

combination of complementary enrichment strategies such as

recently reported for Bacillus subtilis [101] could become stand-

ard in the near future. Like in B. cereus , shotgun proteomics

has enabled the detection of novel secreted virulence factors

from Salmonella enterica enterica , serovar Typhimurium [102] , a

novel secreted antigen of Streptococcus pneumoniae [103] and the

identiﬁ cation of new virulence factors from the catalogue of exo-

proteins generated from Corynebacterium pseudotuberculosis [10 4] .

More recently, exoproteome analysis of Campylobacter ureolyticus

revealed three putative virulence and colonization factors, which

may contribute to its pathogenic potential and its involvement in

the Crohn’s disease [105] . Finally, all these studies revealed that

the exoproteome of a pathogen is more complex than previously

reported from 2D-PAGE approaches [10 6 , 107] . In this regard, the

in-depth study of the exoproteome of a pathogen using shotgun

experiments represents an open door for further exploration

[108 –114] : the identiﬁ cation of novel targets for rational drug and/

or a source of antigens for vaccine design (as an example, a recent

comparative exoproteome analysis of M. tuberculosis identiﬁ ed

Lipoprotein Diacylglyceryl Transferase as a valuable target for

generation of antituberculosis drugs) [115] ; the identiﬁ cation of

mechanisms that contribute to the various virulence pheno-

types of pathogens; the comprehensive study of secretion mecha-

nisms used to export proteins (as a pioneer work, the authors

cite the recent study on the enteropathogenic E. coli [38] where

its type III secretome was characterized, the repertoire of type

III secreted effectors for the attaching and effacing pathogens

was expanded, and new insights into the mode of function for

LifA/Efa1/ToxB/Z4332, an important family of virulence fac-

tors, were provided); the existence of a selection procedure in the

excretion of cytoplasmic proteins as suggested [12] ; the dynamic

of exoproteome within the different environments encountered

by the pathogen throughout its life cycle; the function of a

large number of proteins annotated as uncharacterized; and the

unique opportunity to address the biology of genetically intrac-

table pathogens, such as the obligate intracellular bacterium

Chlamydia trachomatis [116] . In conclusion, shotgun proteomics

greatly enhances the toolbox to understand the pathogenicity

of micro-organisms.

Exoproteomics: exploring the world around biological systems

Expert Rev. Proteomics 9(5), (2012)

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Exploring the exoproteomes of environmental

organisms

Novel functions to be characterized & biotechnological

applications

While proteomics has been restricted for years to just a few

microbial models because of the need for a sequenced genome

to interpret MS/MS spectra, next-generation sequencing tools

have opened up a new era for microbial proteomics [35] . The huge

diversity of environmental organisms can now be investigated

with modern proteomic tools, as a result of the huge increase in

sequenced organisms. A wealth of discoveries is to hand, as exem-

pliﬁ ed by the most recent reports on microbial exo proteomes

[117–121] . Exoproteomics has highlighted the importance, at

least in terms of quantitation, of speciﬁ c protein families. The

Ruegeria pomeroyi DSS-3 marine bacterium belonging to the

Roseobacter clade was shown to accumulate extremely high quan-

tities of a protein in its extracellular environment. This protein,

namely, PaxA, accounted for 61% of the total MS/MS spectra

recorded for the sample in a global shotgun analysis [122] , despite

the fact that it would certainly not have been noticed in the list

of predicted secreted proteins if only the genome was inspected.

PaxA showed strong sequence identities with RTX-like toxins,

which are virulence factors found in several human and ani-

mal pathogens [123] . In fact, the perception of an organism can

change when the theoretical or experimental exo proteomes are

taken into consideration, as illustrated in FIGURE 2 . The Roseobacter

clade is known for its high capacity to scavenge nutrients from

its environment, as indicated by its large number of catabolic

pathways and nutrient transporters [124] . As shown in FIGURE 2 ,

76.5% of the proteins encoded onto the genomes of 12 repre-

sentative members of the Roseobacter clade and predicted to be

secreted are involved in transport. In terms of quantity, the mass

spectrometry detected proteins corresponds only to 51.7% of the

exoproteome. Shotgun proteomics revealed that, in terms of the

secreted proteins found in its milieu, other functions such as

toxicity, motility or adhesion are more important than initially

expected by the theoretical analysis of the genomes (FIGURE 2) .

The secretion of RTX-like proteins seems to be a recurrent fac-

tor for Roseobacter members, as shown by the analysis of the

exoproteomes of 12 strains [117] . Toxin-like proteins represented

from 6 to 45% of the total exoproteome detected. While these

proteins are commonly found in the exoproteomes of marine bac-

teria, their functions and targets remain unknown and warrant

further investigation.

The functions of many secreted proteins are unknown.

Estimates based on comparative genomics have indicated one

third of the genes in most genomes to be annotated as either

‘hypothetical protein’ or ‘conserved protein with unknown

function’. The authors have noted that usually over 40% of pro-

teins fall into this category when considering only the predicted

exoproteome [117, 125 –127] . Indeed, environmental organisms rep-

resent a huge reservoir of enzymatic elements which could be

useful bricks for the ﬁ eld of green chemistry, aiming to produce

novel biotechnological products of interest or to improve chemi-

cal means-of-production. Exoenzymes, targeted bactericides,

antifouling compounds, organism attractants/repellants or

proteins with potential uses in bioremediation, industry or in

the production of alternative energies, are illustrations of the

many possible applications. Many exoenzymes are already being

exploited in industry, for example proteases and lipases used as

additives in detergents or for food processing. In recent years, the

exoproteomes of fungi, the main producers of lignocellulolytic

enzymes, have been documented

[108 , 109, 118, 120] . The hydrolytic

pathway of the complex polymer, lignocellulose, producing more-

easily fermentable sugars, could be useful to obtain alternative

fuels from plant biomasses. The repertoire of useful biotechno-

logical proteins should increase in the near future with a more in-

depth characterization of exoproteomes coupled with systematic

functional screening.

