Proteomics 2010, 10, 3935–3956 DOI 10.1002/pmic.

201000289 3935

REVIEW

Subcellular fractionation methods and strategies

for proteomics

Yie Hou Lee1,2, Hwee Tong Tan1 and Maxey C. M. Chung1,3

1
Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, Singapore
2
Singapore-MIT Alliance for Research and Technology, National University of Singapore, Singapore
3
Department of Biological Sciences, Faculty of Science, National University of Singapore, Singapore

Developments in subcellular fractionation strategies have provided the means to profile and Received: April 30, 2010
analyze the protein composition of organelles and cellular structures by proteomics. Here, we Revised: July 14, 2010
review the application of classical (e.g. density gradient centrifugation) and emerging Accepted: August 3, 2010
sophisticated techniques (fluorescent-assisted organelle sorting) in the fractionation, and
statistical/bioinformatics tools for the prediction of protein localization in subcellular
proteomics. We also review the validation methods currently used (such as microscopy, RNA
interference and multiple reaction monitoring) and discuss the importance of verification of
the results obtained in subcellular proteomics. Finally, the numerous challenges facing
subcellular proteomics including the dynamics of organelles are being examined. However,
complementary approaches such as modern statistics, bioinformatics and large-scale inte-
grative analysis are beginning to emerge as powerful tools to proteomics for analyzing
subcellular organelles and structures.

Keywords:
Cell biology / Organelle / Subcellular fractionation

1 Introduction investigator to frame specific biological and functional

Organellar proteins determine the structure and function of
the subcellular components in which they reside in.
Compared to the highly complex proteome of whole cells or
tissues, the protein composition of organelles and cellular
structures is of reduced complexity [1]. This thus allows the
Correspondence: Professor Maxey C. M. Chung, Department of
Biochemistry, Yong Loo Lin School of Medicine, National
University of Singapore, MD7, 8 Medical Drive, Singapore
117597, Singapore
E-mail: bchcm@nus.edu.sg
Fax: 165-779-1453
Abbreviations: ANN, artificial neural networks; DDF, differential
detergent fractionation; EM, electron microscopy; ER, endoplas-
mic reticulum; FAOS, fluorescent-assisted organelle sorting;
FFE, free flow electrophoresis; GFP, green fluorescent protein;
HMM, hidden Markov models; LOPIT, localization of organelle
proteins by isotope tagging; PCP, protein correlation profiling;
RNAi, RNA-mediated interference; SVM, support vector
machines
questions in the context of the organelle.
In organellar proteomics, organelles or cellular
compartments that are enriched by subcellular fractionation
procedures are usually analyzed with proteomics tools for a
more comprehensive identification of their resident proteins
[2]. In particular, marked improvements in protein/peptide
separation methods (e.g. 1-DE, 2-DE, 1-D and 2-D LC)
coupled with biological MS have resulted in the identifica-
tion and characterization of a much greater number of
resident proteins found in these organelles. A major
concern with MS analysis is the incomplete sampling of
peptide ions. Often, it is the more abundant proteins that
are detected more readily, and this has often resulted in the
loss of peptide identification belonging to the lower abun-
dant proteins, and which in turn makes comparison within
and between subcellular proteomes difficult [3, 4]. In an
effort to alleviate the problem of incomplete sampling of
ions, repeated injections of the same sample were necessary
to uncover a larger portion of proteome [3, 5].
However, the quality of the proteomic readout of orga-
nelles is still heavily dependent on the preceding isolation

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3936 Y. H. Lee et al.
steps of the organelles. This is particularly crucial in the
detection of proteins that are present in low abundance
against the complex background of whole cells or crude
tissue homogenates [3–5]. Classical methods of enriching
organelles and cellular structures such as density-gradient
centrifugation have been used extensively but these orga-
nellar preparations often contain contaminating proteins.
This major limitation provided the opportunities for cell
biologists and biochemists to improve on the enrichment
techniques, which can then lead to an increase in the
proteome coverage and at the same time being able to
provide reliable data on the distribution of bona fide orga-
nellar proteins. Indeed, much progress has been made in
the recent years in uncovering many novel organellar
proteins with their spatial distributions and new biological
paradigms. Such novel discoveries can potentially lead to the
systematic identification of new causative proteins for
various human diseases [6, 7]. Thus in this review, we aim to
discuss, evaluate and summarize the various strategies and
technologies, conventional as well as emerging innovative
approaches that have been used to verify and validate the
proteome of eukaryotic organelles.
2 Strategies for subcellular fractionation
Figure 1 shows the organelles and cellular structures
commonly found within higher organisms and plants. The
approaches used to isolate target organelles from the rest of
the cellular compartments will be important prior to
proteomics. Classically, organellar proteomics depend on
biochemical methods to isolate the diverse organelles found
in cells. However, the complete global profiling of organelle
proteomes demands robust sample preparation and orga-
nelle enrichment methods to ensure the correct annotation
of protein distribution. Such classical approaches have
unfortunately, been plagued by the problem of co-purifying
contaminants. While this may be true, classical methods
remain easy to adopt and modify in most laboratories and
they also lay the ground for more sophisticated approaches.
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Proteomics 2010, 10, 3935–3956
Recent developments in methodological and technological
advances have the potential to yield organelles of higher
purity, and as technologies progress, the effectiveness will
improve, albeit at a cost of higher complexity or prohibitive
resource requirements. Here we discuss both classical and
emerging techniques that may be stand-alone or comple-
mentary to conventional methods to improve subcellular
fractionation for high-resolution organellar proteomics.
2.1 Conventional methods
Specific proteomes of cellular compartments and organelles
can be isolated via several approaches, and the most
commonly used strategy is subcellular fractionation [8, 9].
The concept of subcellular fractionation was pioneered by De
Duve and coworkers in 1955 using rat liver tissue [10]. The
procedure for subcellular fractionation first involved disrup-
tion of cells and tissues using physical methods such as
mechanical or liquid shear or non-physical methods such as
detergents or hypo-osmotic shock. The cell disruption step
aimed to obtain high protein yield, and maintain structural
and functional integrity of intracellular organelles. The effi-
ciency of cell disruption is often monitored with immuno-
chemical and microscopic studies. Conventional methods
involved in subcellular fractionation, typically by differential
and density-gradient centrifugation, employed a series of
centrifugation steps to separate different populations of
cellular compartments or organelles from cell homogenates
based on their mass and/or density [2].
2.1.1 Differential centrifugation
Differential centrifugation operates via sequential centrifu-
gation of the cell or tissue homogenate to obtain subcellular
organelles such as nuclei, mitochondria and lysosomes.
This separation method is based on differences in size and
density, whereby larger and denser organelles sediment at
lower centrifugal forces. However, this method has poor
Figure 1. Selected organelle compo-
sition in eukaryotic cells. Different
organelles resident in eukaryotic
systems that can be subjected to
proteomic analyses. Some organelles
such as zymogen granules, centrioles
and COPI vesicles are not graphically
represented due to space limitation.
Neither are they exhaustive in
terms of the diverse organelles
and cellular structure that can be
found in eukaryotes. ERGIC, endo-
plasmic reticulum–Golgi intermediate
compartment.
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Proteomics 2010, 10, 3935–3956
resolving power and thus may result in fractions containing
a different organelle type that has similar sedimentation
velocities.
2.1.2 Density-gradient centrifugation
The more commonly applied density-gradient centrifuga-
tion method separates organelles based on continuous or
discontinuous gradients using various media, such as
sucrose of different osmolarities, viscosities or densities. In
general, the cell homogenate or post-nuclear supernatant is
first added to the top of the medium and centrifuged. Upon
centrifugation, the organelle focuses in the gradient where
its density equals the density of the surrounding medium
(isopycnic point). The rate of sedimentation of an organelle
in density-gradient centrifugation thus depends on the
differences in organelle density relative to medium, and that
in turn is determined by the organelle content, lipid/protein
ratio, size and shape [11]. For example, high-density mito-
chondria and endoplasmic reticulum have high protein
content, whereas endosomes have a low density due
to their lipid-rich membranes. Sucrose is the most
commonly used medium for density-gradient centrifugation
as sucrose is biologically inert, inexpensive and dialyzable.
Other media include Ficoll, Percoll, Nycodenz and
Metrizamide [12].
