Global profiling of the cell surface proteome of cancer cells uncovers an abundance of proteins with chaperone function

There is currently limited data available pertaining to the global characterization of the cell surface proteome. We have implemented a strategy for the comprehensive profiling and identification of surface membrane proteins. This strategy has been applied to cancer cells, including the SH-SY5Y neuroblastoma, the A549 lung adenocarcinoma, the LoVo colon adenocarcinoma, and the Sup-B15 acute lymphoblastic leukemia (B cell) cell lines and ovarian tumor cells. Surface membrane proteins of viable, intact cells were subjected to biotinylation then affinity-captured and purified on monomeric avidin columns. The biotinylated proteins were eluted from the monomeric avidin columns as intact proteins and were subsequently separated by two-dimensional PAGE, transferred to polyvinylidene difluoride membranes, and visualized by hybridization with streptavidin-horseradish peroxidase. Highly reproducible, but distinct, two-dimensional patterns consisting of several hundred biotinylated proteins were obtained for the different cell populations analyzed. Identification of a subset of biotinylated proteins among the different cell populations analyzed using matrix-assisted laser desorption ionization and tandem mass spectrometry uncovered proteins with a restricted expression pattern in some cell line(s), such as CD87 and the activin receptor type IIB. We also identified more widely expressed proteins, such as CD98, and a sushi repeat-containing protein, a member of the selectin family. Remarkably, a set of proteins identified as chaperone proteins were found to be highly abundant on the cell surface, including GRP78, GRP75, HSP70, HSP60, HSP54, HSP27, and protein disulfide isomerase. Comprehensive profiling of the cell surface proteome provides an effective approach for the identification of commonly occurring proteins as well as proteins with restricted expression patterns in this compartment.     

The F(1)F(0) ATP synthase and mitochondrial respiratory chain complexes are present on the plasma membrane of an osteosarcoma cell line: An immunocytochemical study

F(1)F(0) ATP synthase is ectopically expressed on the surface of several cell types, including endothelium and cancer cells. This study uses immunocytochemical detection methods via highly specific monoclonal antibodies to explore the possibility of plasma membrane localization of other mitochondrial proteins using an osteosarcoma cell line in which the location of the mitochondrial reticulum can be clearly traced by green fluorescent protein tagging of the organelle. We found that subunits of three of the four respiratory chain complexes were present on the surface of these cells. Additionally, we show for the first time that F(0) subunits d and OSCP of the ATP synthase are ectopically expressed. In all cases the OXPHOS proteins show a punctate distribution, consistent with data from proteome analysis of isolated lipid rafts that place the various mitochondrial proteins in plasma membrane microdomains. We also examined the cell surface for marker membrane proteins from several other intracellular organelles including ER, golgi and nuclear envelope. They were not found on the surface of the osteosarcoma cells. We conclude that mitochondrial membrane proteins are ectopically expressed, but not proteins from other cellular organelles. A specific mechanism by which the mitochondrion and plasma membrane fuse to deliver organellar proteins is suggested.     

Design of recombinant antibody microarrays for cell surface membrane proteomics

Generating proteomic maps of membrane proteins, common targets for therapeutic interventions and disease diagnostics, has turned out to be a major challenge. Antibody-based microarrays are among the novel rapidly evolving proteomic technologies that may enable global proteome analysis to be performed. Here, we have designed the first generation of a scaleable human recombinant scFv antibody microarray technology platform for cell surface membrane proteomics as well as glycomics targeting intact cells. The results showed that rapid and multiplexed profiling of the cell surface proteome (and glycome) could be performed in a highly specific and sensitive manner and that differential expression patterns due to external stimuli could be monitored.     

Plasmodium lipid rafts contain proteins implicated in vesicular trafficking and signalling as well as members of the PIR superfamily, potentially implicated in host immune system interactions

Plasmodium parasites, the causal agents of malaria, dramatically modify the infected erythrocyte by exporting parasite proteins into one or multiple erythrocyte compartments, the cytoplasm and the plasma membrane or beyond. Despite advances in defining signals and specific cellular compartments implicated in protein trafficking in Plasmodium-infected erythrocytes, the contribution of lipid-mediated sorting to this cellular process has been poorly investigated. In this study, we examined the proteome of cholesterol-rich membrane microdomains or lipid rafts, purified from erythrocytes infected by the rodent parasite Plasmodium berghei. Besides structural proteins associated with invasive forms, we detected chaperones, proteins implicated in vesicular trafficking, membrane fusion events and signalling. Interestingly, the raft proteome of mixed P. berghei blood stages included proteins encoded by members of a large family (bir) of putative variant antigens potentially implicated in host immune system interactions and targeted to the surface of the host erythrocytes. The generation of transgenic parasites expressing BIR/GFP fusions confirmed the dynamic association of members of this protein family with membrane microdomains. Our results indicated that lipid rafts in Plasmodium-infected erythrocytes might constitute a route to sort and fold parasite proteins directed to various host cell compartments including the cell surface.     

