Insights into a plasma membrane signature

SHERYL HARVEY, YAN ZHANG, FRANCE LANDRY, COLEEN MILLER and JEFFREY W. SMITH

Program on Cell Adhesion, Cancer Research Center, The Burnham Institute, La Jolla, California 92037


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The plasma membrane (PM) is an organized biological system that serves as a structural barrier and communication interface with the extracellular environment. Many basic questions regarding the PM as a system remain unanswered. In particular, we do not understand the scope of similarity and differences in protein expression at the PM. This study takes an initial step toward addressing these questions by comparing the PM proteomes of fibroblasts and mammary carcinoma cells. Three sets of proteins were revealed by the study. The first set comprises between 9 and 23% of all proteins at the PM and appears to be common to both fibroblasts and mammary carcinoma. A second group of proteins, comprising ~40% of the proteins at the PM, is tightly linked to cell lineage. The third set of proteins is unique to each cell line and is independent of cell lineage. It is reasonable to hypothesize then, that this third group of proteins is responsible for unique aspects of cell behavior. In an effort to find proteins linked to the metastatic phenotype, we identified several proteins that are uniquely expressed at the PM of the metastatic MDA-MB-435 cells. These proteins have functions ranging from cell adhesion to the regulation of translation and the control of oxidant stress.

proteome; expression profiling; breast cancer; metastasis


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
AS EARLY as 1953, Nordling (19) suggested that the clinical manifestations of cancer result from the cumulative effect of multiple genetic mutations. The multifactorial nature of tumor progression precludes its explanation by any single factor or even a small group of factors. It has yet to be determined how many alterations must occur to convert a normal mammary epithelial cell to carcinoma or how many additional changes are necessary for the carcinoma to progress to invasive disease. Answers to these questions require global examinations of biological systems.

This study focuses on the plasma membrane (PM) as a system. The PM regulates many aspects of tumor progression including cellular differentiation, cell proliferation, adhesion and migration, pericellular proteolysis, and even the escape from immune surveillance. Each of these events represents a point for potential therapeutic intervention, making the PM a rich source of drug targets. Many of the molecules at the PM are also exposed to the extracellular milieu, providing an additional therapeutic advantage. As recognized more than 25 years ago by Garth Nicolson, there is a great need to define the PM as a system: "It would be convenient if these (biological) properties were definable by unique cell surface characteristics which were readily identifiable so that highly specific molecular approaches to fighting cancer could be developed" (18).

Presented with this long-recognized but unmet need, the present study was aimed at gaining some basic information on the PM proteome of mammary carcinoma. We compared the PM proteomes of mammary carcinoma to that of normal human fibroblasts. Indeed, the study reveals large groups of proteins that can be considered signatures of cell lineage. There are obvious and striking differences between the PM proteomes of fibroblasts and mammary carcinoma. In addition, we are able to identify a smaller set of proteins that is unique to the PM proteome of MDA-MB-435 tumor cells, a highly metastatic mammary carcinoma line. These proteins may represent part of the metastatic signature.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Cells
All cell lines were obtained from American Type Culture Collection (ATCC), except the MDA-MB-435 cells, which were obtained from Janet Price (University of Texas). MDA-MB-435 cells were cultured in MEM Earle’s (Irvine Scientific) with 10% fetal bovine serum (Irvine Scientific), 2 mM L-glutamine (GIBCO-BRL), 1 mM sodium pyruvate (Irvine Scientific), MEM vitamins (GIBCO-BRL), and nonessential amino acids (Irvine Scientific). All other cells were cultured according to specifications set forth by ATCC. Cells were grown in 15-cm dishes at 37°C with 5% CO2. Cells were harvested at 80–90% confluence.