Microbial ecology

Life has deﬁ ned the chemistry of the Earth that we know today.

Because of its origin, life is based, fundamentally, on exchanges

and interactions between the enormous diversity of existing

prokaryotic and eukaryotic organisms and their environments.

This continuous exchange of compounds with the environment

is vital for their persistence and evolution. It is not restricted

to the uptake of nutrients and secretion of cell waste, as many

compounds that cells secrete are directed to modifying or inﬂ u-

encing the environment, for example antibiotics, toxins, viru-

lence factors, catabolic enzymes, pheromones, alarmones and

quorum-sensing molecules. Most cellular processes are carried

out by proteins. Thus, clues about the ecological strategies of an

organism and, as a result, its functions within its environment,

may be inferred by analyzing its exoproteome and conﬁ rmed

by further experimentation. As an example, the symbiotic ele-

ments found in the exoproteome of the ecto mycorrhizal fun-

gus, Laccaria bicolor , detected by different shotgun proteomic

approaches, show the ecological association between the fun-

gus and the plant [121] . Four adaptive strategies may be deﬁ ned

within the microbial world: competition for existing resources

by the use of effective and diverse nutrient uptake systems and

exoenzymes; sensing and moving towards or away from stim-

uli; elimination of competitors or avoidance of predation; and

direct interaction with other community members, in a parasitic

or symbiotic manner. These different lifestyles were shown to

exist within different members of the same group of marine

bacteria, the Roseobacter clade, by means of exoproteomic data

[117] . Nevertheless, while some strains showed a clear preference

for one of these four strategies, others presented a combination,

as attested by the abundance of their secreted proteins. A single

organism may adopt different trophic strategies depending on

the context or its physiological state. Bacillus licheniformis , a

bacterium commonly found in the soil, secreted a higher amount

of proteins when grown in rich media, although speciﬁ c pro-

teins to counteract starvation were found in limited media, as

shown by a 2D-PAGE approach [128] . Such exoproteomic result

is expected as starved cells induce a stringent global response

characterized by a decrease in protein synthesis upon shifting

from growth and proliferation to survival only. In another study,

Armengaud, Christie-Oleza, Clair, Malard & Duport

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the actinobacteria Frankia spp., an actino-

rhizal plant symbiont, showed completely

different exoproteome proﬁ les when it

was grown in culture or in symbiosis, as

determined by nano-LC-MS/MS, with 38

secreted proteins identiﬁ ed in the former

condition while up to 73 proteins could

be detected in ﬁ eld-collected root nodules

of Alnus incana and Elaeagnus angustifolia

[129] . The exoproteome of another marine

organism, Pseudoalteromonas tunicata , was

also screened [130] . This study revealed

how the secreted proﬁ le of the same organ-

ism can vary due to a mutation in a type-II

secretion pathway, for example, nutrient

transport mechanisms or non-protein pig-

mented toxin compounds. Multicellular

organisms also have an enormous range of

proteins that can, potentially, be secreted

into their environment. In this sense,

the rice plant showed a large variety of

proteins in its rhizosphere exoproteome,

with functions such as defense or protec-

tion against environmental stress [131] . The

mucous subproteome from the freshwa-

ter planarian Schmidtea mediterranea , a

ﬂ atworm, was recently documented with

119 detected proteins homologous to

proteins found in human secretions [132] .

This demonstrates how exoproteomics on

such organisms could be informative for

understanding parasitic worm infections.

In fact, all living organisms interact with

their biotic and abiotic surroundings by

means of secreted compounds, such as

proteins. Due to the high energy and

nutrient cost, evolutionary pressure will select for only those

secretions which will bring a ﬁ nal beneﬁ t to the organism, and

this will slowly develop its ecological strategy. Furthermore,

costly functions can be shared within a community reducing

the genomic content even in free-living organisms [133] .

Expert commentary

Proteomic tools are now able to generate comprehensive list of

spare-parts for numerous organisms’ exoproteomes. Nevertheless,

most studies still rely on the prediction of the subcellular locali-

zation of proteins and a wealth of predictors has been proposed.

N-terminomics is a speciﬁ c proteogenomic approach aimed at

conﬁ dently establishing the N-terminus of proteins, among them

the mature secreted proteins and, thus, N-terminomics helps to

characterize their peptide signal and maturation speciﬁ cities [31] .

These experimental data should be helpful for the further devel-

opment of prediction software [31] . In addition to the rather lim-

ited conﬁ dence of the current prediction algorithms, annotation

errors in genomes should also be considered. It has been noted

in recently conducted, large N-terminome studies that for some

genomes more than 15% of the genes are wrongly annotated in

terms of translation initiation sites [134 , 135] . Interestingly, large

efforts to curate the annotations of Shewanella strains manually

have shown that incorrect gene prediction is a major issue for

lipoproteins belonging to the Tat export system [52] . Combining

predictive tools and proteomic data has proven to be effective for

the Shewanella genus [52] , E. coli [136] , M. tuberculosis [137] and

natural microbial communities [138] . Further improvement of

the reliability of predictive programs is expected in the coming

years, taking advantage of the high-throughput proteomic data

currently being collated from many different organisms. The

HUPO initiative on the Human Proteome Project is worth citing

because speciﬁ c emphasis on secreted proteins could be fruitful

[139] . Exoproteomics is expected to expand in many disciplines of

biology, with the objective of further characterizing the fraction

of this speciﬁ c pool of proteins and the molecular mechanisms

associated. Any organism can now be rapidly sequenced and

investigated with modern proteomic tools. Secreted proteins

Theoretical exoproteome

Transport (76.5%)