In a continuous gradient, the density increases linearly
along the tube but in a discontinuous gradient, the gradient
is divided into fixed portions consecutively. During discon-
tinuous gradient centrifugation, different organelles are
enriched at different interphases of the medium. On the
other hand, in continuous gradient centrifugation, equili-
brium separation occurs whereby organelles distribute
throughout the gradient and are focused at their isopycnic
points. This provided a better resolution of organelles than
discontinuous gradients. However, preparation of contin-
uous gradient can be time consuming and required special
apparatus. Density-gradient centrifugation remained as a
popular choice of researchers for subcellular fractionation as
this method is well developed and referenced, and applic-
able for many organellar proteomics studies [13].
2.1.3 Differential detergent fractionation
Besides differential and density-gradient centrifugation, one
approach advocated to reduce contaminating proteins from
other organelles and to avoid time-consuming centrifuga-
tion steps is by using buffers of increasing stringency in the
subcellular fractionation steps. One such method is differ-
ential detergent fractionation (DDF), which employed
sequential extraction of organelle proteins using different
detergent-containing buffers. DDF is a simple method that
can fractionate proteins in their native state according to
four subcellular localizations (cytosolic, membrane and
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membrane organelle-localized, soluble and DNA-associated
nuclear and cytoskeletal proteins) [14, 15], and is commer-
cially available as the ProteoExtract Subcellular Proteome
Extraction kit (Calbiochem, San Diego, CA, USA). Abdol-
zade-Bavil et al. described a robust DDF method with high
efficiency and selectivity, which was supported by 2-D gel
electrophoresis results, morphological, immunological and
enzymatic analyses [15]. Recently, this method has been
used to obtain comprehensive proteomes (including hydro-
phobic membrane proteins) for functional genome annota-
tion [16]. It has also been applied on frozen archival tissues
that showed that there is no loss of proteome coverage or
information about protein localization [17]. DDF has also
been used to fractionate neurons in culture plates to enrich
for cytosolic, membrane-bound or enclosed, nuclear, and
cytoskeletal proteins for differential proteomics analysis [18].
Lastly, Holden and Horton established a protocol to
sequentially extract proteins from cultured mammalian cells
into fractions that were enriched for cytosolic, membrane-
bound organellar, nuclear and insoluble proteins, using
digitonin and detergent [19].
2.2 Emerging methods
2.2.1 Free-flow electrophoresis
Free-flow electrophoresis (FFE) offers an alternative method
to isolate and fractionate organelles such as peroxisomal
membranes [20], mitochondria [21], secretory vesicles [22],
Arabidopsis thaliana tonoplasts, plasma membrane vesicles
and peroxisomes [23, 24]. Organelle isolation by FFE is
based on separation by their net global isoelectric charges or
electrophoretic mobilities. In addition to fractionation of
organelles, FFE has been used for protein separation [25].
During FFE, the carrier buffer is flowing continuously and
laminarly in a narrow gap between two cooling plates, with
an electric field being applied perpendicularly to the laminar
flow of the buffer and sample, thus resulting in the differ-
ential deflections of the sample molecules [26].
Some of the advantages of FFE include the wide choice of
pH, conductivity and composition of electrolytes solutions
[26] and the high sample recoveries (approximately in the
range of 95%) that arose from the injection of samples as
thin film which precluded the use of any matrix. The
continuous application and fractionation of the samples and
collection of the samples at the other end of the FFE
chamber lend itself well for preparative organelle separa-
tions. In addition, the purified organelles retain their
intactness and functionality, which makes FFE an attractive
technique not only for proteomic analyses but also
complementary functional studies. Also, the preservation of
proteins in their native state by FFE is complementary to
native or top-down proteomics [27, 28]. To avoid the problem
of Joule heating, which arose from the heating of the
separation medium by electric currents to focus the larger
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3938 Y. H. Lee et al.
drag force of organelles, micro-FFE that utilized lower
electric currents and higher surface area-to-volume ratio to
dissipate heat, has been employed [29, 30].
One challenge of FFE is when organelles or cellular
structures have similar isoelectric points, which would
result in comigration. For example, it was found that in the
FFE separation of neutrophil secretory vesicles, contamina-
tions with ER and mitochondria were evident, probably as a
result of the similarity in isoelectric points. Similarly,
mitochondrial and microsomal proteins can be found in
peroxisomal preparations [20, 24] and hence other orthogo-
nal approaches are required to resolve them. In spite of this,
FFE was shown to be useful in resolving a minor fraction of
peroxisomes from the heavy mitochondrial fraction, in
addition to the more commonly enriched light mitochon-
drial fraction [31].
2.2.2 Immunoaffinity purification
Immunoaffinity purification is a powerful method to isolate
organelles with specificity and in adequate yields [32]. The
two major methods, affinity purification and immunopre-
cipitation that are used to isolate organelles are based on
binding between ligands (antibodies) immobilized on solid
supports and targets (organelle of interest). For proteomic
analysis, affinity purification is a good option to start for its
ability to do repeated purification using a single sample and
hence enrichment of the organelle. Ligands are no longer
limited to antibodies, but also include chemical ligands,
combinatorial peptide libraries, aptamers and others [33].
Limitations to affinity purification include its high cost and
the increased experimental duration required for effective
purification. Lastly, the signal-to-noise generated in MS may
be inadequately low for complex biological samples, espe-
cially with the initially less well-fractionated samples.
Therefore, to reduce the complexity of the sample, very often
density centrifugation is performed prior to immunopur-
ification. For example, Nycodenz density-gradient centrifu-
gation followed by immunoaffinity purification has been
reported to isolate peroxisomes from rat liver [34]. Likewise,
such a strategy has been used to enrich for secretory
synaptic vesicles from synaptosomes [35].
In a bid to enrich phagosomes, opsonized latex beads
have been used. Cells engulf these targets and the phago-
somic membranes remain adherent and can be isolated by
flotation of the beads. Opsonized latex beads are particularly
useful because they are inert, amenable to laboratory
manipulations and their different sizes can be suited to the
different targets of individual and differing phagocytic
systems [36]. The ER is generally dense so it should not
sediment with the latex beads in density gradients [37].
However, opsonized latex beads enrichment has consis-
tently presented ER proteins in the phagosomal prepara-
tions [36]. The issue surrounding the contribution of ER
membranes to phagosomes is contentious as several
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Proteomics 2010, 10, 3935–3956
different groups have suggested conflicting hypotheses
[38–42]. Hence complementary and supportive biochemical
and microscopic techniques will aid in defining the
contribution of ER to phagosomes [43] in subcellular frac-
tionation. Recently, a different method of purifying phago-
somes has been developed which entailed the use of a
combination of differential and density-gradient sedi-
mentation on sucrose and iodixanol gradient media. The
method did not use latex beads and it showed a high degree
of purification [44].
Recently, more interest has been generated in the char-
acterization of exosomes, given their pleiotropic effects on
paracrine and endocrine processes [45]. Immunoaffinity
purification of exosomes is an attractive way of obtaining
more homogenous exosomal preparations without the
possibility of capturing contaminants of similar density and
size. It should be noted that exosomes and HIV-1 particles
share a number of similar biophysical properties such as
size, density and export/transport processes. To this end,
exosomes purified using immunoaffinity capture has been
shown to be virion free [46, 47]. For verification, flow cyto-
metry combined with exosomal-specific markers was used to
demonstrate the purity of enriched exosomes [48].
2.2.3 Fluorescent-assisted organelle sorting (FAOS)
Besides its use as a verification method, cell sorter flow
cytometry can also be used to isolate and enrich organelles in
a method termed ‘‘fluorescent-assisted organelle sorting’’
(FAOS) [49, 50]. Only simple amendments to the flow
cytometer are required, thus making FAOS readily accessible
to most laboratories attempting subcellular proteomics. This
approach has been used to isolate a number of subcellular
organelles such as phagosomes [51], mitochondria [52],
endocytic vesicles [53, 54] and secretory granules [50].
There are a few things to observe when sorting organelles
of interest. Intact organelles are important for successful
sorting and proteomic analysis. A protein marker that is
specific to the organelle of interest will be mandatory for
their successful sorting. This protein tagged with green
fluorescent protein (GFP) can be transfected into cells,
enabling its fluorescence to be discriminated by the cell
sorter [54]. Alternatively, if organelle-specific surface
markers are available, FAOS can be employed in a fashion
analogous to FACS for cell sorting. Information on the size
of organelles is important and as long as an appropriate
sorting window is set within the known dimensions of the
organelle of interest, their enrichment can be achieved.