PM Isolation
PMs were prepared using a slightly modified version of a procedure described by Chaney and Jacobson (4). Briefly, cells were harvested from the culture plates with phosphate-buffered saline containing 200 µM EDTA. The cells were suspended in coating buffer (20 mM MES, 150 mM NaCl, 400 mM sorbitol, pH 5.3) and were added dropwise through a 21-gauge needle onto a gently swirling solution of 10% cationic colloidal silica (Aldrich). After removal of excess silica by washing, the cells were diluted in coating buffer and were added dropwise through a 21-gauge needle onto a gently swirling solution of 10 mg/ml polyacrylic acid (Fluka). After washing to remove any excess of the anionic polymer, the cells were resuspended in 2.5 mM imidazole, pH 7.0, containing a protease inhibitor cocktail (Sigma) and 1 mM vanadate (Sigma). After a 30-min incubation on ice, the cells were lysed by Dounce homogenization using multiple strokes with a size B pestle. Sheets of PM were then separated from the cellular contents by ultracentrifugation (60,000 g, 30 min) through a gradient of Nycodenz (Sigma). The supernatant was aspirated, and the PM sheets, made dense by the silica and polyacrylic acid, were collected from the bottom of the tubes. After several washes in 2.5 mM imidazole, proteins were solubilized from the membranes with 8 M urea, 2 M thiourea, 2% CHAPS, and 13 mM dithiothreitol (DTT). Protein concentration was determined using a micro-BCA protein assay (Pierce) with bovine serum albumin as a standard.

Two-Dimensional Gel Electrophoresis
Proteins associated with PMs were analyzed by two-dimensional (2D) electrophoresis. Solubilized PM protein, 100 µg in 200 µl of 8 M urea, 2 M thiourea, 2% CHAPS, and 13 mM DTT was used to rehydrate an 11-cm immobilized pH gradient (IPG) strip (Immobiline DryStrip, 4-7L; Amersham Pharmacia Biotech). The strip was rehydrated for 24 h at ambient temperature under oil. The proteins in the strip were focused using a Multiphor II apparatus (Amersham Pharmacia Biotech) for a total of 19,250 V-h. IPG strips were subsequently equilibrated in 6 M urea, 30% (wt/vol) glycerol, 2% (wt/vol) SDS, 1% DTT, and 0.05 M Tris·HCl, pH 8.8, for 15 min before the proteins were separated in the second dimension. Second dimension electrophoresis was performed for 15 min at 190 V and then for an additional 22 h at 65 V using a 12% acrylamide gel. Gels were silver stained under uniform and carefully controlled conditions according to the procedure of Shevchenko et al. (28). Silver-stained gels were scanned at 200 dpi using a Sharp JX-330 desktop scanner (Amersham Pharmacia Biotech).

Image Analysis
Three to four 2D gels were prepared for each cell line. A representative gel was chosen for image analysis, and the remaining gels were stored in 1% acetic acid and used for protein identification by mass spectrometry. Protein spots were selected using the ImageMaster Analysis software (v. 2.01, Amersham Pharmacia Biotech), and each spot was verified by the operator. The volume of each spot was quantified using the ImageMaster algorithm. These values were summed for all spots within the gel. The percentage volume that each spot contributed to the entire gel was determined by dividing the individual spot volume by the total spot volume on the gel. Based on visual inspection of the gels, spots that represented less than 0.04% of the total protein on the gel were not reproducibly detected by the software. Consequently, we constrained our analysis to spots with a volume above this value. When considering all eight test cell lines, we observed an average of 360 ± 57 (mean ± SD) spots with a volume above this cutoff. Protein spot patterns within the gels were compared using the ImageMaster analysis software. Gels were compared in a pairwise manner so that every cell line was compared directly to every other cell line in the study. The degree of similarity between gels was determined by calculating the percentage of protein spots with an identical migration pattern.

Cell Surface Iodination
To track the yield of cell-surface proteins through the electrophoresis procedure, PM proteins were labeled by iodination, using procedures we have reported previously (32). 125I-labeled cells were diluted 1:4 with unlabeled cells, and PM protein was prepared as described above. A quantity of 100 µg of PM protein containing ~37,000 cpm was loaded into each of several IPG strips, and the entire 2D PAGE procedure was carried out. At various points in the procedure, samples were subjected to gamma counting.

Identification of Proteins by Mass Spectrometry
The identity of several polypeptide spots was determined by peptide mass fingerprinting using methods that we have previously described (13). Proteins were identified by comparing observed mass fingerprints to National Center for Biotechnology Information database using a Bayesian algorithm (http://prowl.rockefeller.edu/cgi-bin/ProFound). Five polypeptides unique to the highly metastatic cell line, MDA-MB-435, were examined by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) and mass spectrometry-mass spectrometry (MS-MS) sequencing (15, 16) by Protana (http://www.protana.com).