Toxicity (9.2%)

Motility (11.6%)

Adhesion (2.7%)

Experimental exoproteome

Transport (51.7%)

Toxicity (19.8%)

Motility (19.9%)

Adhesion (8.6%)

Time

Intensity

m/z

Figure 2. Theoretical versus experimental exoproteomes from 12 marine

Roseobacter strains. The ratio of predicted secreted proteins annotated with functions

related to transport, toxicity, motility or adhesion are indicated in terms of numbers of

occurrence in the 12 genomes (theoretical secreted proteins; 14,845 theoretical proteins

predicted to be secreted among 51,686 proteins encoded by the genomes) and in terms

of cumulated quantities when assessed by mass spectrometry (experimental

exoproteome: 874 polypeptides detected). The experimental abundance of exoproteins

found in the extracellular milieu was obtained by shotgun proteomics of each of the

Roseobacter exoproteomes grown in a rich marine broth and quantiﬁ ed by the

normalized spectral abundance factor (data from

[117] ). The 12 Roseobacter clade strains

are: Ruegeria pomeroyi DSS-3, Oceanicola batsensis HTCC2597 , Pelagibaca bermudensis

HTCC2601 , Roseobacter denitriﬁ cans OCh114 , Oceanicola granulosus HTCC2516 ,

Oceanibulbus indolifex HEL45 , Ruegeria lacuscaerulensis ITI1157 , Roseobacter litoralis

OCh149 , Roseobacter spp. MED193 , Roseovarius nubinhibens ISM , Dinoroseobacter

shibae DFL12, and Sagittula stellata E-37.

Exoproteomics: exploring the world around biological systems

Expert Rev. Proteomics 9(5), (2012)

570

Review

can be identiﬁ ed in a larger dynamic range and their quanti-

ties compared in different experimental conditions. In the near

future, such strategies will highlight with more reﬁ nement the

processes of life outside the cell and, of particular interest, the

interactions between cells.

Five-year view

The current generation of mass spectrometers for the analysis of

proteins and peptides present considerable potential. Today, thou-

sands of proteins may be identiﬁ ed and quantiﬁ ed from a single

complex sample. This is exempliﬁ ed by the rather simple fractiona-

tion process needed for a total mammalian proteome prior to analy-

sis with a high-ﬁ eld Orbitrap mass spectrometer. This has resulted

in the identiﬁ cation of 11,731 proteins from a single organism [140] .

The next generation of mass spectrometers, which could be mar-

keted within the next ﬁ ve years, will probably be complex hybrid

conﬁ gurations of different analyzers, as exempliﬁ ed by the current

trend of conﬁ guration including Q-TOF, Q-trap and Q-Orbitrap

coupling, as well as new, high-ﬁ eld Orbitrap and ion-mobility ana-

lyzers. Technical developments will certainly allow improvements

by orders of magnitude in dynamic range, accuracy and speed.

Proteomics will become very comprehensive in the near future. In

the ﬁ eld of human health, exoproteome studies have demonstrated

their usefulness in deﬁ ning candidate cancer biomarkers. It appears

to be unlikely that a single biomarker could diagnose a multistage

cancer or monitor the efﬁ cacy of a given treatment, but rather a

pool of biomarkers could be decisive. Validating these candidates

for translational medicine using more-accurate quantitative pro-

teomic approaches is another ﬁ eld of development for the near

future. During the next ﬁ ve years, we may extrapolate new, impor-

tant developments in the systematic analysis of exoproteomes for

a large number of cellular models, as well as for studying complex

exoproteomes found in diverse ecosystems (human microbiome,

environmental samples) that could be developed into the ‘meta-exo-

proteomic’ concept. In this latter case, the possible universe of exo-

proteins is so huge that novel methodologies should be developed,

as well as advanced bioinformatics and statistics data treatment, in

order to avoid high levels of false-positive identiﬁ cations. The devel-

opment of speciﬁ c public data repositories for exoproteomic data,

as exempliﬁ ed by pioneering databases for fungi

[141, 142] , should

be fostered. Ideally, such information should be incorporated into

proteogenomic programs dedicated to better annotating genomes.

Moreover, integration of multi-omic data is an important issue as

a number of recent studies have shown poor correlation between

transcriptomic and proteomic analyses on one hand, and difference

in terms of complexity of several orders of magnitude between

proteome and genome data due to posttranslational modiﬁ cations

on the other [51, 95] . Taking advantage of improved predictive tools,

correlation between theoretical prediction, literature data mining,

experimental data and well deﬁ ned experimental conditions should

lead to new insights into secretion mechanisms, and functional

characterization of most of the exoproteome components that have

been, until now, poorly annotated.

Financial & competing interests disclosure

This study was supported by the Commissariat à l’Energie Atomique et

aux Energies Alternatives, the EDF company and the Agence Nationale de

la Recherche. JA Christie-Oleza gratefully acknowledges the Marie Curie

Actions of the EC FP7. The authors have no other relevant afﬁ liations or

ﬁ nancial involvement with any organization or entity with a ﬁ nancial

interest in or ﬁ nancial conﬂ ict with the subject matter or materials discussed

in the manuscript apart from those disclosed.

Kate Vassaux provided writing assistance, and this was funded by the

Commissariat à l’Energie Atomique et aux Energies Alternatives.

Key issues

• The fraction of putative secreted proteins encoded by bacterial genomes could be as much as a quarter of the theoretical proteome.