Proteome analysis of the rice etioplast: metabolic and regulatory networks and novel protein functions

We report an extensive proteome analysis of rice etioplasts, which were highly purified from dark-grown leaves by a novel protocol using Nycodenz density gradient centrifugation. Comparative protein profiling of different cell compartments from leaf tissue demonstrated the purity of the etioplast preparation by the absence of diagnostic marker proteins of other cell compartments. Systematic analysis of the etioplast proteome identified 240 unique proteins that provide new insights into heterotrophic plant metabolism and control of gene expression. They include several new proteins that were not previously known to localize to plastids. The etioplast proteins were compared with proteomes from Arabidopsis chloroplasts and plastid from tobacco Bright Yellow 2 cells. Together with computational structure analyses of proteins without functional annotations, this comparative proteome analysis revealed novel etioplast-specific proteins. These include components of the plastid gene expression machinery such as two RNA helicases, an RNase II-like hydrolytic exonuclease, and a site 2 protease-like metalloprotease all of which were not known previously to localize to the plastid and are indicative for so far unknown regulatory mechanisms of plastid gene expression. All etioplast protein identifications and related data were integrated into a data base that is freely available upon request.     

Proteome profiling of human epithelial ovarian cancer cell line TOV-112D

A proteome profiling of the epithelial ovarian cancer cell line TOV-112D was initiated as a protein expression reference in the study of ovarian cancer. Two complementary proteomic approaches were used in order to maximise protein identification: two-dimensional gel electrophoresis (2DE) protein separation coupled to matrix assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS) and one-dimensional gel electrophoresis (1DE) coupled to liquid-chromatography tandem mass spectrometry (LC MS/MS). One hundred and seventy-two proteins have been identified among 288 spots selected on two-dimensional gels and a total of 579 proteins were identified with the 1DE LC MS/MS approach. This proteome profiling covers a wide range of protein expression and identifies several proteins known for their oncogenic properties. Bioinformatics tools were used to mine databases in order to determine whether the identified proteins have previously been implicated in pathways associated with carcinogenesis or cell proliferation. Indeed, several of the proteins have been reported to be specific ovarian cancer markers while others are common to many tumorigenic tissues or proliferating cells. The diversity of proteins found and their association with known oncogenic pathways validate this proteomic approach. The proteome 2D map of the TOV-112D cell line will provide a valuable resource in studies on differential protein expression of human ovarian carcinomas while the 1DE LC MS/MS approach gives a picture of the actual protein profile of the TOV-112D cell line. This work represents one of the most complete ovarian protein expression analysis reports to date and the first comparative study of gene expression profiling and proteomic patterns in ovarian cancer.     

Distinct cell surface proteome profiling by biotin labeling and glycoprotein capturing

We performed here MS-based cell surface proteome profiling of HCT-116 cells by two distinct methods based on biotin labeling and glycoprotein capturing. In total, 742 biotinylated and 219 glycosylated proteins were identified by the biotin labeling and glycoprotein capturing, of which 224 and 138 proteins known to be located on plasma membrane were included, respectively, according to ingenuity pathway analysis. Although 104 plasma membrane proteins were identified by both methods, the rest of 154 were identified only by one. Almost all the identified plasma membrane proteins possessed consensus N-glycosylation sites, and proteins having various numbers of glycosylation sites were identified by both methods. Thus, the discrepancies of the identified proteins obtained from those two methods might not be only due to the number of glycosylation sites, but also to the expression and/or glycosylation level of the cell surface proteins. We also identified 312 N-glycosylated proteins from xenograft samples by glycoprotein capturing of which 135 were known as plasma membrane proteins. Although a number of highly-expressed plasma membrane proteins were common between culture and xenograft cells, some proteins showed culture- or xenograft-specific expression, suggesting that those proteins might contribute to grow in different environment.     

