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 different subsets of cells with distinct properties, for example neurons, germ cells, or epithelial cells. Secondly, within a given cell, functions such as storage of genetic material, degradation of proteins, or the provision of energy-rich metabolites to fuel cellular reactions are compartmentalized. Different subcellular compartments contain different and compartment-specific subsets of gene products in order to provide suitable biochemical environments, in which they exert their particular function. The identification of subsets of proteins at the subcellular level is therefore an initial step towards the understanding of cellular function. There are subsets of proteins that are associated with subcellular structures only in certain physiological states, but localized elsewhere in the cell in other states (for examples, see [1,2]). Among the possible mechanisms that underlie such conditional association, there is protein translocation between different compartments, cycling of proteins between the cell surface and intracellular pools or shuttling between nucleoplasm and cytoplasm. In many cases, initial states of developing diseases are likely to be characterized by translocation events that precede altera- tions in gene expression. For comparative studies, in order to elucidate the molecular basis of biological processes, the analysis of dynamic changes of the subcellular distribution of gene products is necessary. In order to be able to monitor these changes, the classic proteome analysis approach must be modified. Performing proteomics at a subcellular level is an appropriate strategy for this kind of analysis as it is suited to the way in which cells are organized. 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: saihtam@chemie.fu-berlin.de Abbreviations: NPC, nuclear pore complex; NE, nuclear envelope; IGC, interchromatin granule cluster; ICAT, isotope-coded affinity tag; PSD, postsynaptic density; LC, liquid chromatography. (Received 12 September 2002, accepted 12 December 2002) Eur. J. Biochem. 270, 589–599 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03426.x represented proportional to their abundance. Furthermore, the analysis of post-translational modifications like protein phosphorylation requires a complex repertoire of analytical tools [4]. There are also limitations with respect to the dynamic range of proteins that can be displayed on a gel [5]. This problem increases with sample complexity. The classic approach may fail in the discovery of gene products that are major proteins of particular subcellular compartments, but are minor proteins of the whole crude homogenate. Even if sequential extractions of crude homogenate samples are performed to visualize more proteins [6], the approach still remains blind towards the cellular architecture and thus also towards protein translocation events, and therefore inevit- ably will miss significant alterations in the proteome. Characteristics of subcellular proteomics strategies Fractionation techniques to isolate distinct subcellular compartments have been among the standard strategies established in biochemistry-oriented laboratories for dec- ades. The efficiency of the subcellular fractionation was assessed based on the determination of marker enzyme activities, and a major analytical goal was the identification of single new proteins specifically localized to the subcellular structure. However, due to the limited power of protein identification techniques in traditional protein chemistry, the systematic characterization of the protein subsets specific to subcellular compartments was time-comsuming, 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). In contrast to the classic proteome analysis approach, no unifying experimental procedure applies to the analysis at the subcellular level. In most cases, the preparation of subcellular structures is optimized for single structures prepared from distinct sources. Apart from the subcellular structure to be isolated, the rest of the preparation is usually regarded as waste. A standardized preparation protocol, working with every experimental system, does not exist. The preparation conditions may only refer to a particular cell line and may not work in a different one. To give an example: under conditions in which neuroblastoma neuro 2a cells are lysed to prepare intact nuclei devoid of other organelles [7], pheochromoytoma PC12 cells remain largely intact. Under conditions suited for the isolation of nuclei from this cell line [8], neuro 2a nuclei would already be severely damaged. Problems of this kind have to be kept in mind when studies on the same subcellular structures prepared from Fig. 1. Subcellular proteomics as a functional genomics strategy. The comprehensive identi- fication of the proteins present in the prepar- ation may reveal true previously unknown components of the structure investigated at the level of the endogenous gene products, but will also yield a certain amount of false-posi- tives, depending on the degree of impurities derived from other subcellular structures pre- sent in the preparation. Classic cell biological methods as well as sequence analyses by bio- informatics tools are required to validate the 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 particular marker protein but are distinguished from other structures based on the description of there entire proteome. Many proteomic studies deal with tissue samples. A number of proteomic studies have targeted synaptic structures of the CNS. As their study is central to the understanding of the molecular basis of the function of the nervous system, studies on synaptic