The functions of most secreted proteins are still unknown, with more than 40% of experimentally detected bacterial exoproteins

hitherto annotated as either ‘hypothetical protein’ or ‘conserved protein with unknown function’.

• The ‘secretome’ includes the pool of secreted proteins and also the secretion machinery itself. The ‘proteomic surfaceome’ or

‘surface proteome’ comprises the accessible fraction of exported proteins which are inserted into the membrane via the presence of

hydrophobic transmembrane segments or associated with such proteins. The ‘exoproteome’ corresponds to the proteins found in the

extracellular milieu.

• Prediction software has been conceived for predicting the subcellular localization of proteins, the presence of a signal peptide at the

protein N-terminus and its corresponding cleavage site. Further reﬁ nement of these tools is expected thanks to mass spectrometrically

validated experimental exoprotein datasets.

• Proteins secreted by human cell lines represent a valuable source of therapeutic targets and candidate biomarkers. The development of

exoproteome studies should lead to numerous applications ranging from the development of new cancer biomarkers to toxicological

evaluations.

• Methods to characterize the modiﬁ cations that are frequent in secreted proteins are important, but limited and challenging.

Glycosylation, in particular, is important for the activity and stability of many secreted proteins, but is heterogeneous and can be

difﬁ cult to characterize by mass spectrometry. Many other post-translational modiﬁ cations are also relevant and deserve speciﬁ c

methodological developments.

• Shotgun proteomic strategies, and the latest generation of hybrid mass spectrometers, are producing a comprehensive view of

the arsenal of virulence factors present in microbial pathogens. Exoproteomics should contribute to the elucidation of the novel

functions of the numerous hypothetical proteins exported out of the cells, as well as to an exploration of the relationships between

environmental organisms and their habitats.

Armengaud, Christie-Oleza, Clair, Malard & Duport

571

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Exoproteomics: exploring the world around biological systems

Leishmania Vesicle-Depleted Exoproteome: What, Why, and How?

Article

Full-text available

Dec 2022

Leishmaniasis, a vector-borne parasitic protozoan disease, is among the most important neglected tropical diseases. In the absence of vaccines, disease management is challenging. The available chemotherapy is suboptimal, and there are growing concerns about the emergence of drug resistance. Thus, a better understanding of parasite biology is essential to generate new strategies for disease control. In this context, in vitro parasite exoproteome characterization enabled the identification of proteins involved in parasite survival, pathogenesis, and other biologically relevant processes. After 2005, with the availability of genomic information, these studies became increasingly feasible and revealed the true complexity of the parasite exoproteome. After the discovery of Leishmania extracellular vesicles (EVs), most exoproteome studies shifted to the characterization of EVs. The non-EV portion of the exoproteome, named the vesicle-depleted exoproteome (VDE), has been mostly ignored even if it accounts for a significant portion of the total exoproteome proteins. Herein, we summarize the importance of total exoproteome studies followed by a special emphasis on the available information and the biological relevance of the VDE. Finally, we report on how VDE can be studied and disclose how it might contribute to providing biologically relevant targets for diagnosis, drug, and vaccine development.

The impact of Elaeagnus angustifolia root exudates on Parafrankia soli NRRL B-16219 exoproteome

Article

Full-text available

May 2024

Root exudates from host plant species are known to play a critical role in the establishment and maintenance of symbiotic relationships with soil bacteria. In this study, we investigated the impact of root exudates from compatible host plant species; Elaeagnus angustifolia on the exoproteome of Parafrankia soli strain NRRL B-16219. A total of 565 proteins were evidenced as differentially abundant, with 32 upregulated and 533 downregulated in presence of the plant exudates. Analysis of the function of these proteins suggests that the bacterial strain is undergoing a complex metabolic reprogramming towards a new developmental phase elicited in presence of host plant root exudates. The upregulation of Type II/IV secretion system proteins among the differentially expressed proteins indicates their possible role in infecting the host plant, as shown for some rhizobia. Additionally, EF-Tu, proteins upregulated in this study, may function as an effector for the T4SSs and trigger plant defense responses. These findings suggest that Parafrankia soli may use EF-Tu to infect the actinorhizal host plant and pave the way for further investigations of the molecular mechanisms underlying the establishment of symbiotic relationships.

Streptococcus thermophilus: A Source of Postbiotics Displaying Anti-Inflammatory Effects in THP 1 Macrophages

Article

Full-text available

Mar 2024
MOLECULES

In addition to traditional use in fermented dairy products, S. thermophilus also exhibits anti-inflammatory properties both in live and heat-inactivated form. Recent studies have highlighted that some hydrolysates from surface proteins of S. thermophilus could be responsible partially for overall anti-inflammatory activity of this bacterium. It was hypothesized that anti-inflammatory activity could also be attributed to peptides resulting from the digestion of intracellular proteins of S. thermophilus. Therefore, total intracellular proteins (TIP) from two phenotypically different strains, LMD-9 and CNRZ-21N, were recovered by sonication followed by ammonium sulphate precipitation. The molecular masses of the TIP of both strains were very close to each other as observed by SDS-PAGE. The TIP were fractionated by size exclusion fast protein liquid chromatography to obtain a 3–10 kDa intracellular protein (IP) fraction, which was then hydrolysed with pancreatic enzyme preparation, Corolase PP. The hydrolysed IP fraction from each strain exhibited anti-inflammatory activity by modulating pro-inflammatory mediators, particularly IL-1β in LPS-stimulated THP-1 macrophages. However, a decrease in IL-8 secretion was only observed with hydrolysed IP fraction from CNRZ-21N, indicating that strain could be an important parameter in obtaining active hydrolysates. Results showed that peptides from the 3–10 kDa IP fraction of S. thermophilus could therefore be considered as postbiotics with potential beneficial effects on human health. Thus, it can be used as a promising bioactive ingredient for the development of functional foods to prevent low-grade inflammation.