Global profiling of surface plasma membrane proteome of oviductal epithelial cells

In mammalian reproduction, many important events occur within the female reproductive tract, especially within the oviduct. These include transport and final maturation of the female and male gametes, fertilization, embryonic development, and transport of the embryo to the uterus. The plasma membrane molecules of oviductal epithelia that are in direct contact with gametes and embryo(s) and potentially mediate these processes are poorly characterized, and their function is poorly understood. Defining the oviductal cell surface proteome could provide a better understanding of the basis of reproductive processes taking place within the oviduct. We aimed to provide a detailed profile of the surface plasma membrane proteome of the oviductal epithelium by biotinylation of proteins at the cell surface, followed by highly specific purification of these proteins using avidin. This approach for enrichment of oviductal cell surface proteome was validated by immunohistochemistry, gel electrophoresis, and western blot analysis experiments. The enriched molecules were identified using two different technologies: (i) the combination of 2D gel electrophoresis with mass spectrometry and (ii) 1D gel electrophoresis with mass spectrometry (a modified multidimensional protein identification technology (MudPIT) technique). The number of proteins identified using the MudPIT approach was approximately 7 times the number of proteins identified by 2D gel electrophoresis using the same samples (40 versus 276, respectively). Some of the proteins found at the surface of oviductal cells had previously been reported as present in the oviduct and to have known functions in relation to reproductive processes. The other category of proteins that were highly represented in the oviductal surface proteome were various members of the family of heat-shock proteins. To the best of our knowledge, this is the first comprehensive study to identify and characterize proteins at the surface of the epithelium of the mammalian oviduct.     

DIGE compatible labelling of surface proteins on vital cells in vitro and in vivo

Efficient methods for profiling of the cell surface proteome are desirable to get a deeper insight in basic biological processes, to localise proteins and to uncover proteins differentially expressed in diseases. Here we present a strategy to target cell surface exposed proteins via fluorescence labelling using CyDye DIGE fluors. This method has been applied to human cell lines in vitro as well as to a complex biological system in vivo. It allows detection of fluorophore-tagged cell surface proteins and visualisation of the accessible proteome within a single 2-D gel, simplifying subsequent UV MALDI-MS analysis.     

Lessons from the stem cell proteome

The proteome of a cell is a molecular fingerprint directly relating to the gene expression snapshot profile at a certain point of time or developmental stage. Monitoring the expansion and the differentiation state of stem cells by proteomic means seems therefore a very attractive method for diagnostic as well as for therapeutic purposes. We have investigated the protein expression patterns of umbilical cord blood-derived CD34+/AC133+ cells in order to obtain a most comprehensive view of the stem cell proteome. For this purpose, we have applied 2-D gel electrophoresis and 2-D chromatography for most efficient protein/peptide separation and characterisation. The proteins were identified after tryptic digestion by nano-HPLC coupled directly to an ion trap mass spectrometer. An extensive bioinformatic analysis of the protein obtained revealed a dynamic stem cell proteome. This means that the heterogeneity of protein expression patterns obtained from different stem cell preparations refers to a limited set of stem cell-specific house keeping proteins as well as to a large number of proteins which depend on (marginal) stimuli from the environment. Since those are difficult to standardise, snapshot views of the stem cell proteome reflect not only stem cell-intrinsic metabolism but also the strong influence of the sample history on protein expression patterns.     

Towards a human proteome atlas: high-throughput generation of mono-specific antibodies for tissue profiling

A great need exists for the systematic generation of specific antibodies to explore the human proteome. Here, we show that antibodies specific to human proteins can be generated in a high-throughput manner involving stringent affinity purification using recombinant protein epitope signature tags (PrESTs) as immunogens and affinity-ligands. The specificity of the generated affinity reagents, here called mono-specific antibodies (msAb), were validated with a novel protein microarray assay. The success rate for 464 antibodies generated towards human proteins was more than 90% as judged by the protein array assay. The antibodies were used for parallel profiling of patient biopsies using tissue microarrays generated from 48 human tissues. Comparative analysis with well-characterized monoclonal antibodies showed identical or similar specificity and expression patterns. The results suggest that a comprehensive atlas containing extensive protein expression and subcellular localization data of the human proteome can be generated in an efficient manner with mono-specific antibodies.     

Protein profiling of microdomains purified from renal cell carcinoma and normal kidney tissue samples

Renal cell carcinoma (RCC) is representing about 3% of all adult cancers. A promising strategy for cancer biomarker discovery is subcellular comparative proteomics, allowing enriching specific cell compartments and assessing differences in protein expression patterns. We investigated the proteomic profile of a peculiar RCC subcellular compartment, plasma membrane microdomains (MD), involved in cell signalling, transport, proliferation and in many human diseases, such as cancer. Subcellular fractions were prepared by differential centrifugation from surgical samples of RCC and adjacent normal kidney (ANK). MD were isolated from plasma-membrane-enriched fractions after Triton X-100 treatment and sucrose density gradient ultracentrifugation. MD derived from RCC and ANK tissues were analyzed after SDS-PAGE separation by LC-ESI-MS/MS. We identified 93 proteins from MD isolated from RCC tissue, and 98 proteins from ANK MD. About 70% of the identified proteins are membrane-associated and about half of these are known as microdomain-associated. GRAVY scores assignment shows that most identified proteins (about 70%) are in the hydrophobic range. We chose a panel of proteins to validate their differential expression by WB. In conclusion, our work shows that RCC microdomain proteome is reproducibly different from ANK, and suggests that mining into such differences may support new biomarker discovery.     