However, in cases where there is heterogeneity of organelle
sizes, it can be difficult to ascertain if FAOS sorted the
organelles accurately. One recent application of FAOS is in
the identification of more than 150 secretory granule
proteins [50]. It was noted that a high degree of purity was
achieved, including FAOS’s ability to distinguish two other
fluorescing organelles, the ER and Golgi apparatus.
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Proteomics 2010, 10, 3935–3956
2.2.4 Proteomics in combination with statistical
analyses
In order to address the problem of co-purifying proteins,
many groups have looked at the use of statistical analysis
and bioinformatic predictions to aid the assignment of
known and unknown proteins to their respective subcellular
compartments. Multivariate analysis in general offers great
versatility in analyzing proteomic data, especially where
there are a large number of parameters (proteins) and since
dimension reduction is required to predict and annotate
proteins of unknown function and localization.
Multivariate analysis such as principle component
analysis (PCA) and partial least-squares discriminant
analysis (PLS-DA) were applied by Dunkley and Lilley to
predict and classify proteins to their organelles in a method
termed ‘‘localization of organelle protein by isotope tagging’’
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(LOPIT) in A. thaliana [55, 56]. In principle, the LOPIT
workflow can be summarized as follows: (i) preparative
isolation of organelles by density centrifugation, (ii) selec-
tion of fractions for analysis based on known marker protein
distribution across the density gradients (Figs. 2A, B and D)
and (iii) identification and quantitation by iTRAQ labeling
and MS analysis. The strength of LOPIT lies in that it works
around the problem of imperfect methods to isolate pure
organellar preparations. Proteins were sorted according to
their subcellular distributions by PCA and tight clusters of
proteins belonging to similar compartments can be
observed visually on PCA plots with 1 being the perfect
match to a group. For the purpose of classifying proteins of
unknown distributions, PLS-DA predicts group member-
ship by the use of the set of distinctly clustered proteins
already assigned by PCA. Where proteins cannot be discri-
minated, such proteins may be found in (i) multiple
Figure 2. Schematic diagram showing
how emerging approaches are correlated
with density gradient centrifugation to
distinguish genuine organellar proteins.
(A) Very often, biological extracts
containing intact organelles are separated
by density-gradient centrifugation and
fractions probed with specific marker
proteins by (B) immunoblotting to analyze
profiles for each organelle (C, D and E).
Such a method can be adopted and inter-
faced with MS to confidently designate
protein localization within cells. Three
approaches discussed in this review are
described below. (C) PCP. Signal inten-
sities of peptides from the same organellar
protein from fractions B to H are correlated
as a function of time. Analogous to
immunoblotting profiling of centrifugation
fractions, ion intensities for peptides from
marker proteins, measured by mass
spectrometric analysis of each gradient
fraction and PCP, generate the defined
organelles. Proteins belonging to same
organelle will have similar distribution
profiles (e.g. proteins from fractions B and
C). In contrast, contaminating proteins will
have a different profile across the density
fractions (e.g. proteins from fractions B
and C will differ from fractions F, G and H).
(D) Localization of Organelle Proteins by
Isotope Tagging (LOPIT) Fraction C
labeled with iTRAQ 114. Fraction E labeled
with iTRAQ 115. Fraction F labeled with
iTRAQ 116. Fraction G labeled with iTRAQ
117. Compare quantified peptides from
ratios: 115/114, 116/115, 116/114, 117/116,
117/115 and 117/114. Compare these ratios
with ratios of known organelle markers.
(E) Subtractive proteomics. Subtract
proteins in fraction D from fractions B
and C.
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3940 Y. H. Lee et al.
locations, (ii) transiting cellular compartments such as
endosomes or (iii) where few or no proteins of known
compartments are detected in the MS data such that no
distinctive clusters are formed [57].
Another major advancement in subcellular proteomic
strategy that is independent of organelles being purified to
homogeneity and which also utilizes statistical measures is
protein correlation profiling (PCP) [37, 58–60]. In PCP,
organelles are recovered from fractions generated in density
centrifugation and the abundance of each peptide in each
gradient fraction is calculated from the area of its extracted
ion current peak. Adjacent fractions are also taken into
account, hence signals of the same peptide are normalized,
correlated with elution times of different fractions and
compiled together to form protein correlation curves
(Fig. 2C). Using the established resident organellar proteins
peaking in different density fractions, one can define the
distinctive profiles of these markers by using the w2 value
that then acts as a basis to discern subcellular localization of
other proteins [59]. This strategy is similar to the more
commonly used immunoblotting profile of fractions
(Figs. 2A and B). Notably, PCP was able to distinguish
dynamic transiting organelles including endosomes and
ER/Golgi-derived vesicles [37]. PCP can also be used to train
for false-positive sets with proteins known to be localized
outside the organelle of interest [58, 59]. For example, Wiese
et al. applied PCP on kidney peroxisomes fractionation, and
found 15 novel peroxisomal candidates, 5 of which were
validated by immunocytochemistry [58]. Peroxisomes and
mitochondria possess similar densities and such character-
istics compound the problem of correctly assigning proteins
to either of each fraction. As such, unsupervised k-means
clustering was employed as an added criterion to better
differentiate unique peroxisomal, mitochondrial or dual
localized proteins.
2.2.5 Subtractive proteomics
One potential error of organellar proteomics is the inad-
vertent contamination of target organelles with co-purifying
organelles from the commonly used classical methods. This
problem may be circumvented through the use of subtrac-
tive proteomics [6, 61, 62]. In subtractive proteomics
(Fig. 2E), the method compares and ‘subtracts’ the identified
protein constituents of the contaminated fraction containing
the organelle of interest against that of a crude preparation
[1]. With the aim of identifying membrane proteins from the
nuclear envelope, Yates and colleagues first prepared
microsomal membranes from livers that were rich in ER
proteins, contained mitochondrial proteins, but free of
nuclear membranes. Next they collected crude nuclear
envelope fraction that contained contaminating mitochon-
drial membranes. To obtain nuclear envelope-specific
proteins, they subtracted proteins of the microsomal
membrane fraction from the crude nuclear envelope frac-
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Proteomics 2010, 10, 3935–3956
tion. The investigators then restrict their analyses to
uncharacterized open reading frames that were only found
in the subtracted nuclear envelope fractions, thereby anno-
tating 67 previously unknown putative proteins to the
nuclear envelope, in addition to the 13 known proteins [6].
Of these proteins, eight of them were validated using
immunofluorescence. Their findings led them to propose
that 62 of these nuclear envelope proteins are potentially
associated with dystrophies in humans. While these
hypotheses remain to be tested [63], many dystrophy disea-
ses have been linked to mutations in nuclear envelope
proteins [64]. In summary, this highlighted that the
subtractive proteomics approach is capable of assigning and
identifying proteins to their specific subcellular locations
and generating new models to link protein mutations to
human diseases.
2.2.6 Large-scale integrative analyses
Increasingly, integrative analyses are being applied to better
annotate protein localization within organelles. MS-based
proteomics are employed as the main tool of detecting
organellar proteins, integrated with diverse data sets and
robust statistics and bioinformatics to systemically assign
proteins their spatial locations [7, 37, 65, 66].