Computational Tools and Phytopathogenic Fungi Secretome: Unraveling the Protein Arsenal

Article

Full-text available

Dec 2022

Gabriela Piccolo Maitan-Alfenas

Rio Tinto as a niche for acidophilus enzymes of industrial relevance

Article

Full-text available

Feb 2023

Lignocellulosic residues are amongst the most abundant waste products on Earth. Therefore, there is an increasing interest in the utilization of these residues for bioethanol production and for biorefineries to produce compounds of industrial interest. Enzymes that breakdown cellulose and hemicellulose into oligomers and monosaccharides are required in these processes and cellulolytic enzymes with optimum activity at a low pH area are desirable for industrial processes. Here, we explore the fungal biodiversity of Rıo Tinto, the largest acidic ecosystem on Earth, as far as the secretion of cellulolytic enzymes is concerned. Using colorimetric and industrial substrates, we show that a high proportion of the fungi present in this extremophilic environment secrete a wide range of enzymes that are able to hydrolyze cellulose and hemicellulose at acidic pH (4.5–5). Shotgun proteomic analysis of the secretomes of some of these fungi has identified different cellulases and hemicellulolytic enzymes as well as a number of auxiliary enzymes. Supplementation of pre‐industrial cocktails from Myceliophtora with Rio Tinto secretomes increased the amount of monosaccharides released from corn stover or sugar cane straw. We conclude that the Rio Tinto fungi display a good variety of hydrolytic enzymes with high industrial potential.

Responses of carbapenemase-producing and non-producing carbapenem-resistant Pseudomonas aeruginosa strains to meropenem revealed by quantitative tandem mass spectrometry proteomics

Article

Full-text available

Dec 2022

Pseudomonas aeruginosa is an opportunistic pathogen with increasing incidence of multidrug-resistant strains, including resistance to last-resort antibiotics, such as carbapenems. Resistances are often due to complex interplays of naturally and acquired resistance mechanisms that are enhanced by its large regulatory network. This study describes the proteomic responses of two carbapenem-resistant P. aeruginosa strains of high-risk clones ST235 and ST395 to subminimal inhibitory concentrations (sub-MICs) of meropenem by identifying differentially regulated proteins and pathways. Strain CCUG 51971 carries a VIM-4 metallo-β-lactamase or ‘classical’ carbapenemase; strain CCUG 70744 carries no known acquired carbapenem-resistance genes and exhibits ‘non-classical’ carbapenem-resistance. Strains were cultivated with different sub-MICs of meropenem and analyzed, using quantitative shotgun proteomics based on tandem mass tag (TMT) isobaric labeling, nano-liquid chromatography tandem-mass spectrometry and complete genome sequences. Exposure of strains to sub-MICs meropenem resulted in hundreds of differentially regulated proteins, including β-lactamases, proteins associated with transport, peptidoglycan metabolism, cell wall organization, and regulatory proteins. Strain CCUG 51971 showed upregulation of intrinsic β-lactamases and VIM-4 carbapenemase, while CCUG 70744 exhibited a combination of upregulated intrinsic β-lactamases, efflux pumps, penicillinbinding proteins and downregulation of porins. All components of the H1type-VI secretion system were upregulated in strain CCUG 51971. Multiple metabolic pathways were affected in both strains. Sub-MICs of meropenem cause marked changes in the proteomes of carbapenem-resistant strains of P. aeruginosa exhibiting different resistance mechanisms, involving a wide range of proteins, many uncharacterized, which might play a role in the susceptibility of P. aeruginosa to meropenem.

A novel artificial intelligence tool for identification of extracellular proteins doubles current estimate of the marine secretome

Preprint

Full-text available

Nov 2023

Microbes are the engines driving the elemental cycles. In order to interact with their environment and the community, microbes secrete proteins into the environment (known collectively as the secretome), where they remain active for prolonged periods of time. Despite the environmental relevance of microbes and the vast quantities of marine prokaryotic sequences available, our knowledge of the marine secretome remains limited due to the available in silico methods for the study of secreted proteins, which are not suited to their use in large datasets due to their low throughput. An alternative and potentially better approach to characterise the secretome is to combine modern machine learning tools with the evolutionary adaptation changes of the proteome to the marine environment. In this study, we found and described adaptations of marine extracellular proteins, which vary between phyla, resulting in differences in ATP costs, amino acid composition and nitrogen and sulphur content. With these adaptations in mind, we developed ‘Ayu’, a machine prediction tool that does not employ homology-based predictors (as current methods do) and achieves better performance than current state-of-the-art software at a fraction of the time. When applied to oceanic samples (Tara Oceans dataset), our method was able to recover more than double the proteins compared to the most widely used method to identify secreted proteins, indicating that more than half of secreted proteins are presently unconsidered in current secrtome analysis. The application of this powerful novel to open ocean samples allowed to better characterise the real composition of the marine secretome, allowing for a better recognition and understanding of the secretome in marine ecosystems.