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Cell Lines
Four breast carcinoma lines and four fibroblast lines were used to assess similarities among PM proteomes (Table 1). The analysis included the MCF7, MDA-MB-231, and MDA-MB-435 mammary carcinoma lines, all of which have been studied extensively. Each of these lines is also part of the NCI60, a group of cancer lines maintained for study by the National Cancer Institute’s Developmental Therapeutics Program (29). The MDA-MB-435 cell line is especially important to the present study, because this cell line is one of the only breast carcinoma lines to exhibit extensive metastasis from the mouse mammary fat pad (25).


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Table 1. Cell lines used for comparison of PM proteins

 
Analysis of PM Proteomes
Although proteomic profiling has been applied to identify proteins expressed by mammary carcinoma cell lines (31) and sections of mammary tumors (2), no study has focused on the PM. We reasoned that a comparison of the PM proteome between breast carcinoma and fibroblasts would provide the first information on the degree of similarity and distinction within the PM proteomes of different cell types.

We isolated PMs from cells using a method established by Chaney and Jacobson (4, 10). This method enriches for PMs by first tagging the cell surface with cationic colloidal silica and then separating the PM from the remainder of the cell by a density gradient. We found that the 2D polypeptide spot patterns of PM proteins isolated in this manner were highly reproducible within each cell line, with 2D gels from separate membrane isolations yielding spot patterns that are superimposable. In several comparisons of PM proteomes, we found the spot similarity within a cell line to be greater than 90%. We also found that 2D gels of the PM proteome are substantially different from the spot patterns of corresponding whole cell lysates. For example, out of an average of 423 total spots, only 103 migrated to identical positions on the 2D gels when PM proteins were compared with a whole cell lysate (data not shown). On the basis of these findings, we elected to enrich for PM proteins by using this purification strategy.

Another consideration in the analysis is the ability of 2D gels to resolve integral membrane proteins. We followed methods recently described by Rabilloud et al. (26) and Pasquali et al. (22), which show that the inclusion of thiourea in the extraction buffer significantly expands the range of integral membrane proteins that can be analyzed by 2D PAGE. Our preliminary work confirmed that thiourea dramatically increased the number of spots visible on 2D gels of PM preparations (data not shown). However, initial mass fingerprinting of a number of spots on the gels identified few integral membrane proteins. By quantitative tracking of proteins tagged by cell surface iodination, we determined that less than 20% of the proteins on the external side of the membrane are actually resolved onto the second dimension gel (Table 2). Consequently, the first part of our analysis of the PM proteome was performed with the realization that the proteomes being compared consist primarily of proteins associated with the PM and that integral membrane proteins are likely to be under-represented.


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Table 2. Tracking 125I-labeled cell surface proteins through the 2D PAGE procedure

 
Identification of PM Signatures
The PM proteomes of four fibroblast cell lines (Fig. 1, AD) and four breast cancer cell lines (Fig. 1, EH) were analyzed by 2D gel electrophoresis. The polypeptide spot patterns of these gels, which contained an average of 360 spots per gel, were compared using computer-assisted spot matching. Similarity between the PM proteomes was quantified by determining the percentage of matching spots between each gel pair. This pairwise comparison showed that cells of the same lineage exhibited remarkable similarity (Fig. 2), with similarity among the mammary carcinoma cell lines ranging from 38% to 55%. The similarity among the fibroblasts ranged from 18% to 40%, but most of the lines were nearly 30% identical. Conversely, cells of different lineage had far less similarity, with values ranging from only 9% to 23%. Altogether, these findings strongly suggest the presence of a PM "signature" that is tightly linked to cell lineage and that the signature of the mammary carcinoma cells constitutes a little under one-half of the PM proteome.