The mitochondrion is an ideal organelle to start such
integrative efforts. Its many core cellular functions includ-
ing oxidative phosphorylation, fatty acid oxidation and
others are well conserved during evolution. Phylogenetic
evolution is a useful dimension in elucidating the proteome
complement of an organelle – sequence homology of
mitochondrial proteins in other organisms offers a level of
inference of the same organelle localization of proteins in
humans [67]. There are two caveats to this approach, first,
only proteins that have homology to other species or are
conserved can be inferred; second phylogenetic analyses
may be difficult to specify subcellular localization for large
protein families such as SNAREs and Rab GTPases that
have diverse functions, structures and diverged evolution
[68]. It is thought that the mitochondrion was derived from
an endosymbiotic relationship with the primordial eukar-
yotic cell [69]. Rickettsia prowazekii, an a-proteobacterium, is
considered the closest living ancestor to the modern day
mitochondria and the homology of its constituent proteins
provides strong clues to the modern day mitochondrial
proteome (specificity 14%, sensitivity 30%). Similarly, the
genetically tractable Saccharomyces cerevisiae provides
experimental ease for the systematic identification of genes
in its relatively small genome, estimated to be 60%
similar to humans [66]. To achieve significant success in
identifying genes and known functionality in yeast, yeast
orthologs provide strong clues to mitochondrial protein
localization in higher organisms (specificity 35–47%,
sensitivity 40–48%) [70]. In addition, proteins targeted to the
mitochondrion generally possess N-targeting sequences
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Proteomics 2010, 10, 3935–3956
[71] that are co-expressed via transcriptional induction upon
mitochondrial biogenesis [72] or GFP-localization [73], and
these are useful information (data set) in identifying bona fide
mitochondrial proteins. Each data set is heterogeneous and
incomplete yet complementary; thus by integrating diverse
data sets of varying specificity and sensitivity using naı̈ve
Bayes, linear predictors or support vector machine (SVM),
proteins can be scored as a function of its probability as a
mitochondrial protein [67]. A predictive score, termed Maes-
tro was generated that permitted the ranking of the likelihood
of a truly mitochondrial protein. To validate their predictions,
localization of 131 predicted novel mitochondrial proteins
were demonstrated unambiguously to reside within the
mitochondrion [7]. Subtractive proteomics (Fig. 2E) was
employed to distinguish mitochondrial proteins from false
positives (non-mitochondrial co-purifying proteins such as
ER proteins). While the current MS lacks the sensitivity of
detecting lower abundance proteins and that the composition
of the mitochondrial proteome changes with its cell state,
proteomics alone will be insufficient in confirming the true
localization of proteins. Hence, large-scale integrative studies
that leverage various aspects of organelle biology are powerful
strategies to characterize the resident proteins in a highly
confident manner.
The phagosome or autophagosome, a crucial double
membrane-bound organelle in immune responses, apopto-
sis and tissue remodeling, is another organelle whose
protein components were expanded and identified through
the use of a combination of MS, RNA-mediated interference
(RNAi) and bioinformatics [74]. The elegant use of a systems
biology approach offered a powerful framework to under-
stand the dynamic nature of the Drosophila phagosome [36].
This set of data was used to build the interactome, which in
turn generated potential phagosome associating proteins
that were validated functionally. The authors extended the
macromolecular complex, exocyst to the phagosome, thus
further increasing the inventory of phagosomal proteins.
Such an effort enhances our understanding of proteome
composition during host–pathogen response and under-
scores the utility of integrative analysis in combination with
simpler fractionation techniques to confidently ascribe
proteins to their cellular localizations.
3 Bioinformatics prediction
Uncovering protein localization would help to annotate
genomes, infer potential roles and discover diagnostics and
drug targets [75]. However, annotation of protein localiza-
tion has not been keeping pace with the rapidly accumu-
lating genomic databases generated from large-scale
sequencing projects. A large proportion of proteins in the
Swiss-Prot and Gene Ontology databases do not have
experimentally validated localization annotations [76].
Many of the ‘wet-lab’ methods used to determine protein
localization are time consuming, expensive, laborious,
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subject to artifacts and/or provide low coverage of the full
proteome [77]. These experimental limitations have
encouraged the ongoing efforts to develop reliable compu-
tational predictors to analyze large-scale genome sequence
for protein localization [78–83]. These computational
methods are automated, faster and provide wider proteome
coverage and hints to the potential biological roles of
uncharacterized proteins to direct further experiments [84].
3.1 Sequence-based and annotation-based
classifiers
Many in silico prediction tools for subcellular localization
have been developed using different data sets and features,
and they are accessible via the Internet [85]. A comprehen-
sive summary of prediction methods was published by Chou
and Shen [86]. Some predictors predict for all cellular
compartments, but many are specific for one organelle or
organism. Organism-specific prediction tools have been
shown to perform better than those designed for general
eukaryotic organisms [78].
The two important factors in predicting subcellular
localization are the extraction of crucial biological features
relevant to subcellular localization, and the computational
method applied in the system. These computational
predictors are mainly grouped as sequence-based or anno-
tation-based classifiers [87]. Sequence-based predictors are
based on the amino acid sequence of the protein, by iden-
tifying protein sorting signals [88, 89] such as N-terminal
targeting peptides [79, 90–92], mitochondrial targeting
peptides [93], chloroplast transit peptides [94] and nuclear
localization signals [95] or analyzing whole sequence
features [96–98] such as amino acid composition [99, 100],
dipeptide composition [101] and gap amino acid pair
compositions [100]. Annotation-based methods detect func-
tional domains and motifs [81, 102, 103], textual information
in databases [104–108], homology search [109–111] or
phylogenetic profiling [103, 112].
There are hybrid predictor systems [96, 113] that applied
a mixture of strategies such as those based on sequence and
annotation [82] or sorting signals and sequence composition
[114, 115]. Some models incorporate results from different
modules to integrate signal peptide or whole sequence
information with features such as physical and chemical
properties [116] or protein domain information [117]. For
example, PSORT-B is based on several features including
signal peptide, amino acid composition, transmembrane
a-helices and motifs, and homology to proteins of known
location [118].
These hybrid systems are more robust and often achieve
higher prediction performance, and hence are preferred for
novel proteins. The incorporation of evolutionary informa-
tion with machine learning technique in RSLpred often
resulted in a better performance than other methods such as
TargetP, Wolf-PSORT, PA-SUB, Plant-Ploc and ESLpred
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3942 Y. H. Lee et al.
[119]. SherLoc2 is another hybrid subcellular localization
prediction system, which covers 11 main eukaryotic
subcellular locations for animal, fungal and plant proteins
with an estimated accuracy of 93% [120]. Small et al.
recommended the use of high-throughput screening tools
such as Predotar [92] and MITOPRED [121, 122] to identify
potential proteins for subsequent testing using hybrid
programs (e.g. SherLoc and PA-SUB) [123].
3.2 Sequence information
Users of predictors that are based on sequence information
should note the loss of order information as the whole
protein sequences are decomposed into the respective
composition. Methods that integrate sequence order with
amino acid composition would provide higher accuracy by
overcoming problems associated with information loss.
Shen and Chou developed a system based on pseudo-amino
acid composition, which reflects protein sequence order and
the amino acid composition [124]. Likewise, Cherian and
Nair showed that prediction based on biological features,
such as atomic composition, physiochemical properties and
amino acid composition, derived from the full-length
sequence performed better than those based on N-terminal
sequence alone [125].
3.3 Targeting signals
Many in silico prediction models are highly dependent on
targeting signal sequences [126]. Targeting signals are short
sequence of amino acid residues located mainly at the N-
terminal region to direct protein translocation to specific
cellular compartments such as mitochondria, nucleus,
chloroplast or peroxisome [95, 127–129]. Examples of these
predictors include MitoProt [93], ChloroP [94], SignalP [130]
and PredictNLS [95]. TargetP predictor [79] differentiates
secretory proteins, mitochondrial proteins and chloroplast
proteins by detecting the various targeting sequence using
Neural Networks approaches. However, the presence of
sequence motifs commonly used in the computational tools
is error prone, and some proteins contain cryptic targeting
sequences, internal targeting sequences or more than one
targeting signal. For example, mitochondrial proteins with
non-canonical targeting signals have been documented
[127]. Mitochondrial predictors perform well for mitochon-
drial matrix proteins, which contain N-terminal cleavable
leader sequence but predict poorly for inner mitochondrial
membrane proteins with internal or less-defined targeting
signals [131]. In the first extensive study of the whole
chloroplast proteome, only 60% of the identified proteins
were predicted to contain chloroplast transit peptide [132].
The complex protein translocation mechanism is not
completely understood and may involve factors other than
signal peptides, e.g. retention of protein within the Golgi
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apparatus. Not all organelle proteins contain sorting signals,
thus the major drawback of prediction models based on
targeting signals is that there are many subcellular sites for
which targeting sequence is not known, such as the nuclear
membranes and the Golgi apparatus. Post-translational
modifications and protein–protein interactions may also
influence protein localization.
3.4 Sequence homology
Besides targeting motifs, many computational techniques
[80, 133] are based on sequence homology with proteins
of known localization derived from the database. For
example, Comparative BLAST for Organelles (CBOrg) is a
homology-based method to predict subcellular localization
by screening large EST and/or genomic data sets [123].
However, such computational tools are unable to predict for
proteins with poor or no homology to proteins with anno-
tated localization [134].
