Localization of proteins to membrane-enclosed organelles is a central feature of cellular organization. Using protein correlation profiling to track the abundance of thousands of peptides through centrifugation gradients, we mapped 1502 proteins to subcellular locations in mouse liver. Ten major clusters emerged, which corresponded to well-characterized cellular compartments based on a combination of enzymatic assays, marker protein profiles and confocal microscopy. Protein correlation profiles validated genuine organellar components and enabled us to assess the specificity of previously published organellar proteomic inventories.  Remarkably, 41% of all organellar proteins were found in more than one location.  Integration of the organellar proteomic data with atlases of RNA abundance and genome sequence enabled us to identify networks of co-expressed genes, cis-regulatory motifs, and putative transcriptional regulators involved in organelle biogenesis.  Our analysis ties classical biochemistry, cell biology and genomics into a common framework for the analysis of organelles.

Protein quantitation and identification by isotope coded affinity tag (ICAT) labelling and MS/MS is a powerful technique that we have adapted for the proteomic investigation of cell membrane protein shedding and degradation by matrix metalloproteinases (MMPs). We have further investigated cellular responses to perturbations in the protease web induced by antiproteolytic drugs in phase III clinical trials for cancer and arthritis. These approaches have led to the identification of a plethoria of novel targets for these important proteases and with biochemical validation have resulted in the expansion of the MMP substrate degradome by ~30 new substrates. Potential substrates were identified as those having altered protein levels in the conditioned medium and cell layer compared with the E240A inactive MMP mutant transfectants. We hypothesized that proteins cleaved and degraded would show reduced ICAT ratios whereas those shed from the cell membrane would be increased in amount in the conditioned medium of cell transfectants. New substrates were biochemically confirmed by MALDI-TOF MS and sequencing of cleavage fragments after incubation with proteases in vitro. Proteins that were cleaved in the medium were shown to accumulate in the presence of the MMP inhibitor whereas proteins shed from the cell surface or matrix showed reduced amounts in the medium in the presence of the drug with corresponding increases in protein found in the plasma membrane fraction. Thus, the utility and quantitative nature of ICAT-MS/MS is a new degradomic screen for protease substrate discovery that should be generally adaptable to other classes of protease for exploring proteolytic function in complex dynamic biological contexts.

The control of pancreatic beta-cell survival is critical to the pathogenesis of all forms of diabetes. Diabetes is caused by the combination of ‘acquired factors’ such as elevated circulating fatty acids and genetic susceptibility. Calpain-10 was the first gene linked to type 2 diabetes susceptibility by positional cloning. We found that apoptosis induced by the fatty acid palmitate was blocked in pancreatic islets isolated from calpain-10 knockout mice and transgenic mice expressing the endogenous calpain inhibitor calpastatin. Palmitate-induced apoptosis was enhanced in transgenic islets overexpressing calpain-10. We employed Cy-dye 2D gel proteomics to identify downstream targets of palmitate and calpain-10 in a number of model systems. Proteomic analysis of both the MIN6 beta-cell line and human islets identified carboxypeptidase E, previously linked to diabetes in mice and man, as the major down-regulated protein during palmitate-induced apoptosis in both model systems. Multiple metabolic enzymes and cytoskeletal components were deduced as calpain targets using tissue from transgenic or knockout mice, as well as MIN6 cells with stable RNAi-mediated knockdown of calpain-10. Together, these studies point to a previously unexpected network of diabetes-associated genes that control beta-cell apoptosis, and therefore the progression of diabetes.

Conventional two dimensional gel electrophoresis is used to separate proteins from cellular homogenate. Isolated proteins are digested and identified by their peptide products using an Ion Trap mass spectrometer. A database of identified proteins will serve as standards for peak identification during electro-elution and co-migration experiments. Single cell 2D-CE analysis will investigate cell to cell heterogeneity in protein expression during various stages of disease progression.

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 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 1D 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 report to date.