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Fig. 1. Comparison of plasma membrane (PM) proteins by two-dimensional (2D) PAGE. PMs were isolated from four fibroblast (AD; Hs 58.Fs, HE-SK, CCD-1090Sk, and Detroit 551) and four mammary carcinoma cell lines (EH; MCF7, MDA-MB-231, MDA-MB-435, and SK-BR-3). Following membrane isolation, proteins were solubilized with a mixture of chaotropic and reducing agents and detergent as described in MATERIALS AND METHODS. A total of 100 µg of protein from this PM preparation was analyzed by 2D PAGE. Samples were focused in immobilized pH gradient (IPG) strips with a linear pH range of 4–7 and then were separated on a 12% acrylamide gel. Gels were silver stained and scanned, and the resulting image was analyzed using ImageMaster Analysis software. Only spots that constituted greater than 0.04% of the total staining density within the gel were considered for analysis. All of the protein spots used for comparative analysis are shaded dark. Each gel is representative of at least three replicate gels.

 


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Fig. 2. Similarity in the PM proteomes of fibroblasts and mammary carcinoma cells. To assess the similarities among the PM proteomes of fibroblasts (Fib) and mammary carcinoma cells (MCC), the polypeptide spot pattern of each 2D gel was compared with that of every other gel in a pairwise fashion. Computer-assisted comparisons were made with ImageMaster analysis software as described in MATERIALS AND METHODS. The degree of similarity between two cell lines is reported as a percentage (% Identity), calculated by dividing the number of matching spots by the average number of spots on each gel pair.

 
As an additional assessment of the relatedness among cell lines, the relative abundance of a set of 83 proteins common to the PM of the mammary carcinoma lines, but absent from the fibroblasts, was compared. The relative abundance of each protein was estimated by dividing its spot density by the combined spot density of all proteins on the gel. Differences in the relative abundance of individual proteins among the cell lines were generally small, falling within a two- to fourfold range (data not shown). This suggests that most of the proteins in the PM signature of mammary carcinoma are expressed at similar levels.

Identification of Proteins in the Metastatic Signature
Because the MDA-MB-435 cells are far more invasive than the MDA-MB-231 cells and the MCF7 cells, we sought to identify proteins unique to the PM proteome of the MDA-MB-435 cells. The mammary carcinoma cell line, SK-BR-3, was not included in this analysis because its metastatic properties are not defined. We reasoned that this unique set of proteins might be linked to the metastatic phenotype. We found 82 polypeptide spots in the PM proteome of the MDA-MB-435 cells that were not detected in the other two mammary carcinoma cells lines (Fig. 3). The fact that these spots are only observed in the MDA-MB-435 cells indicates that they are either uniquely expressed by these cells or that the MDA-MB-435 cells express a uniquely processed variant of each protein. Only a few of these unique polypeptides were present at sufficient levels for identification by peptide mass fingerprinting (Fig. 3, AE). Nevertheless, the results from mass fingerprinting of these proteins (Table 3) reveal some interesting observations. The potential function of each of the identified proteins in tumor progression is discussed below.



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Fig. 3. Toward the identification of a metastatic proteome. Spot comparisons were performed among the PM proteomes of the mammary carcinoma cells to identify polypeptide spots unique to the 2D gel of the highly metastatic MDA-MB-435 cells. For the purpose of illustration, the position of the unique spots is shown on the 2D gel of MDA-MB-435 cells (in red) compared with a gel of the MDA-MB-231 cells. Further detail of the boxed areas is shown in the insets, with arrows referencing the comparable regions of the two gels. The identities of spots A–E were determined by mass spectrometry (Table 3).

 

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Table 3. Identification of proteins from the MDA-MB-435 cells by mass spectrometry

 
The p47 subunit of eukaryotic initiation factor 3.
Spot B in Fig. 3 was found to be the p47 subunit of eukaryotic initiation factor 3 (eIF3). This protein migrated very near its predicted Mr and isoelectric point (pI), indicating that its presence in the PM proteome is due to upregulation in the MDA-MB-435 cells or to a unique association with the PM in these cells. The eIFs control protein synthesis by regulating the interaction between mRNA, tRNA, and ribosomes (21). eIF3 is a multiprotein complex that binds to the 40S ribosomal subunit and is believed to regulate its association with the 60S ribosome (1). Interestingly, the p47 subunit of the eIF3 complex was only recently cloned and was found to be homologous to a number of other proteins including Mov-34, a component of the 26S proteosome (1). The function of p47 is not known, but its relationship to Mov-34 suggests that it may be involved in regulating subunit interactions within eIF3. Our identification of p47 in PM preparations also suggests that it could have a role outside of the eIF3 complex. Such a novel function could explain its prevalence in PM fractions.