The correlation between cellular size and protein expression levels--normalization for global protein profiling

An automated image analysis system was used for protein quantification of 1862 human proteins in 47 cancer cell lines and 12 clinical cell samples using cell microarrays and immunohistochemistry. The analysis suggests that most proteins are expressed in a cell size dependent manner, and that normalization is required for comparative protein quantification in order to correct for the inherent bias of cell size and systematic ambiguities associated with immunohistochemistry. Two reference standards were evaluated, and normalized protein expression values were found to allow for protein profiling across a panel of morphologically diverse cells, revealing putative patterns of over- and underexpression. Using this approach, proteins with stable expression as well as cell-line specific expression were identified. The results demonstrate the value of large-scale, automated proteome analysis using immunohistochemistry, in revealing functional correlations and establishing methods to interpret and mine proteomic data.     

Global detection and characterization of hypothetical proteins in Shewanella oneidensis MR-1 using LC-MS based proteomics

The availability of whole genome sequences has enabled the application of powerful tools for assaying global expression patterns in environmentally relevant bacteria such as Shewanella oneidensis MR-1. A large number of genes in prokaryote genomes, including MR-1, have been annotated as hypothetical, indicating that no similar protein has yet been identified in other organisms. Using high-sensitivity MS coupled with accurate mass and time (AMT) tag methodology, 1078 tryptic peptides were collectively detected in MR-1 cultures, 671 of which were unique to their parent protein. Using only these unique tryptic peptides and a minimum of two peptides per protein, we identified, with high confidence, the expression of 258 hypothetical proteins. These proteins ranged from 3.5 to 139 kDa, with 47 being 100 amino acid residues or less. Using a combination of information including detection in cells grown under specific culture conditions, presence within a specific cell fraction, and predictive algorithms such as PSORT and PSORT-B, possible/plausible functions are proposed for some hypothetical proteins. Further, by applying this approach a number of proteins were found not only to be expressed, but only expressed under certain culturing conditions, thereby suggesting function while at the same time isolating several proteins to distinct locales of the cell. These results demonstrate the utility of the AMT tag methodology for comprehensive profiling of the microbial proteome while confirming the expression of a large number of hypothetical genes.     

Proteomic analysis of the yeast mitochondrial outer membrane reveals accumulation of a subclass of preproteins

Mitochondria consist of four compartments-outer membrane, intermembrane space, inner membrane, and matrix--with crucial but distinct functions for numerous cellular processes. A comprehensive characterization of the proteome of an individual mitochondrial compartment has not been reported so far. We used a eukaryotic model organism, the yeast Saccharomyces cerevisiae, to determine the proteome of highly purified mitochondrial outer membranes. We obtained a coverage of approximately 85% based on the known outer membrane proteins. The proteome represents a rich source for the analysis of new functions of the outer membrane, including the yeast homologue (Hfd1/Ymr110c) of the human protein causing Sj?gren-Larsson syndrome. Surprisingly, a subclass of proteins known to reside in internal mitochondrial compartments were found in the outer membrane proteome. These seemingly mislocalized proteins included most top scorers of a recent genome-wide analysis for mRNAs that were targeted to mitochondria and coded for proteins of prokaryotic origin. Together with the enrichment of the precursor form of a matrix protein in the outer membrane, we conclude that the mitochondrial outer membrane not only contains resident proteins but also accumulates a conserved subclass of preproteins destined for internal mitochondrial compartments.     

Comprehensive proteomic analysis of breast cancer cell membranes reveals unique proteins with potential roles in clinical cancer

Proteins associated with cancer cell plasma membranes are rich in known drug and antibody targets as well as other proteins known to play key roles in the abnormal signal transduction processes required for carcinogenesis. We describe here a proteomics process that comprehensively annotates the protein content of breast tumor cell membranes and defines the clinical relevance of such proteins. Tumor-derived cell lines were used to ensure an enrichment for cancer cell-specific plasma membrane proteins because it is difficult to purify cancer cells and then obtain good membrane preparations from clinical material. Multiple cell lines with different molecular pathologies were used to represent the clinical heterogeneity of breast cancer. Peptide tandem mass spectra were searched against a comprehensive data base containing known and conceptual proteins derived from many public data bases including the draft human genome sequences. This plasma membrane-enriched proteome analysis created a data base of more than 500 breast cancer cell line proteins, 27% of which were of unknown function. The value of our approach is demonstrated by further detailed analyses of three previously uncharacterized proteins whose clinical relevance has been defined by their unique cancer expression profiles and the identification of protein-binding partners that elucidate potential functionality in cancer.