MINIREVIEW
Proteome analysis at the level of subcellular structures
Mathias Dreger
Institute for Chemistry/Biochemistry, Free University Berlin, Germany
The targeting of proteins to particular subcellular sites is an
important principle of the functional organization of cells at
the molecular level. In turn, knowledge about the subcellular
localization of a protein is a characteristic that may provide a
hint as to the function of the protein. The combination of
classic biochemical fractionation techniques for the enrich-
ment of particular subcellular structures with the large-scale
identification of proteins by mass spectrometry and bio-
informatics provides a powerful strategy that interfaces cell
biology and proteomics, and thus is termed Ôsubcellular
proteomicsÕ. In addition to its exceptional power for the
identification of previously unknown gene products, the
analysis of proteins at the subcellular level is the basis for
monitoring important aspects of dynamic changes in the
proteome such as protein transloction. This review sum-
marizes data from recent subcellular proteomics studies with
an emphasis on the type of data that can retrieved from such
studies depending on the design of the analytical strategy.
Keywords: subcellular proteomics; mass spectrometry;
organelle; synapse; nucleus; membrane protein; functional
genomics.
Introduction
With the increasing degree of complexity, organisms acquire
a broader repertoire of options to meet enviromental
challenges. This increased complexity of organisms is
realized at two levels: firstly, not all cells of the organism
serve the same purpose; the organism contains several

Deficits of the classic proteome analysis approach
What is termed here the Ôclassic approachÕ in proteomics
is characterized by a one-step sample preparation from a
crude homogenate followed by two-dimensional electro-
phoretical protein separation in order to display the whole
body of expressed proteins within the studied system under
the given physiological conditions. This approach bears the
advantage of a very fast and easily reproducible sample
preparation. It theoretically provides a complete overview
over all proteins in the sample based on protein spot
patterns. These patterns may be compared between two
samples obtained from the investigated system under
different physiological conditions. There were three basic
assumptions on which the expectations of the approach
were grounded: (a) the separation system is capable of
representing all proteins of the sample, (b) all proteins may
not only be visualized, but also identified (including their
post-translational modifications), and (c) biological proces-
ses manifest as changes in gene expression and/or identifi-
able post-translational modifications that affect the
migration behaviour of the protein on the 2D gel. Despite
the exceptional analytical power of this approach, system-
atic limitations of the approach at the present state of the
technology became apparent. There are certain classes of
proteins, such as integral membrane proteins, that are not
Correspondence to M. Dreger, Institute for Chemistry/Biochemistry,
Free University Berlin, Thielallee 63, 14195 Berlin, Germany.
Tel.: + 49 30 83852232,
E-mail:
Abbreviations: NPC, nuclear pore complex; NE, nuclear envelope;

of limited sensitivity, or even impossible.
This changed with the introduction of peptide mass
spectrometry along with the availability of comprehensive
protein and DNA databases that made easy and quick
protein identification feasible. The analytical tools that are
available nowadays allow the identification of many
proteins in a single experiment. This enables systematic
studies that are designed to describe the proteome of the
whole subcellular entity. In spite of the large overlap with
traditional approaches with respect to the subcellular
fractionation protocols, this change of the scope of the
protein analytical studies at the subcellular level now
justifies the introduction of the term Ôsubcellular proteo-
micsÕ.
The scheme in Fig. 1 summarizes several characteristics
of the subcellular proteomics approach. As a feature unique
to this experimental approach, subcellular proteomics
allows the mapping of the components of particular
subcellular structures at the level of the endogenous
proteins. In addition, the identification and subcellular
assignment of previously unknown gene products at the
level of the endogenous protein is feasible. However, with
respect to the completeness or ÔcoverageÕ of the proteome,
there will be limitations due to the differential abundance of
proteins similar to the situation in classic proteome analysis
experiments. Due to the presence of gene products derived
from other subcellular structures than the one investigated,
the subcellular assignment of newly discovered gene
products requires validation by independent techniques
such as immunocytochemistry (see below).