Although the eIFs are not considered classic oncogenes, there is now an expanding body of literature indicating that misregulation of individual components of eIF2, eIF3, and eIF4 can lead to neoplastic transformation (6, 7, 17, 20, 27). Although p47 of eIF3 has not been extensively studied, one might hypothesize that it could play a similar role in tumor progression.

An uncharacterized protein, p25.
We also observed a 25-kDa protein (spot C, Fig. 3) that is unique to the PM proteome of the MDA-MB-435 cells. Peptide mass fingerprinting showed this protein to be identical to three nucleotide sequences that were independently deposited in the databases (GenBank accession nos. AAD27784, BAA76626, and AAD40193). The predicted mass of the protein derived from each of these genes matches the migration position of p25 on 2D gels. Expressed sequence tags encoding the protein are found in several tissues. However, to our knowledge this is the first demonstration that the protein product of the gene is expressed. This protein lacks homology to any other known protein or gene in the databases and has no known function.

{alpha}-Synuclein.
Spot D in Fig. 3 was found to be {alpha}-synuclein. This protein also migrated close to its predicted Mr and pI in the 2D gels from MDA-MB-435 cells. Synucleins are small cytoplasmic proteins of ~130 amino acids. Although their precise biological function is still poorly understood, the synucleins ({alpha}, ß, and {gamma}) contain lipid binding helices that bind to acidic phospholipids in vitro (5). The prevailing notion is that synucleins have some function at the inner leaf of the PM. {alpha}-Synuclein was originally identified as the precursor protein for the non-ß amyloid component of Alzheimer’s disease amyloid plaques (30), and a variant of {alpha}-synuclein appears to be responsible for a rare genetic from of Parkinson’s disease (24). However, the mechanistic role of these proteins in the pathology of these diseases is unknown. Synucleins are not expressed in normal breast epithelia, but both ß- and {gamma}-synuclein are expressed in the vast majority of ductal carcinomas of the breast (3). The role of synucleins in tumor progression remains unclear, but it is worth noting that transfection of the MDA-MB-435 cells with {gamma}-synuclein was reported to increase the metastatic potential of this cell line (11). Our finding that {alpha}-synuclein is unique to the PM proteome of the MDA-MB-435 cells underscores the need to understand the function of this family of proteins in metastatic spread.

Galectin-1.
Galectin-1, a mammalian lectin that binds to ß-galactoside, was also found to be unique to the PM proteome of MDA-MB-435 cells (spot E, Fig. 3). Galectin-1 migrated at its predicted Mr and pI in the 2D gel. Galectin-1 can be secreted from cells and then is able to bind back to glycosylated proteins on the PM. Galectin-1 is upregulated in several cancers, and its expression is often higher in more differentiated tumors (33, 34). Galectin-1 exists as a homodimer and can facilitate intramolecular and intermolecular cross-bridging. Such cross-bridging may facilitate cellular signaling through growth factor receptors or may mediate cell-cell adhesion (23). Interestingly, galectin-1 has been shown to mediate the adhesion of MDA-MB-435 cells to human endothelial cells, and this has been proposed as a key route to metastatic spread (8). Our observation that galectin-1 is unique to the PM proteome of MDA-MB-435 cells lends further support to the idea that galectin-1 is connected to metastasis.

Thiol-specific antioxidant protein.
Eighty polypeptide spots were observed to be present in the PM preparations of MDA-MB-231 cells and MCF7 cells but were undetectable in the PM preparation of the metastatic MDA-MB-435 cells. This finding suggests that downregulation of large sets of proteins may make an equally important contribution to the metastatic phenotype. One of these proteins was identified from the MCF7 and MDA-MB-231 cells by mass fingerprinting. It was found to be thiol-specific antioxidant protein (TSA). TSA presumably has a role in protecting cells against oxidative damage (35). This observation is consistent with a prior report showing that the TSA gene is downregulated in MDA-MB-435 cells compared with MDA-MB-231 and MCF7 cells (12).