structures like the postsynaptic density will be covered in this minireview. Finally, exemplary studies will be mentioned in which subcellular fractionation was performed to compare cell proteomes in different physiological states to point out specific problems and potentials when studied at the subcellular level. The gain in information yielded by subcellular proteomics studies, in which protein chemical methods are combined with established cell biological methods such as indirect immunofluorescence or immunoelec- tron microscopy, will be pointed out in this mini- review. Except for the nuclear pore complex (NPC), which can be prepared based on subcellular fractionation without affinity purification, the issue of analysis of multiprotein complexes will be discussed in an accompanying minireview [9a]. Proteome analysis of subnuclear structures: the functional architecture of a complex organelle The functional architecture of the nucleus of eukaryotic cells is one of the central topics in current cell biology. A 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 entire genome is sequenced. A similarly exemplary study was the analysis the spindle pole complex of yeast [16], which was isolated by subcellular fractionation. Here, the power of the combination of mass spectrometric identi- fication of numerous novel gene products followed by immunoelectron microscopic subcellular localization of tagged versions of these gene products was demonstrated. Similar as in the case of the nuclear pore complex analysis published later by Rout et al.[17],astructural model of the yeast spindle pole could be derived from the data. Various subnuclear structures and complexes have been analysed in a number of recent studies which are reviewed in the following sections (Table 1). Fig. 2. Schematic representation of a mammalian cell nucleus. Different subnuclear structures, some of which have been investigated by sub- cellular proteomics studies, are indicated. Ó FEBS 2003 Subcellular structure level proteome analysis (Eur. J. Biochem. 270) 591 Nuclear pore complex (NPC) In a comprehensive study Rout et al. [17] identified probably all core components of the yeast NPC. A preparation highly enriched in yeast nuclear core complexes was separated by three different liquid separation systems as the first separation dimension and SDS/PAGE as the second dimension. Proteins were identified both using mass spectrometric peptide mass fingerprints as well as fragment ion spectra containing partial sequence information of selected peptides. In total, 174 different proteins were identified. A total of 34 gene products that at that time corresponded to uncharacterized open reading frames were 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 proteomic data alone. Nuclear envelope (NE) The nuclear envelope comprises an outer and inner nuclear membrane (ONM and INM, respectively), the pore mem- brane, the NPCes and the nuclear lamina [13]. These subcompartments differ with respect to their protein components, but there is no method by which the nuclear membranes can be separated from each other. Dreger et al. [18] therefore used a strategy of alternative extraction of the raw nuclear envelope preparation from mouse neuroblastoma neuro 2a cells, to prepare different nuclear envelope substructures characterized by the presence or absence of substructure-specific marker proteins (Fig. 3A). The protein subsets present in these fractions were identified separately. The methods applied for separation and Table 1. Selected subnuclear proteomics studies. Subnuclear structure New proteins Total proteins Preparative approach Separation and identification technique Additional techniques Major outcome Nuclear pore 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. 2DE for enrichment monitoring, direct protein digestion, LC/MS. Immunofluorescence with transiently expressed proteins. New IGC proteins. Nuclear envelope (NE) (neuro 2a cells, mouse-derived) [18] 19 147 Subcellular fractionation, alternative extraction of the NE preparation. BAC-SDS/PAGE peptide mass fingerprints. Post-source decay. Immunofluorescence with transiently expressed proteins. Assignment of novel proteins within NE; two new INM proteins. Nucleolus (HeLa cells, human-derived) [21,22] 84 271 Nucleolus preparation by subcellular fractionation. 2D, several 1D 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 nuclear membrane as integral membrane proteins. This was independently confirmed by heterologous expression of tagged versions of the proteins in transiently transfected cells followed by indirect immunofluorescence using confo- cal laserscanning microscopy. However, the accuracy of a Fig. 3. Isolation and characterization of nuclear envelope subfractions. (A) Distribution of the marker proteins calnexin (outer nuclear membrane/ endoplasmic reticulum membrane), lamina-associated polypeptide 2b (LAP 2b, inner nuclear membrane), and lamin B1 (nuclear lamina) throughout the different nuclear envelope subfractions. Calnexin is absent from the TX-100-resistant fraction; lamin B1 is almost absent from the chaotrope-resistant fraction. (B) Distribution of NE proteins in the different fractions. Selected proteins detected in the TX-100-resistant NE fraction (Tx) and in the chaotrope-resistant fraction (U/C) grouped according to their subcellular localization. INM, inner nuclear membrane; ER/ ONM, endoplasmic reticulum/outer nuclear membrane; L/M, nuclear lamina and attached protein scaffold; NPC, nuclear pore complex; CS, cytoskeleton; Mito, mitochondria. Note the differences in the distribution of ER/ONM, L/M and NPC proteins. Ó FEBS 2003 Subcellular structure level proteome analysis (Eur. J. Biochem. 