A bioanalytical method for the proteome wide display and analysis of protein complexes from whole plant cell lysates.

While protein interaction studies and protein network modeling come to the forefront, the isolation and identification of protein complexes in a cellular context remains a major challenge for plant science. To this end, a nondenaturing extraction procedure was optimized for plant whole cell matrices and the combined use of gel filtration and BN-PAGE for the separation of protein complexes was studied. Hyphenation to denaturing electrophoresis and mass spectrometric analysis allows for the simultaneous identification of multiple (previously unidentified) protein interactions in single samples. The reliability and efficacy of the technique was confirmed (i) by the identification of well-studied plant protein complexes, (ii) by the presence of nonplant interologs for several of the novel complexes (iii) by presenting physical evidence of previously hypothetical plant protein interactions and (iv) by the confirmation of found interactions using co-IP. Furthermore practical issues concerning the use of this 2-D BN/SDS-PAGE display method for the analysis of protein-protein interactions are discussed.

A generic approach for "shotgun" analysis of the soluble proteome of plant cell suspension cultures.

Cell suspension cultures from different plant species act as important model systems for studying cellular processes in plant biology and are often used as "green factories" for the production of valuable secondary metabolites and recombinant proteins. While mass spectrometry based proteome analysis techniques are ideally suited to study plant cell metabolism and other fundamental cellular processes from a birds eye perspective, they remain underused in plant studies. We describe a comprehensive sample preparation and multidimensional 'shotgun' proteomics strategy that can be generically applied to plant cell suspension cultures. This strategy was optimized and tested on an Arabidopsis thaliana ecotype Landsberg erecta culture. Furthermore, the implementation of strong cation exchange chromatography as a peptide fractionation step is elaborately tested. Its utility in mass spectrometry based proteome analysis is discussed. Using the presented analytical platform, over 13,000 unique peptides and 2640 proteins could be identified from a single plant cell suspension sample. Finally, the experimental setup is validated using Nicotiana tabacum cv. "Bright Yellow-2" (BY-2) plant cell suspension cultures, thereby demonstrating that the presented analytical platform can also be valuable tool in proteome analysis of non-genomic model systems.

Next generation functional proteomics in non-model plants: A survey on techniques and applications for the analysis of protein complexes and post-translational modifications.

The congruent development of computational technology, bioinformatics and analytical instrumentation makes proteomics ready for the next leap. Present-day state of the art proteomics grew from a descriptive method towards a full stake holder in systems biology. High throughput and genome wide studies are now made at the functional level. These include quantitative aspects, functional aspects with respect to protein interactions as well as post translational modifications and advanced computational methods that aid in predicting protein function and mapping these functionalities across the species border. In this review an overview is given of the current status of these aspects in plant studies with special attention to non-genomic model plants.

Unraveling tobacco BY-2 protein complexes with BN PAGE/LC-MS/MS and clustering methods.

To understand physiological processes, insight into protein complexes is very important. Through a combination of blue native gel electrophoresis and LC-MS/MS, we were able to isolate protein complexes and identify their potential subunits from Nicotiana tabacum cv. Bright Yellow-2. For this purpose, a bioanalytical approach was used that works without a priori knowledge of the interacting proteins. Different clustering methods (e.g., k-means and hierarchical clustering) and a biclustering approach were evaluated according to their ability to group proteins by their migration profile and to correlate the proteins to a specific complex. The biclustering approach was identified as a very powerful tool for the exploration of protein complexes of whole cell lysates since it allows for the promiscuous nature of proteins. Furthermore, it searches for associations between proteins that co-occur frequently throughout the BN gel, which increases the confidence of the putative associations between co-migrating proteins. The statistical significance and biological relevance of the profile clusters were verified using functional gene ontology annotation. The proof of concept for identifying protein complexes by our BN PAGE/LC-MS/MS approach is provided through the analysis of known protein complexes. Both well characterized long-lived protein complexes as well as potential temporary sequential multi-enzyme complexes were characterized.

Molecular targets of TBBPA in zebrafish analysed through integration of genomic and proteomic approaches.

Tetrabromobisphenol-A (TBBPA) is nowadays one of the most frequently used brominated flame retardants (BFRs) and can be considered as a high production volume chemical. Over the last decade, numerous reports of increasing concentrations of BFRs in the environment and humans have been published. However, the toxicological knowledge on TBBPA, and more specifically its molecular mode of action, is rather fragmentary. In this study two populations of adult zebrafish (Danio rerio) were exposed for 14 days to 0.75 microM and 1.5 microM TBBPA. Subsequently, we employed a combined transcriptomic and proteomic approach to evaluate the molecular effects of TBBPA in zebrafish liver. Oligonucleotide microarrays were used to study the effects on gene expression levels. These results were validated through real-time PCR. The proteome of the liver was analysed by means of differential in-gel electrophoresis (DiGE), an innovative application of traditional 2D-PAGE. Combination of the extracted datasets allowed reassembling of individual molecular responses into a comprehensive overview of affected molecular pathways. Interpretation of the results depicted an interference of thyroid and Vitamin A homeostasis in the exposed zebrafish, TBBPA also elicited responses indicating onset of oxidative stress and general stress responses. Additionally, numerous differentially expressed transcripts could be associated with defence mechanisms or corresponded to metabolizing enzymes. Furthermore, cellular metabolism was clearly affected, illustrated as disturbance of e.g. lipid, carbohydrate, and organic acid metabolic processes. Summarizing, these results enabled us to hypothesize several working mechanisms of TBBPA and demonstrated the potential of a combined genome and proteome approach to generate detailed mechanistic toxicological information.

A tandem affinity purification-based technology platform to study the cell cycle interactome in Arabidopsis thaliana.

Defining protein complexes is critical to virtually all aspects of cell biology because many cellular processes are regulated by stable protein complexes, and their identification often provides insights into their function. We describe the development and application of a high throughput tandem affinity purification/mass spectrometry platform for cell suspension cultures to analyze cell cycle-related protein complexes in Arabidopsis thaliana. Elucidation of this protein-protein interaction network is essential to fully understand the functional differences between the highly redundant cyclin-dependent kinase/cyclin modules, which are generally accepted to play a central role in cell cycle control, in all eukaryotes. Cell suspension cultures were chosen because they provide an unlimited supply of protein extracts of actively dividing and undifferentiated cells, which is crucial for a systematic study of the cell cycle interactome in the absence of plant development. Here we report the mapping of a protein interaction network around six known core cell cycle proteins by an integrated approach comprising generic Gateway-based vectors with high cloning flexibility, the fast generation of transgenic suspension cultures, tandem affinity purification adapted for plant cells, matrix-assisted laser desorption ionization tandem mass spectrometry, data analysis, and functional assays. We identified 28 new molecular associations and confirmed 14 previously described interactions. This systemic approach provides new insights into the basic cell cycle control mechanisms and is generally applicable to other pathways in plants.