3.5 Machine-learning approaches
Most of the computational methods applied machine-
learning approach such as artificial neural networks (ANN),
hidden Markov models (HMM) and SVM to predict protein
locations [78]. The aim of prediction tools is to predict well
on novel data that are not used to construct the model. SVM
is most commonly used and performs relatively better than
many others. Supervised learning algorithms such as ANN
[135], k-nearest neighbors [101, 136], Bayesian methods
[81, 111] and SVM [100, 126] that are used in prediction tools
are trained using a data set of experimentally validated
protein localizations, and these are mostly obtained from the
highly curated Swiss-Prot database [137]. Such systems have
the limitation of requiring a large annotated data set for
training of machine-learning technique, which is laborious,
expensive and time consuming. Recently, Xu et al. employed
a semi-supervised learning approach to design the predic-
tion method [138]. A small set of validated data is used first,
followed by refining the model using a large number of
testing data. By using only 30% annotated data, the authors
showed an enhancement of prediction results of SVM
classifiers.
3.6 Training data sets
Different in silico prediction programs are shown to generate
inconsistent and variable results between them. A main
reason for this is the lack of high quality data set to train the
algorithms. To overcome this, Park et al. developed a
consensus localization prediction method ConLoc that is
based on a large-scale, systematic integration of 13 programs
[139]. Using ConLoc, the authors built a localization guided
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Proteomics 2010, 10, 3935–3956
functional interaction network of human proteome to map
known disease associations and identify novel disease-asso-
ciated genes. However, this model can only predict for five
major subcellular localizations due to limited training and
testing data sets for other cellular compartments. Indeed,
the performance of prediction tools depends heavily on the
redundancy of data set used for training. The distribution of
proteins in different cellular locations is imbalanced, which
results in certain locations with a smaller number of train-
ing data. Many of the computational prediction methods
have limited location sites coverage or predicts for many
locations but with low accuracy. For example, due to the
small peroxisomal training data set, the performance of
predictions tools such as PSORT II [80], pTARGET [140] or
PTS1 predictor [141] have generated inconsistent data, even
for the known peroxisomal proteins.
In addition, the predictive performance of any computa-
tional methods is highly associated with the degree of
similarity between the training and test sets. One of the
limitations of prediction models is that the training data sets
encompass proteins with high sequence homology to others
located in the same cellular compartment. For example,
PSORT-B is a widely used prediction method but its training
set contains a strong homology bias. On the other hand,
Gneg-PLoc has a training set where none of the proteins in a
subcellular location has more than 25% sequence homology
to each other [142]. Unlike PSORT-B that covers five location
sites, Gneg-PLoc can predict eight subcellular location sites.
Despite the exclusion of homologous proteins and the
inclusion of more subcellular locations sites, Gneg-PLoc
achieves higher success rates than PSORT-B. Chou et al.
developed Cell-PLoc that contains six predictors: Euk-
mPLoc, Hum-mPLoc, Plant-PLoc, Gpos-PLoc, Gneg-PLoc
and Virus-PLoc, which are specialized for eukaryotic,
human, plant, Gram-positive and negative bacterial and viral
proteins, respectively [76]. None of the training proteins
had425% sequence identity to other protein in the same
subcellular location. A high accuracy was demonstrated by
Cell-PLoc based on cross-validation tests on the benchmark
datasets that involved up to 22 subcellular location sites.
3.7 Protein with multiple localizations
A large number of eukaryotic proteins, up to more than
35%, are localized at multiple subcellular locations [143,
144]. For example, some proteins are dual targeted to
mitochondria and plastids [145]. Proteins with multiple
locations or dynamic localizations are important in both
basic research and drug discovery. Proteome-scale epitope
tagging, immunolocalization strategies and improved
subcellular fractionation procedures have generated direct
evidence for the multiple localizations of many proteins.
One of the limitations of most existing computational
predictors of localizations is the inability to predict proteins
that are targeted to or traffic between two or more location
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sites [146]. Euk-mPLoc is one of the predictors developed
that can analyze eukaryotic proteins with multiple location
sites including those not annotated in the Swiss-Prot data-
base [147]. Recently, Lin et al. [148] described KnowPredsite,
a knowledge-based system, which can be used for proteome-
wide prediction of proteins that are single-localized or multi-
localized. They showed that KnowPredsite performed better
than the ngLOC and Blast-hit method, with an overall
accuracy of 91.7 and 72.1% for single-localized and multi-
localized proteins, respectively.
3.8 Organelle database
With the increasing number of proteins including novel
uncharacterized gene products being reported by subcellular
proteomics, there are currently a large number of publicly
accessible organelle databases [2, 149]. For example, there
have been a number of comprehensive organellar proteomic
studies being performed, and several systematic studies of
subcellular localizations using cellular fractionation have
been reported [150]. The proteome profiles of most organelles
including mitochondria [151], chloroplast [152] and nucleolus
[153] have now been identified [1]. However, many of the
proteome databases are still incomplete. For example, despite
the completion of the human genome sequence, the exact
number of protein-coding genes is unknown. Furthermore,
the exact genomic structure of human protein-coding genes
has only been correctly predicted for 50–60% of the genes
[154, 155]. Incomplete proteome datasets would thwart the
identification of novel organelle proteins from subcellular
proteomics studies. For example, several proteomics studies
of mitochondria such as those of yeast [156], human heart
[157] and mouse [158] have been performed but the exact
number of mammalian mitochondrial proteins is still
unknown as a fraction of the mitochondrial proteome has not
been identified [159, 160]. False positives in databases due to
contaminants from impure subcellular fractions would
further complicate organellar proteome analyses.
Other limitations of public-accessible databases include
variations in data sets and restricted data sets [161]. For
example, MitoProteome [162] is restricted to human records,
and thus has limitations to integrate results derived from
model organisms. On the other hand, MitoMiner is a public
database of experimental information from the published
mitochondrial proteome studies [161], and includes data sets
from seven species including Homo sapiens, S. cerevisiae and
Drosophila melanogaster to allow cross-species comparisons.
MitoP2 is a database of mitochondrial proteins of yeast,
mouse, A. thaliana, neurospora and human [163], and links
mitochondrial proteins to functional data sets and includes
information about mouse models and human diseases [164].
However, the incompleteness in proteome databases would
hinder inferences of proteins’ subcellular localization
between species such as in correlating model organism
proteome with the human proteome.
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3944 Y. H. Lee et al.
4 Validation of organelle-specific
candidate proteins
As discussed earlier, although advancement in methodolo-
gies and technologies has enabled us to obtain organelles in
purer fractions, hitherto no methods are available to frac-
tionate organelles to homogeneity. Thus cross-contamina-
tion of isolated fractions is often expected, and hence one of
the crucial steps involved in a successful subcellular frac-
tionation is the validation of the enrichment of specific
organelles of interest. Generally, this involved both
morphological and biochemical approaches to assess the
efficiency of enrichment [165]. In the biochemical approach,
each fraction is tested by Western blot or enzymatic assays
targeted at organelle-specific markers to determine the
fraction containing the organelle(s) of interest, and to probe
for potential contaminations. For the morphological
approach, detailed light and electron microscopy (EM)
analyses are used to monitor the degree of purity and
structural integrity of isolated organelles, while also visua-
lizing potential presence of contaminating structures. In
addition, the localization of endogenous proteins could be
validated using bioimaging methods, such as fluorescent
detection or immuno-EM [166]. Additionally, the localiza-
tion of novel or poorly characterized proteins must be vali-
dated with specific organelle markers to determine if the
protein of interest is a ‘contaminant’ or can be assigned to a
subcellular location.
4.1 Western blotting of known, established
organelle markers
The location of a protein can be determined by its co-puri-
fication with an established marker specific for the orga-
nelle, which is also known as an organelle marker. Co-
purification of the protein of interest with the marker occurs
during subcellular fractionation, and this can be verified
using Western blotting. Western blot experiments, for
example, are carried out to determine the expression levels
of organelle markers to confirm the partitioning of organelle
across the fractions to assess the purity of each fraction. The
immunoreactivity of known protein markers for organelle of
interest should be enriched in certain fractions, and
matched with reduced immunoreactivity for markers of
other compartments.
In the study of a single fraction, minor proteins could be
contaminants derived from another organelle containing
high abundance of this protein. Hence, when performing
Western blotting, it is also necessary to immunoblot for
proteins that could easily contaminate the fractions of
interest. For example, to show enrichment of ER proteins,
beside ER markers it is common to immunoblot for Golgi
and lysosomal protein markers. It should also be noted that
not all organellar proteomes are the same in different
tissues or cell types. For example, there exist substantial
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differences in cytosolic, nuclear, mitochondrial, membrane
proteome in different tissues [158, 167]. Hence, it may be
necessary to determine the distribution of organelle markers
in all subcellular fractions to fully assess the efficiency of the
fractionation procedures.
