Báo cáo khoa học: Proteome analysis at the level of subcellular structures Mathias Dreger pot

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 as

M I N I R E V I E W

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 andproteomics, andthus is termedÔsubcellular

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 increasedcomplexity of organisms is

realizedat 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 associatedwith

subcellular structures only in certain physiological states,

but localizedelsewhere 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 andintracellular pools or

shuttling between nucleoplasm andcytoplasm In many

cases, initial states of developing diseases are likely to be

characterizedby 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 kindof analysis as it is suitedto the way in which cells are organized

Deficits of the classic proteome analysis approach What is termedhere the Ôclassic approachÕ in proteomics

is characterizedby 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 basedon protein spot patterns These patterns may be comparedbetween two samples obtainedfrom the investigatedsystem 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.

(Received12 September 2002, accepted12 December 2002)

representedproportional 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

performedto visualize more proteins [6], the approach still

remains blindtowards the cellular architecture andthus 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

establishedin biochemistry-orientedlaboratories for d

ec-ades The efficiency of the subcellular fractionation was

assessedbasedon the determination of marker enzyme

activities, anda major analytical goal was the identification

of single new proteins specifically localizedto 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 limitedsensitivity, or even impossible

This changedwith 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 optimizedfor single structures preparedfrom 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 andmay not work in a different one To give an example: under conditions in which neuroblastoma neuro 2a cells are lysedto prepare intact nuclei devoidof 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 wouldalready be severely damaged

Problems of this kindhave to be kept in mindwhen 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 investigatedat the level of the endogenous gene products, but will also yielda 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 requiredto validate the findings.

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 selectedgene

products by indirect immunofluorescence

For studies on dynamic changes of the proteome at

subcellular level, there is a strong needfor the optimization

of preparation protocols, as several subcellular structures

have to be monitoredin 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 listedin a

recent review by Jung andHochstrasser [9]

Insteadof 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 usedto 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 basedon 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 performedto compare cell

proteomes in different physiological states to point out

specific problems andpotentials when studiedat the

subcellular level

The gain in information yielded by subcellular

proteomics studies, in which protein chemical methods

are combinedwith establishedcell biological methods

such as indirect immunofluorescence or

immunoelec-tron microscopy, will be pointedout in this

mini-review

Except for the nuclear pore complex (NPC), which can

be preparedbasedon 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

simplifiedschematic representation of a cell nucleus is

shown in Fig 2 Insteadof representing a nonstructured

container for the chromatin, nuclei contain functionally distinct substructures like the nucleolus, the nuclear speckles, coiledbodies andsome more (for a review see [10]), many of which were discoveredbasedon electron microscopy andthe distribution of single specific marker proteins The nuclear architecture is thought to

be relatedto 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, andDNA [13] Furthermore, the nuclear envelope contains the NPCs, multiprotein complexes that enable the cell to exchange molecules between nucleus andcytoplasm [14]

Both subnuclear structures andnuclear multiprotein complexes have been subject to proteomic analysis The analysis of the mammalian spliceosome ([15], see also accompanying minireview by Bauer andKu¨ster) repre-sentedan exemplary study for the whole fieldof 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 isolatedby 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 publishedlater by Rout et al [17], a structural model of the yeast spindle pole could be derived from the data

Various subnuclear structures andcomplexes have been analysedin 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 investigatedby sub-cellular proteomics studies, are indicated.