findings.
590 M. Dreger (Eur. J. Biochem. 270) Ó FEBS 2003
different sources are to be compared. This problem also
highlights the need for independent validation methods in
subcellular proteomics studies. This may be achieved, e.g. by
assessing the subcellular localization of selected gene
products by indirect immunofluorescence.
For studies on dynamic changes of the proteome at
subcellular level, there is a strong need for the optimization
of preparation protocols, as several subcellular structures
have to be monitored in parallel.
The scope of this minireview is to present data obtained
from exemplary studies that can be described as Ôsubcellular
proteomicsÕ.
Not all recent studies dealing with the identification of
proteins of subcellular structures can be mentioned, nor can
there be a reasonable effort to review all the classic papers
that describe subcellular fractionation protocols, as there
are hundreds, if not more. A number of studies that address
the proteomes of subcellular compartments are listed in a
recent review by Jung and Hochstrasser [9].
Instead of pointing out unifying strategies, this
minireview covers exemplary studies which, depending
on the approach, contain different kinds of information
exceeding the mere identification of proteins. These
comprise of studies on different part of the nucleus of
eukaryotic cells to demonstrate how proteome analysis
can be used to elucidate the functional architecture of
cell nuclei. These also comprise of studies on vesicle-like
organelles, including structures that up to now lack one

simplified schematic representation of a cell nucleus is
shown in Fig. 2. Instead of representing a nonstructured
container for the chromatin, nuclei contain functionally
distinct substructures like the nucleolus, the nuclear
speckles, coiled bodies and some more (for a review
see [10]), many of which were discovered based on
electron microscopy and the distribution of single specific
marker proteins. The nuclear architecture is thought to
be related to the epigenetic control of gene expression.
Some of the structures seem to be dynamic, and the
overall nuclear structure appears distorted in transformed
cells [12]. The nuclear envelope not only represents a
barrier which separates the genetic information from the
cytosol, but also may take part in the regulation of
chromatin structure through binary or ternary contacts
between proteins of the inner nuclear membrane, of the
nuclear lamina, and DNA [13]. Furthermore, the nuclear
envelope contains the NPCs, multiprotein complexes that
enable the cell to exchange molecules between nucleus
and cytoplasm [14].
Both subnuclear structures and nuclear multiprotein
complexes have been subject to proteomic analysis. The
analysis of the mammalian spliceosome ([15], see also
accompanying minireview by Bauer and Ku
¨
ster) repre-
sented an exemplary study for the whole field of
subcellular proteomics as it demonstrates the analytical
power of the approach, especially the efficiency of protein
identification by mass spectrometry in an organism whose

expressed and localized by indirect immunofluorescence. In
total, 40 gene products were assigned to be associated with
the NPC. Others represented proteins that were either
assumed to be contaminants derived from other structures,
or protein with unknown relation to the NPC. The
localization of 27 tagged nucleoporins within the NPC
structure was determined by immunoelectron microscopy.
Aided by literature data, a detailed structural model for the
yeast NPC was proposed.
Apart from the gene products assessed in more detail,
Rout et al. interpreted the significance of the identification
of the other proteins in three ways: firstly, there are proteins
that according to the literature are known NPC interactors,
e.g. transport factors with a role in nucleocytoplasmic
transport. Second, there are mere contaminants like
subunits of the mitochondrial ATP synthase. Third, there
are proteins that likely will turn out to be new transient
nucleoporin interactors, but this issue cannot be addressed
on the basis of the reported proteome analysis alone.
This interpretation highlights important features of
informations retrieved by a subcellular proteomics
approach: Firstly, there are findings on known proteins
that confirm literature data. Secondly, there are findings on
known proteins that are not covered by the literature, but
that are additionally validated in the respective study by
classic cell biological tools. Thirdly, there remains a body of
information of unknown or speculative significance. This is
likely to contain new significant information on the
subcellular structure investigated, but also likely contains
artifacts. Therefore no decision can be taken based on the

complex (NPC)
(yeast) [17]
34 174 NPC preparation by
subcellular fractionation.
Alternative LC
SDS/PAGE as
second dimension,
peptide mass
fingerprints. CID.
Protein tagging,
immunoelectron
microscopy.
Structural model
of NPC.
Spindle pole (yeast)
[16]
11 23 Subcellular fractionation. SDS/PAGE,
peptide mass
fingerprints.
Protein tagging,
immunoelectron
microscopy.
Structural model
of spindle pole.
Interchromatin
granule clusters (IGC)
(mouse liver) [20]
3 36 Subcellular fractionation,
WB: enrichment of
markers.