The selected organelle markers must have exclusive
subcellular locations. Examples of organelle markers
commonly used to determine the distribution of cellular
organelles in each fraction based on their protein content
and/or enzymatic activities include succinate dehy-
drogenase, voltage-dependent anion channel and TOM20
for mitochondria, glucose-6-phosphatase and BiPP/GRP75
for ER, alkaline phosphatase for plasma membrane,
glyceraldehyde 3-phosphate dehydrogenase for cytosol,
Lamp-1 and acidic phosphatase for lysosomes, catalase for
peroxisomes and histone H3 for nucleus. At times, multiple
markers for different sites of each organelle may be
performed to determine the integrity and purity of orga-
nelles. For example, Song et al. applied Western blot using
anti-VDAC (mitochondrial outer membrane), anti-cyto-
chrome c (intermembrane lumen) and anti-COXIV (inner
membrane) for mitochondria and anti-calnexin and anti-
KDEL for integral membrane and lumen of the ER,
respectively [168]. The authors assessed the purity, efficiency
and integrity of isolated organelles by using a variety of
methods including protein yield, immunoblotting and
transmission EM.
4.2 Multiple reaction monitoring
The chief limitation of immunological-based assays such as
Western blotting is the lack of commercial antibodies for
many proteins. Coupled with their semi-quantitative nature
and high costs for large-scale verification and validation
studies, multiple reaction monitoring (MRM) has recently
been suggested as an alternative for verifying the identities
of many proteins [169, 170]. Thus MRM is now well posi-
tioned for the detection and quantification of proteins that
are distributed across subcellular compartments and which
are often obscured by the more abundant proteins present
in complex biological matrixes. Transcription factors are
low-abundant nuclear proteins, and MRM has been used to
detect transcription factors such as NF-kB2 and STAT1 in
the breast cancer cell-line, MCF-7 [171]. Likewise, in the
complex human brain, MRM has been used to verify the
presence of L-ferritin in neuromelanin granules [172].
Importantly, MRM also allows the rapid transfer between
discovery and verification. For example, Agilent 6400 series
Triple Quadrupoles and 6500 series Quadrupole TOF
(Agilent Technologies, Santa Clara, CA) mass spectrometers
use the same ion optics and collision cells to derive the same
peptide fragmentation patterns, thus facilitating the
successful transfer of information across platforms as one
move from subcellular discovery/profiling assays to valida-
tion [173].
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Proteomics 2010, 10, 3935–3956
4.3 Assay for enzymatic activity of organelle
markers
Besides Western blotting, the other widely used practice of
evaluating the composition of subcellular fractions is the
measurement of enzymatic activities. Biochemical moni-
toring of subcellular fractionation requires at least one
reliable marker enzyme per organelle to determine the
biochemical homogeneity of each fractions. Evaluating the
amount and activity of characteristic enzyme markers in
different cellular compartments will allow quantitative
assessment of the fractions obtained. It should be noted that
measurement of enzymatic activity is semi-quantitative and
subjected to possible interference from in vitro environment.
Likewise, the criterion for marker selection is similar to
immunoblotting verification. However, the presence of
activity may not indicate intact organelles. For example, the
presence of succinate dehydrogenase activity could indicate
intact mitochondria or fragments of mitochondrial inner
membrane. In order to confirm the presence of intact
mitochondria, morphological evidences or assay of markers
of outer mitochondria and intermembrane space must also
be performed.
Recently, Andreyev et al. fractionated RAW264.7 cells
into six fractions that represent the major organelles (nuclei,
mitochondria, cytoplasm, endoplasmic reticulum, plasma
membrane and a dense microsomal fraction) [174]. The
authors validated these subcellular fractions by analyzing
the distribution of enzymatic markers. Subsequently, they
identified 50-member organelle-specific ‘‘marker ensem-
bles’’ for each subcellular compartments using quantitative
LC-MS proteomics. The quantitative composition of the
fractions was based on distribution of the marker ensem-
bles. The use of panels of markers avoided the problem of
multiple locations of some markers, thereby preventing bias
from single ‘‘best’’ protein, and the difficulty in obtaining
completely pure organelle fractions.
4.4 Fluorescent microscopy
Novel localization assignments by organellar proteomics can
be validated by visualizing the subcellular protein locations
using the orthogonal technique of fluorescence microscopy.
This method can be applied to show co-localization of target
protein with the known organellar markers. Endogenous or
recombinantly expressed proteins can be detected immu-
nochemically or by tagging with a recombinant epitope or
fluorescent protein. One of the most widely used methods
for this is transfection of cells with cDNA clones fused to a
fluorescent reporter such as the GFP [175]. GFP is widely
used in bioimaging as this protein can be easily tagged to
almost any protein to determine its localization. The loca-
tion of proteins fused to non-fluorescent tag, such as FLAG,
could be determined using a fluorescent-labeled antibody
targeted to the tag, known as indirect immunofluorescence.
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While fluorescence microscopy is of low throughput, since it
is mostly targeted to one protein of interest each time and
being semi-quantitative, and thus less suitable for large-
scale quantitative proteomics, it is useful in validating a
small number of protein localizations. For example, GFP
fusions and confocal microscopy was used to image the
localization of several uncharacterized target proteins to
validate subcellular assignments obtained from organellar
proteomics [6, 158, 167].
More importantly, some studies have demonstrated
that the presence of tags can induce artifacts such as
mislocalization by obscuring targeting motif, or interfering
with folding or protein–protein interactions [176–179]. The
tag may contain localization signal that overwhelms
the original targeting sequence information of the
protein [180, 181] or become activated in different species
[182, 183]. The addition of tag may result in differential
processing or interfere with post-translational modifications
of protein, thus changing the localization [184]. Moreover,
overexpression of tagged fusion proteins to determine
subcellular localization of gene products has been
reported [178, 185] which often results in mislocalization.
Changes in protein abundance that are dependent on
different stimuli or conditions are also likely to be lost with
overexpression.
To circumvent the problem of overexpression, the fluor-
escence-based verification technique termed CD-tagging can
be applied to verify proteomic data. CD-tagging is a random
tagging approach whereby the coding sequence of GFP is
inserted randomly into a genomic DNA, generating a large
library of subcellular images after all proteins are tagged
[186]. In contrast to cDNA modification that uses constitu-
tive promoter and results in higher expression level, this
method retains the regulatory sequence and expression level
of the protein [187]. Chromosomally integrated GFP
constructs under the control of endogenous promoter would
lead to low protein expression [75, 188]. Limitations of CD-
tagging include the need for complete coding sequence of
cDNA, such as inclusion of signal peptides for correct
localization and the generation of antibodies for novel gene
products can be difficult and time consuming.
In addition to CD-tagging, there are other methods
designed to minimize the problems of overexpression.
New generation vectors have been designed to include:
(i) Flp recombination target site that facilitates rapid
isogenic integration in cell lines containing a single FRT
(flanked with heterologous recombinase target) site [189];
(ii) Tetracycline induction that allows the control of
expression levels [190]; (iii) A tag that facilitates protein
detection. These new generation vectors combined with
large ‘‘orfeome’’ collections are a very powerful system
[191–193]. The use of these vectors circumvents the pitfalls
of CD tagging, that requires the generation of new anti-
bodies, while at the same time allowing the scientist to
express the tagged protein uniformly and at close to
physiological levels.
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3946 Y. H. Lee et al.
Strategies that could avoid the possible artifacts of protein
fusions include the use of fluorescent probes or antibodies
conjugated with fluorescent dyes to visualize protein locali-
zation within organelles. There are several available fluor-
escent markers for cell imaging such as the ERTracker,
MitoTracker, LysoTracker, DAPI or phallicidin for ER,
mitochondria, lysosome, nuclei and filamentous actin
cytoskeleton, respectively. However, these approaches
require cells that are fixed and permeabilized for probe
entry, which thus disallow temporal study of protein loca-
lization. The fixation step may mask antigens or alter the
conformation of proteins. Fluorescence-based methods also
have the disadvantages of pH- and concentration-dependent
fluorophore quenching and photobleaching. Other
problems include non-specific binding and aggregation of
organelles by antibodies.