S-layer is a key element in metabolic response and entry into the stationary phase in Bacillus cereus AH187

Article

Full-text available

Sep 2023

Effect of NaCl stress on exoproteome profiles of Bacillus amyloliquefaciens EB2003A and Lactobacillus helveticus EL2006H

Article

Full-text available

Aug 2023

Salt stress can affect survival, multiplication and ability of plant growth promoting microorganisms to enhance plant growth. Changes in a microbe’s proteome profile is one of the mechanisms employed by PGPM to enhance tolerance of salt stress. This study was focused on understanding changes in the exoproteome profile of Bacillus amyloliquefaciens EB2003A and Lactobacillus helveticus EL2006H when exposed to salt stress. The strains were cultured in 100 mL M13 ( B. amyloliquefaciens ) and 100 mL De man, Rogosa and Sharpe (MRS) ( L. helveticus ) media, supplemented with 200 and 0 mM NaCl (control), at pH 7.0. The strains were then incubated for 48 h (late exponential growth phase), at 120 rpm and 30 ( B. amyloliquefaciens ) and 37 ( L. helveticus ) °C. The microbial cultures were then centrifuged and filtered sterilized, to obtain cell free supernatants whose proteome profiles were studied using LC–MS/MS analysis and quantified using scaffold. Results of the study revealed that treatment with 200 mM NaCl negatively affected the quantity of identified proteins in comparison to the control, for both strains. There was upregulation and downregulation of some proteins, even up to 100%, which resulted in identification of proteins significantly unique between the control or 200 mM NaCl ( p ≤ 0.05), for both microbial species. Proteins unique to 200 mM NaCl were mostly those involved in cell wall metabolism, substrate transport, oxidative stress tolerance, gene expression and DNA replication and repair. Some of the identified unique proteins have also been reported to enhance plant growth. In conclusion, based on the results of the work described here, PGPM alter their exoproteome profile when exposed to salt stress, potentially upregulating proteins that enhance their tolerance to this stress.

Ecotoxicological significance of bio-corona formation on micro/nanoplastics in aquatic organisms

Article

Full-text available

Jul 2023

The unsustainable manufacturing, utilization and inadequate handling of plastics have led to a surge in global plastic pollution. In recent times, there has been increasing concern about the plausible hazards associated with exposure to micro/nanoplastics (M/NPs). As aquatic systems are considered to be the likely sink for M/NPs, it is crucial to comprehend their environmental behavior. The bioavailability, toxicity and fate of M/NPs in the environment are predominantly dictated by their surface characteristics. In the aquatic environment, M/NPs are prone to be internalized by aquatic organisms. This may facilitate their interaction with a diverse array of biomolecules within the organism, resulting in the formation of a biocorona (BC). The development of BC causes modifications in the physicochemical attributes of the M/NPs including changes to their size, stability, surface charge and other properties. This review details the concept of BC formation and its underlying mechanism. It provides insight on the analytical techniques employed for characterizing BC formation and addresses the associated challenges. Further, the eco-toxicological implications of M/NPs and the role of BC in modifying their potential toxicity on aquatic organisms is specified. The impact of BC formation on the fate and transport of M/NPs is discussed. A concise outlook on the future perspectives is also presented.

Two-dimensional electrophoresis analysis of the extracellular proteome of Bacillus cereus reveals the importance of the PIcR regulon

Article

Full-text available

Jun 2002
PROTEOMICS

Many virulence factors are secreted by the gram-positive, spore forming bacterium Bacillus cereus. Most of them are regulated by the transcriptional activator, PlcR, which is maximally expressed at the beginning of the stationary phase. We used a proteomic approach to study the impact of the PlcR regulon on the secreted proteins of B. cereus, by comparing the extracellular proteomes of strains ATCC 14579 and ATCC 14579 Δ plcR, in which plcR has been disrupted. Our study indicated that, quantitatively, most of the proteins secreted at the onset of the stationary phase are putative virulence factors, all of which are regulated, directly or indirectly, by PlcR. The inactivation of plcR abolished the secretion of some of these virulence factors, and strongly decreased that of others. The genes encoding proteins that are not secreted in the ΔplcR mutant possessed a regulatory sequence, the PlcR box, upstream from their coding sequence. These proteins include collagenase, phospholipases, haemolysins, proteases and enterotoxins. Proteins for which the secretion was strongly decreased, but not abolished, in the ΔplcR mutant did not display the PlcR box upstream from their genes. These proteins include flagellins and InhA2. InhA2 is a homologue of InhA, a Bacillus thuringiensis metalloprotease that specifically degrades antibacterial peptides. The mechanism by which PlcR affects the production of flagellins and InhA2 is not known.

Assessment of the IgA immunoassay diagnostic potential of the Mycobacterium tuberculosis MT10.3-MPT64 fusion protein in tuberculous pleural fluid (Clinical and Vaccine Immunology (2010) 17, 12 (1963-1699))

Article

Full-text available

Feb 2011

Genomic and proteomic analysis of the coral pathogen Vibrio coralliilyticus reveal a diverse viruence repertoire

Article

Full-text available

Mar 2011

Vibrio coralliilyticus has been implicated as an important pathogen of coral species worldwide. In this study, the nearly complete genome of Vibrio coralliilyticus strain P1 (LMG23696) was sequenced and proteases implicated in virulence of the strain were specifically investigated. The genome sequence of P1 (5 513 256 bp in size) consisted of 5222 coding sequences and 58 RNA genes (53 tRNAs and at least 5 rRNAs). Seventeen metalloprotease and effector (vgrG, hlyA and hcp) genes were identified in the genome and expressed proteases were also detected in the secretome of P1. As the VcpA zinc-metalloprotease has been considered an important virulence factor of V. coralliilyticus, a vcpA deletion mutant was constructed to evaluate the effect of this gene in animal pathogenesis. Both wild-type and mutant (ΔvcpA) strains exhibited similar virulence characteristics that resulted in high mortality in Artemia and Drosophila pathogenicity bioassays and strong photosystem II inactivation of the coral dinoflagellate endosymbiont (Symbiodinium). In contrast, the ΔvcpA mutant demonstrated higher hemolytic activity and secreted 18 proteins not secreted by the wild type. These proteins included four types of metalloproteases, a chitinase, a hemolysin-related protein RbmC, the Hcp protein and 12 hypothetical proteins. Overall, the results of this study indicate that V. coralliilyticus strain P1 has a diverse virulence repertoire that possibly enables this bacterium to be an efficient animal pathogen.Keywords: V. coralliilyticus; coral disease; zinc-metalloprotease; genome; secretome