270) 593 prediction made based on the proteomic data is consider- ably reduced by the presence of contaminants derived from other subcellular structures, as well as by the Ôresolution of the studyÕ which is determined by the availability of different subfractionation procedures. The use of independent meth- odstovalidatetheresultsisalwaysrequired. Interchromatin granule clusters Interchromatin granule clusters (IGCs) are microscopically defined subnuclear structures associated with enhanced transcriptional activity [10]. Mintz et al. [20] addressed these structures in a proteome analysis approach using either a particular subfractionation procedure or immunoaffinity isolation of presumed IGC-related protein complexes with a known IGC protein as the bait. 2D gel electrophoresis was used for visualization of proteins enriched in the IGC preparation as compared to other subnuclear fractions. Thus, the display of the protein pattern was used to monitor 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 identified proteins. The efficiency of this analytic approach is demonstrated by the very high number of 271 identified proteins in the preparation of which only a very low percentage had to be assigned to contaminants. More than 30% of the identified gene products were previously unknown or uncharacterized, 82 of them were termed Ônovel nucleolar proteinÕ. The subcellular localization of several of them was assessed by indirect immunofluores- cence of cells transiently transfected with DNA encoding tagged versions of these gene products. Two of the newly discovered gene products, as assessed by indirect immuno- fluorescence and immunoelectron microscopy using anti- bodies that recognize the endogenous proteins, defined a new subnuclear structure, termed Ôparaspeckle compart- mentÕ [22]. This finding represents an example for the identification of novel subcellular structures driven initially by a proteomic approach. Proteomic analysis of small organelles and vesicles Golgi apparatus A number of proteomic studies have been conducted on other cellular organelles such as the Golgi apparatus and peroxisomes. The work on the Golgi apparatus is mentioned here as it has been subject to several proteomic studies designed to create a Golgi complex protein map [24,25]. The particular problem of the preparation of the Golgi appar- atus, as compared to the relatively straightforward prepar- ation of nuclei, is that the procedure comprises a series of density centrifugation steps as the physical properties of the material differ minimaly from those of, e.g. microsomal 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 preparative electrophoresis followed by two-dimensional separation of the eluted fractions of the preparative gel was reported by Werhahn and Braun [30]. Using sucrose density centrifugation as a first dimension, Hanson et al.[31]aimed to create a Ôthree dimensional protein mapÕ of the mito- chondrial proteome. Both methods are either restricted by limited resolution or limited use for very complex samples. However, they share the basic idea that the information 594 M. Dreger (Eur. J. Biochem. 270) Ó FEBS 2003 content of proteomic screens could be extended by addressing protein interactions in one of the separation dimensions. This differs from the analysis of multiprotein complexes subsequent to affinity purification. Vesicles charcterized on the basis of comprehensive proteome analysis There are a number of studies on vesicular structures that are characterized not by containing specifically localized proteins, but are characterized by a particular protein population as determined by proteomic approaches. One example is the analysis of phagosomes [32], organelles that occur upon phagocytic internalization of foreign material by macrophages. In this analysis, in addition to the description of the phagosome proteome, the maturation of the organelle was monitored by comparative analysis of phagosomes in different stages. The authors demonstrated that the phagosomes acquire cathepsins, key catabolic enzymes of mature phagosomes, in a sequential manner during pahgosome maturation. There has also been a proteomics approach to charac- terize exosomes, secreted organelles that, among potential 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 preparation starts from the isolation of synaptosomes, vesicles that form spontaneously upon homogenization of nervous tissue and that contain pre- and postsynaptic structures. The final PSD preparation contains postsynaptic neurotransmitter receptors as well as their anchoring proteins together with the underlying cytoskeleton and docked signalling molecules. The enrichment of the PSD is achieved by different density centrifugation steps subse- quent to the lysis of the synaptosomes, and by detergent extraction of membrane proteins not bound to the PSD. A total of 24 different proteins were identified by mass spectrometry subsequent to 1D gel electrophoretic separ- ation of the PSD fraction. However, at least one presynap- tically localized protein as well as a few mitochondrial contaminants were identified in addition to known key postsynaptic proteins. A similar analysis was preformed by Satoh et al. [37] who separated their PSD fraction in two dimensions and detected difference spots depending on synaptic activity. In total, 47 different proteins were identified. However, subunits of ionotropic glutamate receptors, which are key PSD proteins, were not detected, in contrast to the aforementioned study and in line with the assumed underrepresentation of integral membrane pro- teins on 2D gels. Phillips et al. [38] reported the preparation of specific presynaptic structures and the preparative separation of the presynaptic membrane from the postsynaptic membrane. Only a few selected proteins have been identified in this study, many of which can be assigned to the presynaptic side of the synapse. It will be interesting to observe what the 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 by application of the mixture directly or subsequent to further prefractionation to an affinity column loaded with monomeric avidin. Bound peptides are then eluted from the column and analysed by LC-MS. Peptides with the same amino-acid sequence derived from the two samples will Ó FEBS 2003 Subcellular structure level proteome analysis (Eur. J. Biochem. 