Of note, in SILAC-based experiment on adenovirus-
infected HELA cells showed that a subset of proteins enri-
ched in the nucleolus was detectable by proteomics but not
detected by fluorescence microscopy [194]. This highlighted
that some proteins may be present in such low abundance
that they are below the limit of the current microscopic
detection methods. This could be due to the fact that
fluorescence-based microscopy focuses on single cells,
whereas proteomics analyses are based on purified orga-
nelles obtained from millions of cells. Despite certain
limitations most laboratories have access to fluorescence
microscopy to perform spatial confirmation of at least
several proteins.
4.5 Large-scale protein localization
Large-scale protein localization experiments based on GFP
tagging or immunofluorescence are an important step
forward in complementing and validating cellular spatial
organization of proteins [185, 195] despite its high cost. In
the first comprehensive study of proteome-wide protein
localization of a eukaryotic system using systematic GFP
tagging and microscopy, 4156 S. cerevisiae proteins were
localized to one or more of 22 subcellular organelles or
compartments [75]. High-throughput screening of GFP-
tagged proteins has also been reported in yeast on a
genome-wide scale [196]. Natter et al. studied localization
pattern of proteins involved in lipid metabolism in S. cere-
visiae by tagging 493 proteins with GFP and using high-
resolution fluorescence microscopy [197]. The results for
many proteins with known localization were consistent with
other reports using cell fractionation or large-scale localiza-
tion approaches. Barbe et al. reported the pilot large-scale
antibody-based study of 466 proteins in 3 human cell lines
using fluorescent-labeled antibodies and confocal micro-
scopy [198]. They generated approximately 3000 high-reso-
lution images, which are available on the Human Protein
Atlas portal. Using these techniques, they showed that
480% of these proteins could be classified in one or
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multiple subcellular compartment(s), and many corre-
sponded to Gene Ontology localization prediction model.
They used three organelle probes: a DNA stain, DAPI for
the nucleus, tubulin for cytoskeleton as well for internal
control for fixation quality and homogeneity and calreticulin
for the endoplasmic reticulum. In summary, these large-
scale protein localization approaches are well adapted to
evaluate the large volumes of data generated from proteo-
mics and to validate their cellular locations.
4.6 Automated assignment of localization using
fluorescent microscopy
Traditionally, protein locations are assigned using visual
inspection and manual evaluation which often contained
bias and varied with the experimenter’s training and
experience. Automated, high-throughput fluorescent
microscopy can generate images for tens of thousands of
fluorescent-tagged proteins, which is well suited for the
validation of high volumes of data generated from large-
scale proteome studies [199–201]. In addition, publicly
available databases of images for protein localizations would
provide information about organellar locations and help to
assign locations to unknown proteins in organelle proteo-
mic experiments.
Systematic study of protein locations in cultured cells has
been accomplished by coupling fluorescence microscopy
with pattern recognition and machine-learning techniques,
such as ANN and SVM [202, 203]. Methods for systematic
study of protein locations commonly extract subcellular
location features from images to recognize subcellular
patterns [199]. There are pattern recognition systems avail-
able to describe new location patterns or subtle changes.
Cluster analysis of images derived from a library of tagged
protein clones would group proteins into location families.
Clustering of images has also been used to group drugs
effects upon subcellular patterns [204]. This method has also
allowed building of generative models to create protein
localization images for the proteome, unlike the conven-
tional microscopy that focused on a few proteins each time
[205]. High-resolution microscopy images and training sets
with sufficient number of well-annotated proteins are
required to allow proper determination of protein location.
In particular, the recent technological advances in auto-
mated imaging have dramatically improved the speed of
bioimaging [206, 207]. Newberg et al. developed a system to
assign automatically subcellular locations on a proteome-
wide scale for collections of tissue images such as the
Human Protein Atlas [208]. The Human Protein Atlas
currently contained images for 46000 proteins [209].
One of the current challenges is to apply such compu-
tational method for all proteins in all cell types in different
conditions. Time-lapse images are difficult to acquire since
many microscopes do not maintain a viable environment for
cell cultures. Repeated excitations of the fluorescent dyes
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Proteomics 2010, 10, 3935–3956
would also cause photobleaching, reduced signal and
phototoxicity to the cells. To circumvent such challenges, a
system to infer correlation of subcellular protein localization
with cell cycle by analyzing only static, asynchronous images
using a one-dimensional manifold computed on simple
nuclear image features was developed. For example, Buck
et al. associated protein pattern variation in asynchronous,
static cell images with cell cycle progression [210]. Helmuth
et al. also presented a novel, model-based image analysis
algorithm to reconstruct outlines of subcellular structures
using a sub-pixel representation [211]. They validated the
reconstruction performance on synthetic data and applied to
fluorescence microscopy images of endosomes. This new
algorithm allowed quantification of endosome morphology
and capture the dynamics of endosome fusion. While large-
scale antibody-based protein localization study provides
valuable temporal and spatial information, such work is
often limited by antibody specificity [212].
4.7 Electron microscopy
EM allows the direct morphological characterization of the
samples, providing valuable information on the purity and
integrity of the enriched organelle of interest. EM uses
particle beam of electrons to illuminate and produce a
magnified image of a specimen. EM enables examination of
samples with a high magnification and a resolution of
approximately 1 nm. Additionally, the location of a protein
can be localized to the organelles using immuno-EM to
confirm fluorescence microscopic data.
One application of EM in organellar studies is to analyze
the isolated mitochondria with regards to structural integrity
of the inner and outer membranes and mitochondrial
matrix, so as to distinguish between intact and damaged
mitochondria [213]. Owing to its high resolution, EM can
detect the presence of double layer membrane and matrix.
Subsequently, functional integrity of the organelle was
determined based on assay for JC-1 dye uptake. Immuno-
gold particles staining and EM was also used to confirm the
protein localization along and/or in the mitochondrial inner
and outer membranes.
Electron microscopic visualization of target proteins in
the sample is achieved by electron optical contrast, which
involves selective electron scattering of the atoms of sample
and stain. Several staining procedures have been employed
in EM such as immunoantibody staining using colloidal
gold or enzymes such as horse-radish peroxidase, and
enzyme cytochemistry which target site of enzyme activity in
situ. Although EM allows a direct assessment of organelle
ultrastructure, this method is often criticized for being time
consuming and inconvenient as it requires skilled operators
and access to sophisticated equipments, and permitting the
study of small quantities of sample each time. Artifacts of
EM, such as shrinkage and precipitation are also widely
known. However, due to its direct, visual comparison of
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samples, EM will remain an important technique to
conclusively demonstrate sufficient purity of enriched
organelles.
4.8 RNAi
The correlation of decreased protein expression in specific
organelles upon RNAi knockdown of target proteins can
also be used to determine the validity of the protein location
assignment. Coene et al. studied the localization of BRCA1
using confocal and immune-EM to show that BRCA1 is
present in mitochondria of several cancer cell lines as well
as primary breast and nasal epithelial cells [214]. They
showed that siRNA knockdown of BRCA1 in human cancer
cells led to decreased nuclear, cytoplasmic and mitochon-
drial immunofluorescence staining, validating the data
obtained from the microscopic analyses. The authors also
applied subcellular fractionation, dephosphorylation and
enzyme protection experiments to show the enrichment of a
phosphorylated isoform of BRCA1 in mitochondrial and
nuclear subcellular fractions. Using these independent
approaches, they showed that phosphorylation relocalization
of BRCA1 plays a role in the maintenance of genome
integrity in mitochondria and nucleus.
Many studies have reported nuclear cytoplasmic shuttl-
ing of tumor suppressor APC. Using immunofluorescence
microscopy, cell fractionation, siRNA and Cre/Fox silencing
technology, Brocardo et al. found that some commonly used
antibodies against APC showed non-specific nuclear stain-
ing pattern in immunofluorescence despite being effective
in immunoblotting [215]. Despite inactivation of APC
expression by siRNA or Cre/Flox, staining of APC was still
observed by some antibodies in immunofluorescence
microscopy. Their results refuted the hypothesis that APC
nuclear export is inhibited by truncation of C-terminal NES,
but demonstrated that this mutant of APC shuttle between
nucleus and cytoplasm. This study demonstrated the need
for several independent validations and cautions in inter-
preting localization from immunofluorescence microscopy
alone.
5 Perspectives: Challenges of organelle
purification exclusiveness or dynamic
localization?