Nuclear pore complex (NPC)

In a comprehensive study Rout et al [17] identified

probably all core components of the yeast NPC A

preparation highly enrichedin yeast nuclear core complexes

was separatedby three different liquidseparation 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

correspondedto uncharacterizedopen reading frames were

expressedandlocalizedby indirect immunofluorescence In

total, 40 gene products were assigned to be associated with

the NPC Others representedproteins that were either

assumedto be contaminants derivedfrom other structures,

or protein with unknown relation to the NPC The

localization of 27 taggednucleoporins 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 interpretedthe 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 reportedproteome analysis alone This interpretation highlights important features of informations retrievedby 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 coveredby 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 andinner nuclear membrane (ONM andINM, respectively), the pore mem-brane, the NPCes andthe nuclear lamina [13] These subcompartments differ with respect to their protein components, but there is no methodby which the nuclear membranes can be separatedfrom each other Dreger et al [18] therefore useda strategy of alternative extraction of the raw nuclear envelope preparation from mouse neuroblastoma neuro 2a cells, to prepare different nuclear envelope substructures characterizedby 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 seconddimension, 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 expressedproteins.

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 expressedproteins.

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 expressedproteins.

Many new nucleolar proteins; discovery

of new compartment ÔparaspecklesÕ.

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

andpost source decay fragmentation of selectedpeptides

Within each fraction, identified known proteins were

grouped according to literature data on their subcellular

localization (Fig 3B) andaccording 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 couldbe predictedaccordingly LUMA andmurine 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), andcontainedputative 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 taggedversions of the proteins in transiently transfected cells followedby 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), andlamin 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) andin the chaotrope-resistant fraction (U/C) groupedaccording to their subcellular localization INM, inner nuclear membrane; ER/ ONM, endoplasmic reticulum/outer nuclear membrane; L/M, nuclear lamina andattachedprotein scaffold; NPC, nuclear pore complex; CS, cytoskeleton; Mito, mitochondria Note the differences in the distribution of ER/ONM, L/M and NPC proteins.

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-od s to valid ate the results is always required

Interchromatin granule clusters

Interchromatin granule clusters (IGCs) are microscopically

definedsubnuclear structures associatedwith 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 presumedIGC-relatedprotein complexes with a

known IGC protein as the bait 2D gel electrophoresis was

usedfor visualization of proteins enrichedin the IGC

preparation as comparedto other subnuclear fractions

Thus, the display of the protein pattern was used to monitor

proteins that were coenrichedandwere 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 transfectedcells

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 andassembly of ribosomal subunits

Andersen et al preparedhighly purifiednucleoli from

human HeLa cells Proteins were separatedandanalysed

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 anddifferent buffer systems were usedto separate the

proteins This was followedby gel slicing, tryptic digestion

andnano-LC/MS analysis Here proteins couldbe covered

that escapedanalysis 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 hadto be assignedto 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 assessedby indirect

immunofluores-cence of cells transiently transfectedwith DNA encoding

taggedversions of these gene products Two of the newly

discovered gene products, as assessed by indirect immuno-fluorescence andimmunoelectron 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 comparedto the relatively straightforwardprepar-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 performedby triton X-114 phase partioning, with the triton-soluble fraction in the focus of the analysis Both Bell

et al [23] andTaylor et al [24] succeeded in the identifica-tion of new gene products of which one, termed either GPP34 [23] or GMx33 [25], was unamibigiously localizedto the Golgi apparatus as a peripheral membrane protein using immunoelectron microscopy In addition, Wu et al [27] reportedupregulation of a number of Golgi proteins in Golgi preparations from rat mammary glandcells in the state of maximal secretion at lactation as comparedto 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 andmass 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 andJagow useda 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 followedby two-dimensional separation of the elutedfractions of the preparative gel was reportedby Werhahn andBraun [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 limitedresolution or limiteduse for very complex samples However, they share the basic idea that the information

content of proteomic screens couldbe extendedby

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 characterizednot by containing specifically localized

proteins, but are characterizedby 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 monitoredby 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, secretedorganelles 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 separatedfrom other vesicular organelles by means of

free-flow electrophoresis, andthat the whole population of

identifiedproteins servedto 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 secretedvesicles 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 comparedto

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 aimedat

the comparative analysis of tissue samples, e.g prepared

from CNS structures Tissue samples are more complex

than samples from culturedcells 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

targetedby proteomic analysis in various studies Walikonis

et al [36] analysedproteins 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 andthat contain pre- andpostsynaptic