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The primary objective of this study was to obtain basic information on the similarities and differences among PM proteomes. One might hypothesize that cells with similar lineage, or phenotype, would exhibit a high degree of similarity at the PM. However, to our knowledge, there has been no information published on this topic. The findings of the present study indicate that there are indeed signatures of proteins at the PM. In general, the proteins at the PM can be segregated into three groups. One group of proteins is likely to be common to most cell types. We found that between 9% and 23% of all proteins at the PMs of fibroblasts and mammary carcinoma cells migrate to the same position on 2D gels and are likely to be identical. Given the functional distinction between these two types of cells, it seems reasonable to suggest that this common set of PM proteins is widely expressed among most cell types.

A second set of PM proteins appears to be linked with cell lineage. From our 2D gel study, we found that between 38% and 55% of the proteins at the PM of mammary carcinoma are identical. The similarity among fibroblasts was somewhat lower, ranging from 18% to 40%. The lower degree of similarity among the fibroblasts may be explained by the fact that fibroblasts are less differentiated and have functions that are less specialized than mammary epithelial cells. One might expect that this set of proteins confer the overall character of the cell. For example, in the case of mammary carcinoma cells, these proteins might be responsible for epithelial structure and function. This structure-function relationship, then, would clearly distinguish these cells from fibroblasts.

A third group of proteins appears to be expressed uniquely on each individual cell line and be independent of cell lineage. These proteins composed roughly 22% of all proteins in the PM preparations. When considering this set of proteins, it is important to emphasize the distinction between cell lineage and cellular phenotype. Lineage, or the tissue source of a cell, clearly has a significant impact on the profile of proteins expressed by a cell. However, cells of similar lineage, like the MCF7 and MDA-MB-435 cells, can exhibit distinct phenotypes. This is particularly evident with cancer cells, which can represent any number of stages of tumor progression. For example, the MCF7 cells will only grow as a primary tumor in mice when estrogen is supplemented. In contrast, the MDA-MB-435 cells grow tumors independent of estrogen. Furthermore, the MCF7 cells fail to metastasize, whereas the MDA-MB-435 cells exhibit extensive macroscopic metastases to multiple organs (25). Although a clear lineage-specific signature is common among these two cell lines, it is not surprising that another set of proteins is different between the two. Nearly one-fourth of all PM proteins appeared to be unique to individual cell lines. Our identification of a few of these proteins from the MDA-MB-435 cells, which show wide variation in their proposed functions, illustrates the scope of biochemical pathways that could be influenced by these differences. For example, compared with the MCF7 and MDA-MB-231 cells, the MDA-MB-435 cells have undetectable levels of TSA, a key regulator of oxidant stress. The MDA-MB-435 cells also differentially express proteins that could dramatically influence the kinetics of large groups of mRNAs, as well as proteins that could alter the character of the inner leaf of the PM. Altogether, the functional scope of the cell-specific proteins appears to be immense and is consistent with the widely disparate phenotype of these mammary carcinoma cell lines.

In the area of protein profiling there is a need to perform large-scale comparisons that provide both protein identity and protein abundance. The present report illustrates some of the difficulties and limitations encountered by a typical biochemistry laboratory in meeting these hurdles. Although 2D gels represent the best technology available for protein separation, they clearly fall short in the separation of integral membrane proteins as well as true measurements of protein abundance. Recent advances suggest that labeling of proteins within a mixture with isotope tags can be combined with mass spectrometry to obtain protein identity and abundance on a broad scale (9). Independent work indicates that combining protein digestion with highly sophisticated peptide mass analysis could yield a similar level of information (14). Despite the appeal of each of these strategies, they all require significant technological expertise and resources. Given the value of the profiling information to cell biology and biochemistry, a strong argument for a national effort to completely define the composition of cellular and subcellular proteomes can be made.


    ACKNOWLEDGMENTS
 
This study was supported by National Institutes of Health Grants CA-69306 and HL-58925 (to J. W. Smith) and by Cancer Center Support Grant CA-30199.


    FOOTNOTES
 
Article published online before print. See web site for date of publication (http://physiolgenomics.physiology.org).

Address for reprint requests and other correspondence: J. W. Smith, The Burnham Institute, 10901 North Torrey Pines Road, La Jolla, CA 92037 (E-mail: jsmith{at}burnham.org).


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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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