Computational Prediction and Experimental Validation of Signal Peptide Cleavages in the Extracellular Proteome of a Natural Microbial Community

Article

Full-text available

Apr 2010
J PROTEOME RES

An integrated computational/experimental approach was used to predict and identify signal peptide cleavages among microbial proteins of environmental biofilm communities growing in acid mine drainage (AMD). SignalP-3.0 was employed to computationally query the AMD protein database of >16,000 proteins, which resulted in 1,480 predicted signal peptide cleaved proteins. LC−MS/MS analyses of extracellular (secretome) microbial preparations from different locations and developmental states empirically confirmed 531 of these signal peptide cleaved proteins. The majority of signal-cleavage proteins (58.4%) are annotated to have unknown functions; however, Pfam domain analysis revealed that many may be involved in extracellular functions expected within the AMD system. Examination of the abundances of signal-cleaved proteins across 28 proteomes from biofilms collected over a 4-year period demonstrated a strong correlation with the developmental state of the biofilm. For example, class I cytochromes are abundant in early growth states, whereas cytochrome oxidases from the same organism increase in abundance later in development. These results likely reflect shifts in metabolism that occur as biofilms thicken and communities diversify. In total, these results provide experimental confirmation of proteins that are designed to function in the extreme acidic extracellular environment and will serve as targets for future biochemical analysis.Keywords: Mass spectrometry; multidimensional liquid chromatography; shotgun proteomics; signal peptides; microbial communities

Functional Analyses of Mycobacterial Lipoprotein Diacylglyceryl Transferase and Comparative Secretome Analysis of a Mycobacterial lgt Mutant

Article

Full-text available

Jul 2012
J BACTERIOL

Preprolipopoprotein diacylglyceryl transferase (Lgt) is the gating enzyme of lipoprotein biosynthesis, and it attaches a lipid structure to the N-terminal part of preprolipoproteins. Using Lgt from Escherichia coli in a BLASTp search, we identified the corresponding Lgt homologue in Mycobacterium tuberculosis and two homologous (MSMEG_3222 and MSMEG_5408) Lgt in Mycobacterium smegmatis. M. tuberculosis lgt was shown to be essential, but an M. smegmatis ΔMSMEG_3222 mutant could be generated. Using Triton X-114 phase separation and [14C]palmitic acid incorporation, we demonstrate that MSMEG_3222 is the major Lgt in M. smegmatis. Recombinant M. tuberculosis lipoproteins Mpt83 and LppX are shown to be localized in the cell envelope of parental M. smegmatis but were absent from the cell membrane and cell wall in the M. smegmatis ΔMSMEG_3222 strain. In a proteomic study, 106 proteins were identified and quantified in the secretome of wild-type M. smegmatis, including 20 lipoproteins. All lipoproteins were secreted at higher levels in the ΔMSMEG_3222 mutant. We identify the major Lgt in M. smegmatis, show that lipoproteins lacking the lipid anchor are secreted into the culture filtrate, and demonstrate that M. tuberculosis lgt is essential and thus a validated drug target.

Chapter 32 Translocation of proteins across the bacterial cytoplasmic membrane

Article

Dec 1996

Arnold J.M. Driessen

Bacteria export proteins to the periplasmic space and outer membrane via a mechanism that involves a multisubunit integral membrane protein complex termed translocase. Proteins to be secreted are synthesized in the cytosol as cleavable signal sequence bearing precursor proteins. The signal sequence acts as a targeting and recognition signal for the translocase. The translocase mediates the energy-dependent translocation of the precursor protein across the cytoplasmic membrane. The mechanism of protein translocation has rapidly advanced due to the ability to use powerful genetic and biochemical techniques in Escherichia coli. Extensive genetic screening methods have identified the genes, termed “sec(retion)-genes,” which code for the components involved in this process. Many of the genes are essential for viability, and conditional lethal mutants are selected that cause pleiotropic protein export defects. The products of these genes are overproduced and purified to homogeneity. Authentic energy-dependent protein translocation as it occurs in vivo is reconstituted into proteoliposomes with purified components. This chapter discusses the biochemical role of the individual components that catalyze the actual translocation reaction in bacteria. The enzymology of signal peptidases, and alternative and dedicated protein translocation pathways are also discussed.

Comprehensive analysis of exported proteins from Mycobacterium tuberculosis H37Rv

Article

May 2007
PROTEOMICS

Proteins secreted by Mycobacterium tuberculosis play an essential role in the pathogenesis of tuberculosis. The culture filtrates of M. tuberculosis H37Rv made by Sadamu Nagai (Japan), are considerably enriched for secreted proteins compared to other culture filtrates. Complementary approaches were used to identify the secreted proteins in these culture filtrates: (i) 2-DE combined with MALDI-TOF MS and (ii) LC coupled MS/MS. Peptides derived from a total of 257 proteins were identified of which 144 were identified by more than one peptide. Several members of the immunologically important early secretory antigenic target-6 (ESAT-6) family of proteins were found to be major components. The majority of the identified proteins, 159 (62%), were predicted to be exported through the general secretory pathway. We experimentally verified that the signal peptides, which mediate translocation through the cell membrane, had been removed in 41 of the identified proteins, and in 35 of those, there was an AXA motif N-terminally to the cleavage site, showing that this motif is important for the recognition and cleavage of signal peptides in mycobacteria. A large fraction of the secreted proteins were unknown, suggesting that we have mapped an unexplored part of the exported proteome of M. tuberculosis.∁