It is becoming increasingly clear that organelles are dynamic
cellular entities, remodeling and interfacing with other
organelles at different stages of development, physiological
conditions and tissues [146]. Translocation of certain
proteins may also be crucial during the development of
diseases, and thus comparative analysis of the dynamic
changes in organellar proteome would aid in understanding
the molecular mechanism of diseases. It has been observed
that proteins can be dual or multiple localized in different
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3948 Y. H. Lee et al.
organelles. It has been reported that 39% of proteins are not
unique to one cellular compartment but exhibit multiple
localizations [37, 216] and this distribution of proteins
complicates organellar proteomics.
Indeed, one of the factors that contributed to the
conflicting results obtained from different subcellular
proteomics studies is shuttling of proteins between cellular
compartments. This includes proteins that translocate
between organelles, shuttle between cytoplasm and nucleo-
plasm or cycle between subcellular pools. A well-known
dynamic proteome system is the endomembrane system
where proteins trafficking en route to their final destination
are in a constant state of flux through several organelles.
Intracellular trafficking of endosomes and secretory vesicles
occurs continuously between the Golgi and ER to achieve
dynamic equilibrium [217, 218]. Isolation of these organelles
involved in the endomembrane system is challenging due to
the similar buoyant densities of vesicular structures. The
dynamic exchange of materials between vesicles would also
alter their densities [219]. Some plasma membrane proteins
are endocytosed and then retrieved to the plasma
membrane. For example, subtypes of glucose transporters
shuttle from intracellular pools to cell surface upon insulin
signaling [220]. Isolation and analysis of a single organelle is
thus unable to fully understand the dynamic processes
between organelles. To understand the dynamic change of
the proteome at the subcellular level, several subcellular
structures should be monitored in parallel [8].
In fact, comparative analysis at the subcellular level is
necessary to confirm spatial rearrangement of proteins to
provide insights into their biogenesis and cross-talk. It is a
challenge to study the biological significance of proteins
found in multiple localizations in different conditions,
particularly with a complex intracellular communication
between organelles [156]. There have been new develop-
ments in proteomics to facilitate the study of organelle
dynamics in a high-throughput manner. For example,
Andersen and colleagues applied SILAC on the human
nucleoli and in vivo fluorescent imaging to characterize the
flux of endogenous nucleolar proteins in response to three
metabolic inhibitors known to affect nucleolar morphology
[221]. They demonstrated that the nucleolar proteome
exhibited temporal compositional and kinetic changes in
response to different cellular growth conditions. These data
suggested that there is no unique proteome for any orga-
nelle (nucleoli at least); instead the proteomes overlap in
different cell states or conditions and this showed the
significance of determining the relative abundance of
proteins in different cellular organelles in different physio-
logical states. Qattan et al. combined sucrose density-gradi-
ent subcellular fractionation with quantitative, MS/MS-
based shotgun proteomics to study the distribution pattern
of proteins fractionated from MCF-7 breast cancer cells
[222]. Out of the 2184 identified proteins, 1249 proteins
possessed multiple location sites of which many of these
multi-localized proteins were implicated in breast cancer.
& 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Proteomics 2010, 10, 3935–3956
This led to the authors proposing that the dynamic change
in protein localization in different compartments may have
functional roles in breast cancer.
However, different studies sometimes throw up inter-
esting and conflicting results and alternative conclusions are
suggested. For instance, PCP argues that GAPDH and
aldolase, both predominantly cytosolic glycolytic enzymes
are not localized to the mitochondrion [37]. In contrast,
other proteomic studies (MudPIT, 2-D native PAGE and 3-D
denaturing PAGE) have suggested that these two enzymes
and glucokinase (another glycolytic enzyme) physically
interact with the mitochondrion [157, 223]. Not surprisingly,
organelle preparation may be the early causes of such
arguments. Alternatively, the different physiological states
at which the samples were sampled might cause this
discrepancy.
Another mitochondria-related controversy is catalase.
Catalase is thought to be confined to the peroxisomal matrix.
Indeed in the most complete mitochondrial proteome
compendium catalogued, catalase is considered a non-
mitochondrial protein [7]. However, catalase has been found
in rat heart mitochondria [224]. More interestingly, yeast
catalase (catalase A) has been noted to distribute to the
peroxisome as well as mitochondrion, a phenomenon
dependent on the carbon source [225]. Do take note that
yeast homology is considered part of the strategy in several
mitochondrial protein annotative studies [7, 65]. Therefore
this reiterates that the state of the cell’s physiology at a
particular point in time determines the fraction of proteins
that are distributed across the various compartments.
The interconnectivity of organelles and their subsequent
exchange of proteins is increasingly thought to heavily
influence cell function, and their dysregulations can lead to
pathological conditions. To facilitate Ca21 exchange, the
mitochondria are organized in networks physically linked to
the ER [226]. The interaction between the ER and mito-
chondria is important in modulating the Ca21 signaling for
apoptosis and cell metabolism, and mutations in Ca21
signaling can lead to Charcot-Marie-Tooth-type 2a neuro-
pathy, type 2 diabetes and obesity [227, 228]. As such, this
highlighted the need to consider the pathological and
biological state when analyzing organellar proteomics data
and do not simply regard the presence of other organellar
proteins as contaminants. Autophagy is another highly
complex biological mechanism that reflected the dynamism
of protein localization. Since autophagy engaged the lyso-
some and vacuole with the target organelle or cellular
structure destined for degradation [229], it is foreseable that
when autophagy is induced, an intertwining of the phago-
cytic and the to-be-degraded organelle proteomes would
occur. Selective autophagy of organelles is known to
degrade peroxisomes, secretory granules and mitochondria
[230, 231]. For example, Brunner and colleagues observed a
large number of lysosomal proteins and attributed their
presence to the degradation and sorting of old and imma-
ture insulin granules respectively [175]. It has been
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Proteomics 2010, 10, 3935–3956
demonstrated in vitro that mitochondrial autophagy has a
role during hepatocyte remodeling [232]. Proteomic studies
in this area is still lacking, perhaps due to the difficulty in
‘locking’ this dynamic biological event for sampling, but we
expect this area of organellar proteomics to grow rapidly
since there is increasing evidence of the contribution of
dysfunction lysosomal activities in human diseases [233].
The dynamic localization of proteins could also be
resulted from post-translational modifications (PTMs) of
proteins. Spatial and temporal PTMs of proteins are
important aspects in the intracellular signaling systems
associated with human physiology. For example, PTMs of
mitochondrial proteome due to changes in cell’s redox
status and increase in reactive oxygen and nitrogen species
are known to play key roles in signal transduction, and also
disruption of mitochondrial functions in cancer and
Parkinson’s disease [234]. Thus, proteomics analysis of
oxidative and nitrosative stress on mitochondria would
identify redox-based mitochondrial proteome alterations
that are associated with the disease progression [235–237].
Phosphorylation of proteins is another well-established PTM
that results in redistribution of the modified proteins into
different subcellular compartments, as well as regulation of
signal transduction [238, 239]. The development of proteo-
mics technologies to detect PTMs of organelle proteome that
contributes to the translocation of proteins between
subcellular locations is an important aspect to be addressed.
6 Concluding remarks
Organellar proteomics aims to determine protein localiza-
tion and the associated biological functions of these
proteins. Classical subcellular fractionation methods that
have been widely applied in many studies to isolate cellular
organelles have limited resolution, thus resulting in erro-
neous assignment due to cross-contaminating proteins.
Despite the developments in bioinformatics and computa-
tional tools to predict localization of proteins, these are not
totally reliable and may not be applicable for all gene
products. It is thus necessary to validate assignment of
protein localization derived from subcellular fractionation
and in silico predictions using independent techniques.
However, the lack of high-throughput methods for this
purpose is a bottleneck for the validation of proteomics data.
It has been decades since cellular organelles were studied
using microscopy and subcellular fractionation but protein
cataloguing of every cellular compartment is not yet
complete. One of the challenges in this field is to fully
characterize all subcellular proteomes, and understand the
dynamic changes in protein localization in response to
various perturbations. With the recent advancement of new
proteomics technologies and strategies, such as FFE,
immunopurification, FAOS, LOPIT, PCP and subtractive
proteomics, which are designed specifically for the
systematic isolation and characterization of various subcel-
& 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
3949
lular proteomes, it is expected that new and valuable data
will be obtained soon.
The authors have declared no conflict of interest.
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