As part of the display capability development we have developed small, high copy E. coli/Caulobacter shuttle vectors, enabling rapid production of display libraries of any type.  Of particular relevance to proteomic applications is the development of a vector variant enabling display of random genomic fragments or cDNAs.  The high copy number and the fact that it is a bacterial display system enables adaptation to flow cytometry as a rapid means of detecting and then immediately sorting clones of interest.  The concept is to develop libraries of cells that can be stored frozen, amplified whenever desired and then probed with fluorescent labeled ligands.  “Hits” are readily retrieved for sequence analysis.  If desired, one can also convert selected clone plasmids into protein secretion versions, enabling the production of the selected proteins or peptides.

Affinity tagging of proteins in combination with mass spectrometric protein identification is a common tool in the study of protein-protein interactions. Stringent washing conditions are often necessary to reduce ubiquitous background, which can result in the loss of weak and transient interactions during purification. We have developed a novel approach that uses protein tagging and treatment of live cells with formaldehyde to induce chemical cross-linking of interacting proteins. After quenching and cell lysis, the tagged protein of interest and all proteins that are covalently attached to it due to cross-linking are affinity-purified under stringent conditions. The cross-links are reversed to release to captured proteins, which are separated by gel electrophoresis and identified by in-gel digestion and peptide sequencing using LC-MS/MS. This concept has been used successfully to identify interaction partners of a constitutively active mutant of the small GTPase M-Ras (Q71L). In addition to known interaction partners of M-Ras, several additional candidates have been identified this way and have become subject of further studies. This has led to the demonstration that IQGAP, a RasGAP-like protein, is a novel interaction partner of M-Ras. The method described here is widely applicable and will serve as a new tool in the study of protein-protein interactions in vivo.

Fish cell lines are relatively easy to develop and most have simple growth requirements that make cross contamination a potential problem. Cell line contamination is not an uncommon incident in laboratories handling more than one cell line and many reports have been made on cross contamination of mammalian cell lines. Although problems of misidentification and cross-contamination of fish cell lines have rarely been reported, these are issues of concern for cell culturists that can make scientific results and their reproducibility unreliable. Proper identification of cell lines is thus crucial and protocols for routine and rapid screening are preferred.  Cytogenetic evaluation, DNA fingerprinting, microsatellite analysis and PCR methods have been published for inter-species identification of many cell lines, but discerning intra-species contamination has been challenging. More complex DNA fingerprinting and hybridization techniques coupled with isoenzyme analysis have been developed to discriminate intra-species contamination, however, these require complex and time consuming procedures to enable cell identification thus are difficult to apply for routine use. A simple proteomic approach has been made to identify several fish cell lines derived from tissues of the same or differing species. Protein expression signatures (PES) of the evaluated fish cell lines have been developed using 2D gel electrophoresis and image analysis. A higher degree of concordance was seen among cell lines derived from rainbow trout, than from other fish species. This proteomic approach could thus serve as an additional, valuable and reliable technique for the identification of fish cell lines. (NSERC)

The expressed protein fingerprints of MCF7 breast cancer cell homogenates were mapped using both one-dimensional and two-dimensional capillary electrophoresis (CE).  While one-dimensional CE can resolve dozens of proteins, two-dimensional CE has the potential to resolve hundreds or thousands of proteins with broad dynamic range. Homogenate proteins are labeled by coupling lysine residues with the fluorogenic reagent 3-(2-furoyl)quinoline-2-carboxaldehyde.  Labeled proteins are detected by laser-induced fluorescence in a sheath flow cuvette.  In each two-dimensional separation two of the following modes are interfaced: capillary sieving electrophoresis, capillary zone electrophoresis, or micellar electrokinetic chromatography.  Coupling two of these orthogonal separation modes increases the number of resolved proteins from MCF7 cellular homogenates.  While two-dimensional gel electrophoresis remains the most commonly used technique for protein separation, it also has several drawbacks including: limited dynamic range, difficulty focusing highly basic or acidic proteins, poor quantitation, long experiment duration, and unautomatibility.  We suggest that two-dimensional CE is a powerful alternative to standard gel electrophoresis.  Two-dimensional CE lacks many of the drawbacks of traditional gel electrophoresis, and we have demonstrated that the technique can automatically and rapidly produce protein fingerprints from complex cellular samples.  We use this methodology to study breast cancer because it has been shown that breast cancer prognosis is correlated with cancer cell protein expression.  By correlating protein expression with prognosis it is possible that patients could receive more reliable prognoses.