Shotgun nanoLC-MS/MS proteogenomics to document MALDI-TOF biomarkers for screening new members of the

Article

Jun 2012
ENVIRON MICROBIOL

The identification of bacteria by means of matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry directly using whole cells has become a standard method in clinical diagnosis due to its rapidity and simplicity. Nevertheless, the analysis of environmental samples with this approach still represents a challenge due to the enormous microbial diversity existing on earth and the lack of a comprehensive database. Most of the environmentally relevant species comprise only one unique strain, while pathogens such as Escherichia coli, with 667 described strains, are well documented. In such case, identification of the proteins responsible for the peak signals within MALDI-TOF spectra can give crucial information for species discrimination. To give higher confidence in MALDI-TOF biomarker description we exploited information from proteins identified by shotgun nanoLC-MS/MS, consisting of the identification and quantification of low-molecular-weight proteins after SDS-PAGE, in-gel trypsin proteolysis and analysis of tryptic peptides. We also proposed the standardization of the inclusion of internal calibrants in the bacterial sample to improve the accuracy of the MALDI-TOF measurements. In this way, nine candidate biomarkers were tentatively proposed for Ruegeria lacuscaerulensis ITI-1157. The conserved biomarkers were theoretically deduced for all other Ruegeria strains whose genomes have been sequenced and their corresponding m/z MALDI-TOF signals were estimated. Among these, DNA-binding protein, HU, and ribosomal proteins, L29, L30, L32 and S17, were shown experimentally to be also the most prominent and conserved signals in the other strain tested, Ruegeria pomeroyi DSS-3. Thus, we suggested that these five biomarkers, which give rise to 10 m/z peak signals derived from the mono- and doubly protonated proteins, are the best candidates for identifying bacteria belonging to the Ruegeria genus, and quickly assessed their phylogenetic proximity to described species. As an application of these biomarkers, we quickly screened 30 seawater bacterial isolates by MALDI-TOF and found one belonging to the Ruegeria genus, as further confirmed by 16S RNA sequencing. Due to its simplicity and effectiveness, this technique could be of immense value in monitoring bacteria in the environment in the near future.

Microbiology and proteomics, getting the best of both worlds!

Article

Jun 2012
ENVIRON MICROBIOL

Jean Armengaud

High-throughput identification of proteins with the latest generation of hybrid high-resolution mass spectrometers is opening new perspectives in microbiology. I present, here, an overview of tandem mass spectrometry technology and bioinformatics for shotgun proteomics that make 2D-PAGE approaches obsolete. Non-labelling quantitative approaches have become more popular than labelling techniques on most proteomic platforms because they are easier to carry out while their quantitative outcome is rather robust. Parameters for recording mass spectrometry data, however, need to be chosen carefully and statistics to assess the confidence of the results should not be neglected. Interestingly, next-generation sequencing methodologies make any microbial model quickly amenable to proteomics, leading to the documentation of a wide range of organisms from diverse environments. Some recent discoveries made using microbial proteomics have challenged some biological dogma, such as: (i) initiation of the translation does not occur predominantly from ATG codons in some microorganisms, (ii) non-canonical initiation codons are used to regulate the production of specific but important proteins and (iii) a gene may code for multiple polypeptide species, heterogeneous in terms of sequences. Microbial diversity and microbial physiology can now be revisited by means of exhaustive comparative proteomic surveys where thousands of proteins are detected and quantified. Proteogenomics, consisting of better annotating of genomes with the help of proteomic evidence, is paving the way for integrated multi-omic approaches in microbiology. Finally, meta-proteomic tools and approaches are emerging for tackling the high complexity of the microbial world as a whole, opening new perspectives for assessing how microbial communities function.

MetaLocGramN: A meta-predictor of protein subcellular localization for Gram-negative bacteria

Article

Jun 2012
Biochim Biophys Acta

Subcellular localization is a key functional characteristic of proteins. It is determined by signals encoded in the protein sequence. The experimental determination of subcellular localization is laborious. Thus, a number of computational methods have been developed to predict the protein location from sequence. However predictions made by different methods often disagree with each other and it is not always clear which algorithm performs best for the given cellular compartment. We benchmarked primary subcellular localization predictors for proteins from Gram-negative bacteria, PSORTb3, PSLpred, CELLO, and SOSUI-GramN, on a common dataset that included 1056 proteins. We found that PSORTb3 performs best on the average, but is outperformed by other methods in predictions of extracellular proteins. This motivated us to develop a meta-predictor, which combines the primary methods by using the logistic regression models, to take advantage of their combined strengths, and to eliminate their individual weaknesses. MetaLocGramN runs the primary methods, and based on their output classifies protein sequences into one of five major localizations of the Gram-negative bacterial cell: cytoplasm, plasma membrane, periplasm, outer membrane, and extracellular space. MetaLocGramN achieves the average Matthews correlation coefficient of 0.806, i.e. 12% better than the best individual primary method. MetaLocGramN is a meta-predictor specialized in predicting subcellular localization for proteins from Gram-negative bacteria. According to our benchmark, it performs better than all other tools run independently. MetaLocGramN is a web and SOAP server available for free use by all academic users at the URL http://iimcb.genesilico.pl/MetaLocGramN. This article is part of a Special Issue entitled: Computational Methods for Protein Interaction and Structural Prediction.

Exoproteomics: Exploring the world around biological systems

Abstract

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