270) 595 differ in mass due to the mass difference between the nondeuterated and the deuterated ICAT reagent. As these ICAT pair peptides behave chemically the same during chromatography and mass spectrometric analysis, the ratio of their intensities in the mass spectra is a semiquantitative measure for the abundance of the proteins they are derived from. Microsomes from control cells or differentiated cells were isolated, the proteins were labelled by the ICAT reagents, proteolytically cleaved and analysed by liquid chromato- graphy/MS (Fig. 4A). The analysis of ICAT pairs yielded semiquantitative information on more than 400 microsomal proteins, of which several displayed a differential abundance in the control as compared with the phorbol ester-stimula- ted sample. This study highlighted some important aspects concerning the interpretation of data obtained from com- parative proteomics at the subcellular level. Firstly, virtually all classes of proteins were represented, including regulatory proteins like protein kinases and multispanning integral membrane proteins (Fig. 4B), which are thought to be 596 M. Dreger (Eur. J. Biochem. 270) Ó FEBS 2003 underrepresented on classic 2D gels [3] (see [41] for contrasting data). Secondly, the question arises, which ratio of abundance of a particular protein is considered as a true 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 structures, the combination of subcellular fractionation and protein identification, in other terms Ôsubcellular proteomicsÕ, can be used as a multifunctional tool in cell biology. The first line of information (and the best- established approach) is the discovery of novel gene products and their assignment to subcellular structures. A second line of information is the characterization of subcellular structures based on their entire protein popula- tion in addition to known physical and biochemical properties of these structures. As it starts from subcellular fractions and is based on the identification of endogenous proteins in functional contexts, this approach is comple- mentary to recent molecular biology-based studies to systematically probe the subcellular localization of large numbers of gene products. As examples for such molecular biology-based approaches, see [43] for the systematic assessment of the subcellular localization of gene products based on the heterologous overexpression of GFP fusion proteins derived from cDNA libraries, and [44] for the systematic assessment of the subcellular localization of yeast gene products based on overexpression of tagged gene products. In order to detect the subnuclear localization of gene products at the endogenous expression level, a gene trap approach with the introduction of a reporter tag into endogenous genes in embryonic stem cells has been used [45]. Each method has its potentials and drawbacks, so it will be interesting to compare data on the same subcellular structure obtained by different approaches. A strategy to acquire a third line of information derived from subcellular proteomics studies is still in the beginning: 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 http://npd.hgu.mrc.ac.uk/, 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 light and heavy ICAT reagents, respectively; an ICAT reagent is cov- alently attached to each cysteinyl residue in every protein. The protein mixtures are combined, proteolyzed to peptides, and ICAT-labeled peptides are isolated utilizing the biotin tag. These peptides are separ- ated by microcapillary high performance liquid chromatography. A pair of ICAT-labeled peptides are chemically identical and are easily visualized because they essentially co-elute and there is an eight dalton mass difference measured in a scanning mass spectrometer (four m/z units difference for a doubly charged ion). The ratios of the original amounts of proteins from the two cell states are strictly maintained in the peptide fragments. The relative quantification is determined by the ratio of the peptide pairs. Every other scan is devoted to fragmenting and then recording sequence information about an eluting peptide (tandem mass spectrum). The protein is identified by computer searching for the recorded sequence information against large protein databases. In theory, every peptide pair in the mixture is, in turn, measured and fragmented resulting in the relative quantitation and identification of mixture proteins in a single analysis. (B) Categories of proteins identified from HL-60 cell microsomal fraction. The 491 proteins identified and quantified in this study were classified by broad functional criteria. The numbers in parentheses indicate the percentage fraction of identified proteins represented by each category. Some proteins are represented in more than one category. Ó FEBS 2003 Subcellular structure level proteome analysis (Eur. J. Biochem. 270) 597 Acknowledgements I would like to thank Dr Chris Weise and Stephanie Williams for critically reading this manuscript. 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