systems
Immunofluorescence
with transiently
expressed proteins.
Many new nucleolar
proteins; discovery
of new compartment
ÔparaspecklesÕ.
592 M. Dreger (Eur. J. Biochem. 270) Ó FEBS 2003
identification of the proteins were the two-dimensional
protein separation by the 16-BAC-/SDS/PAGE system [19],
followed by standard methods of mass spectrometric
protein identification based on peptide mass fingerprinting
and post source decay fragmentation of selected peptides.
Within each fraction, identified known proteins were
grouped according to literature data on their subcellular
localization (Fig. 3B) and according to features of their
primary structures as determined by bioinformatic analysis
tools. The distribution of identified proteins over the
different fractions analyzed allowed a tentative assignment
of nuclear envelope proteins to NE substructures without a
physical preparation of the substructure. The subcellular
localization of novel identified gene products in this study
could be predicted accordingly. LUMA and murine
KIAA0810 were the only previously unknown gene pro-
ducts that behaved like integral membrane proteins (chao-
trope-resistance), nuclear lamina-interacting proteins
(Triton X-100-resistance), and contained putative trans-
membrane regions within their primary structures. These
proteins were thus predicted to reside within the inner

proteins that were coenriched and were candidates for
colocalization within the same subnuclear structure. Using
Western blot analysis subsequent to 1D-separation of the
proteins, the enrichment of known IGC residents was
monitored. Protein identification was performed by an
LC-MS strategy subsequent to direct proteolytic digest of
the preparation and 36 different gene products were
identified. Among these, three previously unknown IGC-
associated protein were identified. The subcellular localiza-
tion was validated by indirect immunofluorescence of
transiently transfected cells.
Nucleolus
Numerous different separation and analysis methods have
been used in the recent study by Andersen et al.[21]to
explore the proteome of the nucleolus, a subnuclear
structure which is known to be the site of synthesis of the
ribosomal RNA and assembly of ribosomal subunits.
Andersen et al. prepared highly purified nucleoli from
human HeLa cells. Proteins were separated and analysed
according to two major strategies: first, classic 2D gel
electrophoresis was conducted, spots were picked and the
respective proteins identified by peptide mass fingerprinting
of the tryptic digests. Second, different 1D SDS/PAGE
methods using different gradients of acrylamide concentra-
tion and different buffer systems were used to separate the
proteins. This was followed by gel slicing, tryptic digestion
and nano-LC/MS analysis. Here proteins could be covered
that escaped analysis on classic 2D gel electrophoresis, e.g.
because of their basic pI values. The use of different
separation systems yielded partially nonoverlapping sets of

material [26]. Further fractionation of the Golgi preparation
was performed by triton X-114 phase partioning, with the
triton-soluble fraction in the focus of the analysis. Both Bell
et al. [23] and Taylor et al. [24] succeeded in the identifica-
tion of new gene products of which one, termed either
GPP34 [23] or GMx33 [25], was unamibigiously localized to
the Golgi apparatus as a peripheral membrane protein using
immunoelectron microscopy. In addition, Wu et al.[27]
reported upregulation of a number of Golgi proteins in
Golgi preparations from rat mammary gland cells in the
state of maximal secretion at lactation as compared to that
in a state of basal secretion. This upregulation was observed
at the protein level by comparison of protein patterns
displayed by classic 2D gel electrophoretic separation of
proteins from the Golgi preparation.
Mitochondria
A number of studies have been performed using 2D gel
electrophoresis and mass spectrometric protein identifica-
tion to create two-dimensional protein maps for mitochon-
dria (for a review see [28]). However, in a number of studies
concerning the mitochondrial proteome strategies were used
that address additional aspects of the proteome. As early as
1991, Scha
¨
gger and Jagow used a native gel system for the
separation of intact protein complexes in the first dimension
and SDS/PAGE under denaturing conditions as the second
dimension to display the components of the complexes
[29]. A similar approach with three separation dimensions
using Blue native electrophoresis as the first dimension in

other functions, may play a role in the immune response
[33]. A special feature of this analysis was that the exosomes
were separated from other vesicular organelles by means of
free-flow electrophoresis, and that the whole population of
identified proteins served to distinguish exosomes from
apoptotic vesicles.
There have been a number of other proteome analysis
studies to characterize vesicular organelles based on their
entire proteome. One example is the proteomic character-
ization of prespore secreted vesicles of Dictyostelium discoi-
dum [34,35].
A common theme of these studies is the requirement of a
comprehensive proteome analysis in order to acquire an
image of the organelle investigated. This highlights the
unique potential of subcellular proteomics as compared to
other, more traditional approaches, where the analysis was
designed to identify single specifically localized proteins.
Subcellular proteomics at the tissue level:
tackling the synapse
Many current proteome analysis projects are aimed at
the comparative analysis of tissue samples, e.g. prepared
from CNS structures. Tissue samples are more complex
than samples from cultured cells as any tissue contains
many different cell types and contains structural material
like connective tissue that may not be the target of the
analysis.
Samples derived from synaptic structures have been
targeted by proteomic analysis in various studies. Walikonis
et al. [36] analysed proteins present in the classic post-
synaptic density (PSD) preparation from rat brain. This