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 achievedby different density centrifugation steps subse-quent to the lysis of the synaptosomes, andby detergent extraction of membrane proteins not boundto 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 localizedprotein as well as a few mitochondrial contaminants were identified in addition to known key postsynaptic proteins A similar analysis was preformedby Satoh et al [37] who separatedtheir 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 aforementionedstudy andin line with the assumedunderrepresentation of integral membrane pro-teins on 2D gels

Phillips et al [38] reportedthe preparation of specific presynaptic structures andthe preparative separation of the presynaptic membrane from the postsynaptic membrane Only a few selectedproteins have been identifiedin 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 yieldsignificant insight into biological mechanisms In this section I wouldlike to point out analytical aspects andpotentials specific to the analysis at the subcellular level

Microsomal fractions are comprisedof membrane vesi-cles that spontaneously form during cell homogenization They do not represent distinct cellular organelles; they are

of heterogenous origin andmay 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] usedmicrosomes from HL60 cells, a human acute myeloidleukemia cell line that is culturedin 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 andthe test sample are alkylatedby 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 pooledandproteo-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 andanalysedby LC-MS Peptides with the same amino-acidsequence derivedfrom the two samples will

differ in mass due to the mass difference between the

nondeuteratedandthe deuteratedICAT reagent As these

ICAT pair peptides behave chemically the same during

chromatography andmass 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 cleavedandanalysedby

liquidchromato-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 comparedwith the phorbol ester-stimula-tedsample This study highlightedsome 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 andmultispanning integral membrane proteins (Fig 4B), which are thought to be

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 Decreasedabundance of proteins may

be due to reduced protein synthesis, but also due to

signal-induced protein degradation or signal-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 monitoredin parallel the nucleoplasmic

andthe 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

obtainedby classic two-dimensional gel electrophoresis

Concluding remarks With the option to identify large numbers of proteins rather than single proteins specifically localizedto particular structures, the combination of subcellular fractionation andprotein identification, in other terms Ôsubcellular proteomicsÕ, can be usedas a multifunctional tool in cell biology The first line of information (andthe best-establishedapproach) is the discovery of novel gene products and their assignment to subcellular structures A secondline of information is the characterization of subcellular structures basedon their entire protein popula-tion in addipopula-tion to known physical and biochemical properties of these structures As it starts from subcellular fractions andis basedon the identification of endogenous proteins in functional contexts, this approach is comple-mentary to recent molecular biology-basedstudies to systematically probe the subcellular localization of large numbers of gene products As examples for such molecular biology-basedapproaches, see [43] for the systematic assessment of the subcellular localization of gene products basedon 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 basedon overexpression of taggedgene 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 methodhas its potentials anddrawbacks, so it will be interesting to compare data on the same subcellular structure obtainedby different approaches

A strategy to acquire a thirdline 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 andalteredprotein–protein interactions Major requirements are the simultaneous preparation andanalysis of different subcellular structures andthe 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 andtime-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 shouldbe collectedin 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 andidentifying 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 andheavy ICAT reagents, respectively; an ICAT reagent is

cov-alently attachedto 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-atedby microcapillary high performance liquidchromatography A

pair of ICAT-labeled peptides are chemically identical and are easily

visualizedbecause they essentially co-elute andthere 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 maintainedin

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,

measuredandfragmentedresulting 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 identifiedandquantifiedin this study were classifiedby broad

functional criteria The numbers in parentheses indicate the percentage

fraction of identified proteins represented by each category Some

proteins are representedin more than one category.

I wouldlike to thank Dr Chris Weise andStephanie Williams for

critically reading this manuscript I would also like to thank Dr Ruedi

Aebersoldfor providing graphic material for Fig 4 This work was

supportedby the German ministry for research andeducation andby

the Deutsche Forschungsgemeinschaft.

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