outcome of a detailed proteome analysis of this fraction will
be.
Special aspects of comparative studies
at the subcellular level
In addition to the description of the proteome of a
subcellular entity, the analysis of dynamic proteome chan-
ges at a subcellular level promises to yield significant insight
into biological mechanisms. In this section I would like to
point out analytical aspects and potentials specific to the
analysis at the subcellular level.
Microsomal fractions are comprised of membrane vesi-
cles that spontaneously form during cell homogenization.
They do not represent distinct cellular organelles; they are
of heterogenous origin and may contain, e.g. material from
the endoplasmic reticulum and other cytosolic organelles.
However, they are a source for membrane proteins that can
be easily and quickly prepared. In a comparative study, Han
et al. [39] used microsomes from HL60 cells, a human acute
myeloid leukemia cell line that is cultured in suspension, but
that upon certain stimuli (e.g. phorbol ester) differentiates
into an adherent form, to detect alterations in the micro-
somal fraction upon cell differentiation by the application of
the isotope-coded affinity tag (ICAT) technique. In this
technique, the proteins of the control sample and the test
sample are alkylated by the cysteine-specific biotinylated
ICAT reagent in its nondeuterated or in an eightfold
deuterated form, respectively [40]. Subsequent to alkylation,
the proteins from both samples are pooled and proteo-
lytically cleaved. Peptides that carry the cysteine-specific
modification can be isolated from the whole peptide mixture

quantitative difference. Many of the identified proteins
differ by a ratio of around two, which is not considered a
significant difference by the authors. Thirdly, as one
particular subcellular fraction has been analysed, Han et al.
point out several mechanisms that can account for the
increased or decreased abundance of particular proteins in
the preparation dependent on the status of cellular differ-
entiation. There may be upregulation due to increased
protein synthesis, but there may also be signal-induced
translocation of proteins towards cellular membranes,
which accounts for the occurrence of these proteins in the
microsomal fraction. Decreased abundance of proteins may
be due to reduced protein synthesis, but also due to signal-
induced protein degradation or signal-induced detachment
of proteins from the microsomal membranes.
If the biochemical mechanism of the alterations in the
subcellular proteome is to be addressed, it is necessary to
monitor several different subcellular fractions in parallel.
An example for such a study is given by Gerner et al.[42]in
their study of Fas-induced apoptosis in Jurkat T-lympho-
cytes. The authors monitored in parallel the nucleoplasmic
and the cytosolic fraction of the cells. Their data suggested
signal-induced entrance of the protein TCP-1a into the
nucleus as well as translocation of nuclear annexin IV from
the nucleus to the cytosol, as deduced from the comparative
analysis of the protein pattern of the respective fractions
obtained by classic two-dimensional gel electrophoresis.
Concluding remarks
With the option to identify large numbers of proteins rather
than single proteins specifically localized to particular

the study of dynamic changes at the subcellular level, e.g.
upon protein translocation and altered protein–protein
interactions. Major requirements are the simultaneous
preparation and analysis of different subcellular structures
and the development of strategies for the simultaneous
display of many different protein interactions at an appro-
priate resolution.
With an increasing number of subcellular proteomic
studies, most of them directed to the discovery of novel
gene products, the need arises for storage of data in
organelle databases. In typical studies, more than one
hundred different proteins are identified. As the functional
investigation of novel gene products is much more difficult
and time-consuming than protein identification, only a few
will be subject to further research by the research group
that identified the gene product. To prevent loss of
information on the other detected gene products, this
information should be collected in a publicly accessible
database. One such example is the Nuclear Protein
Database at which contains
information on nuclear proteins from many different
studies.
In summary, subcellular proteomics may be more than
separating proteins on gels and identifying them by mass
spectrometry. Depending on the design of the study,
functional insight into cellular processes may be obtained.
Fig. 4. Comparative subcellular proteome analysis of microsomal
membranes using the ICAT method. (A) The ICAT strategy for quan-
titating differential protein expression. Two protein mixtures repre-
senting two different cell states have been treated with the isotopically

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